The present invention relates to modified variants of human alpha-1 -microglobulin pro- tein with improved properties and the use of such variants in medical treatment and diagnostics. The inventors have surprisingly found that introduction of specific amino acid substitutions and / or addition of specific N-terminal extensions confer improved properties to alpha-1 -microglobulin with regard to stability, solubility, and binding of heme. Background of the invention

A1 M (a-i-microglobulin) is a low molecular weight protein with an extracellular tissue- cleaning function (Ekstrom et al., 1977; Akerstrom and Gram, 2014). It is present in all tissues and organs in fish, birds, rodents, mammals and other vertebrates. A1 M is synthesized mainly in the liver but also at a lower rate in most other cells in the body. It is encoded by the a-i-microglobulin-bikunin precursor gene (AMBP) and translated in all cells and species as a continuous peptide precursor together with another protein, bikunin (Kaumeyer et al., 1986). However, the two proteins are separated by protease cleavage, processed and secreted into the blood as two different proteins with different functions (Lindqvist et al., 1992; Bratt et al., 1993). The reason for the ubiquitous co- synthesis of A1 M and bikunin is still unknown. In the blood, about 50% of A1 M is found in free, monomeric form, and the remaining 50% as high-molecular weight complexes covalently bound with immunoglobulin A, albumin and prothrombin (Berggard et al., 1997). A1 M is a one-domain, 183-amino acid, glycosylated protein. The crystal structure of a large fragment of A1 M expressed in E.coli was recently published (Meining and Skerra, 2012). Based on its structure, A1 M belongs to a protein family, Lipocalins, with 50 or more members from animals, plants and bacteria (Flower, 1996; Akerstrom et al., 2006). The lipocalins have a common three-dimensional structure which consists of eight antiparallel β-strands forming a barrel with one closed end (bottom) and an open end (top). The barrel functions as a pocket for hydrophobic ligands in most lipocalins. Four loops (loop 1 -4), which make up the rim of the open end of the barrel, vary highly in length and composition between the various lipocalins. In A1 M, a handful of amino acid side-groups located on these loops, or on the inside of the pocket, have been shown to be important for the identified functions of the protein. Thus, a free cysteine, C34, located on a short helix on loop 1 , has a negative reduction potential and gives A1 M reductase properties (Allhorn et al., 2005). Two tyrosine residues, Y22 and 132, were shown to be covalently modified by radical oxidation products in vitro (Akerstrom et al., 2007). Four lysine residues, K69, 92, 1 18 and 130, regulate the reductase activity (Allhorn et al., 2005), influence the binding of free heme groups (Rutardottir et al., 2015), and are covalently modified on A1 M purified from human urine and amniotic fluid with low molecular weight yellow-brown, heterogeneous substances (Berggard et al., 1999; Sala et al., 2004).

Employing the reductase activity, radical scavenging and heme-binding properties, A1 M acts an antioxidant that protects cells and tissues from oxidative damage. A1 M was shown to protect in vitro blood cell cultures, placenta tissue and skin against oxidative damage from hemoglobin, heme and reactive oxygen species (ROS) (Olsson et al., 2008; May et al., 201 1 ; Olsson et al., PloS One 201 1 ; Olsson et al., ARS 2012). A1 M also showed in vivo protective effects in rats and rabbits against placenta and kid- ney tissue damage after hemoglobin infusion (Sverrison et al., 2014; Naav et al., 2015). In a series of reports, hemoglobin and oxidative stress were shown to be involved in the pathogenesis of preeclampsia, a serious complication of pregnancy (Centlow et al., 2008; Hansson et al., 2013), and the levels of A1 M-mRNA and protein in liver, placenta and plasma were elevated in pregnant women with preeclampsia (Olsson et al., 2010; Anderson et al., 201 1 ). Therefore, it was suggested that A1 M may be employed as a therapeutic agent to treat pregnant women with preeclampsia to ameliorate the oxidative damage and thus the clinical symptoms of the disease (Olsson et al., 2010).

A production process of recombinant human A1 M was developed in E.coli and shown to possess the reductase, radical-binding and heme-binding properties (Kwasek et al., Allhorn et al., 2002; 2005; Akerstrom et al., 2007). Indeed, this recombinant A1 M variant showed in vivo therapeutic effects in a sheep model of preeclampsia (Wester- Rosenlof et al., 2014). However, although functional, E.coli-expressed recombinant A1 M lacks glycosylation, and has poor solubility and stability compared to human A1 M purified from urine or plasma. The lack of stability and solubility of the protein limits its use as a drug for human use, mainly due to difficulties to obtain highly concentrated solutions and long-term storage conditions in buffers at physiological pH and salt conditions. Accordingly, there is a need for developing a protein with structural similarities with human A1 M, but with improved properties regarding stability and solubility. Moreover, the protein should be relatively easy to prepare in amounts suitable for therapeutic use. Detailed description of the invention

The present invention relates to novel alpha-1 -microglobulin proteins with improved stability and solubility profiles.

Site-directed and additive mutagenesis was used to engineer A1 M-species with re- tained, or possibly enhanced functional properties, but with improved protein stability and solubility that allow long-term storage at high concentrations in physiological buffers.

As it appears from the Examples herein, four lines of reasoning were followed when se- lecting the positions and identities of mutated amino acid side-groups:

1 ) Animal homologues from a variety of species with different expected environmental pressure in terms of oxidative stress, temperature, oxygen pressure were expressed (N=12);

2) Single amino acid substitutions that occur frequently among the 56 sequenced A1 M- homologues at positions located in loops 1 -4 or the interior surface of the hydrophobic pocket, were introduced into the human gene construct and expressed (N=3); and

3) Addition or removal of favourably located lysyl or tyrosyl residues, based on the hypothesis that these may infuence pKa of the C35 thiolyl (Allhorn et al., 2005) or serve as radical-trapping sites (Berggard et al., 1999; Sala et al., 2004; Akerstrom et al.

2007) (N=5); 4).

In addition, the influence of N-terminal, charged and hydrophilic extensions were tested on some A1 M-variants. The rationale behind the design of the tested N-terminal extensions was to add 1 ) a tag for purification (e.g. His-tag), 2) a linker to separate the tag from the core of the A1 M protein, 3) several (1-5) charged amino acid side-groups conferring hydrophilic properties to the protein in order to gain maximal stability and solubility in water-solutions, 4) without compromising the physiological functions of A1 M.

The project was divided into three major phases:

Phase I) Expression of the 27 A1 M-variants described above followed by analysis of solubility, stability and function; Phase II) Design, expression and analysis of a few A1 M-variants with expected optimal properties based on the outcome of phase 1 ; and

Phase III) Design, expression and analysis of non-mutated wildtype (wt)-A1 M and the most successful mutated A1 M-variant equipped with or without N-terminal, charged and hydrophilic extensions.

As will be explained in more details herein, the present invention provides an A1 M-de- rived protein having the amino acid sequence of formula I: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ -X ¹² -X ¹³ -X ¹⁴ -GPVPTPPDN IQVQENF-X ¹⁵ -IS

RIYGKWYNLA IGSTCPWLKK l-X ¹⁶ -DRMTVSTL VLGEGATEAE ISMTST-X ¹⁷ - WRK GVCEETSGAY EKTDTDGKFL YHKSKW-X ¹⁸ -ITM ESYWHTNYD EY- AIFLTKKF SRHHGPTITA KLYGRAPQLR ETLLQDFRW AQGVGIPEDS IFT- MADRGEC VPGEQEPEPI LIPR (formula I) wherein at least one X is present and (in parentheses are suggestions for further substitutions)

X ^I is absent or represents Met or N-formyl Met;

X ² is absent or represents His;

X ³ is absent or represents His;

X ⁴ is absent or represents His;

X ⁵ is absent or represents His;

X ⁶ is absent or represents His;

X ⁷ is absent or represents His;

X ⁸ is absent or represents His;

X ⁹ is absent or represents His;

X ¹⁰ is absent or selected from Asp and Glu, Lys and Arg;

X ^{I I} is absent or selected from Asp and Glu, Lys and Arg;

X ¹² is absent or selected from Asp and Glu, Lys and Arg;

X ¹³ is absent or selected from Asp and Glu, Lys and Arg;

X ¹⁴ is absent or represents Lys Glu, Asp or Arg or Met or N-formyl Met;

X ¹⁵ represents Asp or Asn or Glu;

X ¹⁶ represents Met or Lys or Arg;

X ¹⁷ represents Arg or His or Lys;

X ¹⁸ represents Asp or Asn or Glu;

or a pharmaceutically acceptable salt thereof, with the proviso that when all X ¹ -X ¹⁴ are absent, X ¹⁵ represents Asn, X ¹⁶ represents Met, and X ¹⁷ represents Arg, then X ¹⁸ cannot represent Asn. The invention also provides a derivative of A1 M, i.e. X ¹ -X ¹⁴ -A1 M, wherein A1 M may be any A1 M obtained from the species mentioned in Table 2 (i.e. human, mouse, naked mole-rat, frog, chicken, rabbit, squirrel monkey, walrus, manatee, plaice and orangutan). The present inventors have found that inclusion of X ¹ -X ¹⁴ imparts improved properties at least to human A1 M, and accordingly, it is contemplated that this start se- quence also can impart important improved properties to A1 M from other species or to species-recombinant A1 M.

The present invention also provides an A1 M-derived protein having the amino acid sequence of formula II:

X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ -X ¹² -X ¹³ -X ¹⁴ -GPVPTPPDN IQVQENF-X ¹⁵ -IS RIYGKWYNLA IGSTCPWLKK l-X ¹⁶ -DRMTVSTL VLGEGATEAE ISMTST-X ¹⁷ - WRK GVCEETSGAY EKTDTDGKFL YHKSKW-X ¹⁸ -ITM ESYWHTNYD EY- AIFLTKKF SRHHGPTITA KLYGRAPQLR ETLLQDFRW AQGVGIPEDS IFT- MADRGEC VPGEQEPEPI (formula II)

wherein at least one X is present and (in parentheses are suggestions for further substitutions)

X ^I is absent or represents Met or N-formyl Met;

X ² is absent or represents His;

X ³ is absent or represents His;

X ⁴ is absent or represents His;

X ⁵ is absent or represents His;

X ⁶ is absent or represents His;

X ⁷ is absent or represents His;

X ⁸ is absent or represents His;

X ⁹ is absent or represents His;

X ¹⁰ is absent or selected from Asp and Glu, Lys and Arg;

X ^{I I} is absent or selected from Asp and Glu, Lys and Arg;

X ¹² is absent or selected from Asp and Glu, Lys and Arg;

X ¹³ is absent or selected from Asp and Glu, Lys and Arg;

X ¹⁴ is absent or represents Lys or Glu, Asp or Arg or Met or N-formyl Met; X ¹⁵ represents Asp or Asn or Glu;

X ¹⁶ represents Met or Lys or Arg;

X ¹⁷ represents Arg or His or Lys;

X ¹⁸ represents Asp or Asn or Glu;

or a pharmaceutically acceptable salt thereof, with the proviso that when all X ¹ -X ¹⁴ are absent, X ¹⁵ represents Asn, X ¹⁶ represents Met, and X ¹⁷ represents Arg, then X ¹⁸ cannot represent Asn. The invention also provides a derivative of A1 M, i.e. X ¹ -X ¹⁴ -A1 M, wherein A1 M may be any A1 M obtained from the species mentioned in Table 2 (i.e. human, mouse, naked mole-rat, frog, chicken, rabbit, squirrel monkey, walrus, manatee, plaice and orangutan), wherein A1 M is truncated C-terminally, so that the C-terminal tetrapeptide sequence LI PR does not form part of the protein.

As it appears from the Examples herein, the present inventors have found that:

The initial sequence X ¹ -X ¹⁴ seems to impart improved properties to A1 M, Point mutation M41 K, R66H or N 17,96D of the A1 M molecule imparts improved stability with maintained function of A1 M,

Mutations (M41 K + R66H), (M41 K + N17.96D), (R66H + N17.96D), and/or (M41 K + R66H + N17,96D) show increased solubility and/or stability with maintained function,

Mutation (R66H + N17,96D) had best overall performance in the experiments performed,

A1 M with mutations (R66H + N17,96D) and initial sequences

MHHHHHHHHGGGGGIEGR (M8H5GIEGR); MHHHHHHHHDDDDK (M8H4DK), MHHHHHHDDDDK (M6H4DK) or MHHHHHHHH (M8H) as N- terminal sequences have been tested, and the protein variants with M8H4DK as N-terminal sequence showed higher solubility and/or stability compared with the other N-terminal extensions.

Truncation of the C-terminal of A1 M seems to impart improved heme binding and degradation.

It is interesting to note that this N-terminal extension sequence resembles a His-tag with an enterokinase cleavage site, but it is without the ability to cleave the His-tag from A1 M as the amino acid Ala is not included. The presence of Ala in a DDDKA indicate the enterokinase cleavage site. Thus, it is contemplated that the N-terminal sequence itself imparts improved protein stability and solubility properties to A1 M when the repeated His-residues are followed by five charged amino acids.

Based on these observations, it is contemplated that variation of an A1 M protein along the lines indicated above will provide proteins with A1 M functionality, but with improved characteristics regarding stability and/or solubility. Thus, the present invention relates to all possible combinations of A1 M containing X ¹ - X ¹⁸ as described above.

More specifically, the following A1 M derived proteins are within the scope of the present invention, such that the all proteins may be full-length corresponding to human wild type A1 M; or may be truncated C-terminally, i.e. without LIPR:

P017240DK1 8

Position in hAlM

Tag Mutations Compound XI X2 X3 X4

Broadest claim 1 Met/Absent His/absent His/absent His/absent

6-His 'in sequence' mutations can be any 2 (f)Met His His His

8-His in sequence' mutations can be any 3 (f)Met His His His

No tag; in sequence' mutations can be any 4 Absent Absent Absent Absent

Any tag M41K 5 Met/Absent His/absent His/absent His/absent

Any tag N17,96D 6 Met/Absent His/absent His/absent His/absent

No tag N17D 7 Absent Absent Absent Absent

No tag N17,96D 8 Absent Absent Absent Absent

No tag N96D 9 Absent Absent Absent Absent

No tag N17,96D, R66H 10 Absent Absent Absent Absent

No tag M41K 11 Absent Absent Absent Absent

No tag R66H 12 Absent Absent Absent Absent

No tag N17,96D, M41K, R66H 13 Absent Absent Absent Absent

No tag N17D, R66H 14 Absent Absent Absent Absent

No tag R66H, N96D 15 Absent Absent Absent Absent

No tag M41K, R66H 16 Absent Absent Absent Absent

No tag N17,96D, M41K 17 Absent Absent Absent Absent

No tag N17D, M41K 18 Absent Absent Absent Absent

No tag M41K, N96D 19 Absent Absent Absent Absent

Any tag N17,96D, R66H 20 Met/Absent His/absent His/absent His/absent

No tag N17D, M41K, R66H 21 Absent Absent Absent Absent

No tag M41K, R66H, N96D 22 Absent Absent Absent Absent

6His N17D 23 (f)Met His His His

6His N17,96D 24 (f)Met His His His

6His N96D 25 (f)Met His His His

6His N17,96D, R66H 26 (f)Met His His His

P017240DK1 9

Position in hAlM

Tag Mutations Compound XI X2 X3 X4

6His M41K 27 (f)Met His His His

6His R66H 28 (f)Met His His His

6His N17,96D, M41K, R66H 29 (f)Met His His His

6His N17D, R66H 30 (f)Met His His His

6His R66H, N96D 31 (f)Met His His His

6His M41K, R66H 32 (f)Met His His His

6His N17,96D, M41K 33 (f)Met His His His

6His N17D, M41K 34 (f)Met His His His

6His M41K, N96D 35 (f)Met His His His

6His N17D, M41K, R66H 36 (f)Met His His His

6His M41K, R66H, N96D 37 (f)Met His His His

8His N17D 38 (f)Met His His His

8His N17,96D 39 (f)Met His His His

8His N96D 40 (f)Met His His His

8His N17,96D, R66H 41 (f)Met His His His

8His M41K 42 (f)Met His His His

8His R66H 43 (f)Met His His His

8His N17,96D, M41K, R66H 44 (f)Met His His His

8His N17D, R66H 45 (f)Met His His His

8His R66H, N96D 46 (f)Met His His His

8His M41K, R66H 47 (f)Met His His His

8His N17,96D, M41K 48 (f)Met His His His

8His N17D, M41K 49 (f)Met His His His

8His M41K, N96D 50 (f)Met His His His

8His N17D, M41K, R66H 51 (f)Met His His His

8His M41K, R66H, N96D 52 (f)Met His His His

P017240DK1 10

Position in hAlM Con'd

Tag Mutations Compound X5 X6 X7 X8 X9 X10

Broadest claim 1 His/absent His/absent His/absent His/absent His/absent Asp/a bsent/(

6-His 'in sequence' mutations can be any 2 His His His Absent Absent Asp

8-His in sequence' mutations can be any 3 His His His His His Asp

No tag; in sequence' mutations can be any 4 Absent Absent Absent Absent Absent Absent

Any tag M41K 5 His/absent His/absent His/absent His/absent His/absent Asp/a bsent/Gl

Any tag N17,96D 6 His/absent His/absent His/absent His/absent His/absent Asp/a bsent/Gl

No tag N17D 7 Absent Absent Absent Absent Absent Absent

No tag N17,96D 8 Absent Absent Absent Absent Absent Absent

No tag N96D 9 Absent Absent Absent Absent Absent Absent

No tag N17,96D, R66H 10 Absent Absent Absent Absent Absent Absent

No tag M41K 11 Absent Absent Absent Absent Absent Absent

No tag R66H 12 Absent Absent Absent Absent Absent Absent

No tag N17,96D, M41K, R66H 13 Absent Absent Absent Absent Absent Absent

No tag N17D, R66H 14 Absent Absent Absent Absent Absent Absent

No tag R66H, N96D 15 Absent Absent Absent Absent Absent Absent

No tag M41K, R66H 16 Absent Absent Absent Absent Absent Absent

No tag N17,96D, M41K 17 Absent Absent Absent Absent Absent Absent

No tag N17D, M41K 18 Absent Absent Absent Absent Absent Absent

No tag M41K, N96D 19 Absent Absent Absent Absent Absent Absent

Any tag N17,96D, R66H 20 His/absent His/absent His/absent His/absent His/absent Asp/a bsent/Gl

No tag N17D, M41K, R66H 21 Absent Absent Absent Absent Absent Absent

No tag M41K, R66H, N96D 22 Absent Absent Absent Absent Absent Absent

6His N17D 23 His His His Absent Absent Asp

6His N17,96D 24 His His His Absent Absent Asp

6His N96D 25 His His His Absent Absent Asp

6His N17,96D, R66H 26 His His His Absent Absent Asp

P017240DK1 1 1

Position in hAlM on'd

Tag Mutations Compound X5 X6 X7 X8 X9 XIO

6His M41K 27 His His His Absent Absent Asp

6His R66H 28 His His His Absent Absent Asp

6His N17,96D, M41K, R66H 29 His His His Absent Absent Asp

6His N17D, R66H 30 His His His Absent Absent Asp

6His R66H, N96D 31 His His His Absent Absent Asp

6His M41K, R66H 32 His His His Absent Absent Asp

6His N17,96D, M41K 33 His His His Absent Absent Asp

6His N17D, M41K 34 His His His Absent Absent Asp

6His M41K, N96D 35 His His His Absent Absent Asp

6His N17D, M41K, R66H 36 His His His Absent Absent Asp

6His M41K, R66H, N96D 37 His His His Absent Absent Asp

8His N17D 38 His His His His His Asp

8His N17,96D 39 His His His His His Asp

8His N96D 40 His His His His His Asp

8His N17,96D, R66H 41 His His His His His Asp

8His M41K 42 His His His His His Asp

8His R66H 43 His His His His His Asp

8His N17,96D, M41K, R66H 44 His His His His His Asp

8His N17D, R66H 45 His His His His His Asp

8His R66H, N96D 46 His His His His His Asp

8His M41K, R66H 47 His His His His His Asp

8His N17,96D, M41K 48 His His His His His Asp

8His N17D, M41K 49 His His His His His Asp

8His M41K, N96D 50 His His His His His Asp

8His N17D, M41K, R66H 51 His His His His His Asp

8His M41K, R66H, N96D 52 His His His His His Asp

P017240DK1 12

Position in hAlM Con'd

Tag Mutations Compound Xll X12 X13

Broadest claim 1 Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg

6-His 'in sequence' mutations can be any 2 Asp Asp Asp

8-His in sequence' mutations can be any 3 Asp Asp Asp

No tag; in sequence' mutations can be any 4 Absent Absent Absent

Any tag M41K 5 Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg

Any tag N17,96D 6 Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg

No tag N17D 7 Absent Absent Absent

No tag N17,96D 8 Absent Absent Absent

No tag N96D 9 Absent Absent Absent

No tag N17,96D, R66H 10 Absent Absent Absent

No tag M41K 11 Absent Absent Absent

No tag R66H 12 Absent Absent Absent

No tag N17,96D, M41K, R66H 13 Absent Absent Absent

No tag N17D, R66H 14 Absent Absent Absent

No tag R66H, N96D 15 Absent Absent Absent

No tag M41K, R66H 16 Absent Absent Absent

No tag N17,96D, M41K 17 Absent Absent Absent

No tag N17D, M41K 18 Absent Absent Absent

No tag M41K, N96D 19 Absent Absent Absent

Any tag N17,96D, R66H 20 Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg Asp/a bsent/Glu/Lys/Arg

No tag N17D, M41K, R66H 21 Absent Absent Absent

No tag M41K, R66H, N96D 22 Absent Absent Absent

6His N17D 23 Asp Asp Asp

6His N17,96D 24 Asp Asp Asp

6His N96D 25 Asp Asp Asp

6His N17,96D, R66H 26 Asp Asp Asp

P017240DK1 13

Position in hAlM Con'd

Tag Mutations Compound Xll X12 X13 is M41K 27 Asp Asp Asp is R66H 28 Asp Asp Asp is N17,96D, M41K, R66H 29 Asp Asp Asp is N17D, R66H 30 Asp Asp Asp is R66H, N96D 31 Asp Asp Asp is M41K, R66H 32 Asp Asp Asp is N17,96D, M41K 33 Asp Asp Asp is N17D, M41K 34 Asp Asp Asp is M41K, N96D 35 Asp Asp Asp is N17D, M41K, R66H 36 Asp Asp Asp is M41K, R66H, N96D 37 Asp Asp Asp is N17D 38 Asp Asp Asp is N17,96D 39 Asp Asp Asp is N96D 40 Asp Asp Asp is N17,96D, R66H 41 Asp Asp Asp is M41K 42 Asp Asp Asp is R66H 43 Asp Asp Asp is N17,96D, M41K, R66H 44 Asp Asp Asp is N17D, R66H 45 Asp Asp Asp is R66H, N96D 46 Asp Asp Asp is M41K, R66H 47 Asp Asp Asp is N17,96D, M41K 48 Asp Asp Asp is N17D, M41K 49 Asp Asp Asp is M41K, N96D 50 Asp Asp Asp is N17D, M41K, R66H 51 Asp Asp Asp is M41K, R66H, N96D 52 Asp Asp Asp

P017240DK1 14

Position in hAlM Con'd

Tag Mutations Compound X14 X15 X16 X17

Broadest claim 1 Lys/(f)Met/Absent/Glu/Asp/Arg Asp/Asn Met/Lys/Arg Arg/His/Lys

6-His 'in sequence' mutations can be any 2 Lys Asp/Asn Met/Lys/Arg Arg/His/Lys

8-His in sequence' mutations can be any 3 Lys Asp/Asn Met/Lys/Arg Arg/His/Lys

No tag; in sequence' mutations can be any 4 Absent Asp/Asn Met/Lys/Arg Arg/His/Lys

Any tag M41K 5 Lys/(f)Met/Absent/Glu/Asp/Arg Asn Lys Arg

Any tag N17,96D 6 Lys/(f)Met/Absent Glu/Asp/Arg Asp Met Arg

No tag N17D 7 (f)Met Asp Met Arg

No tag N17,96D 8 (f)Met Asp Met Arg

No tag N96D 9 (f)Met Asn Met Arg

No tag N17,96D, R66H 10 (f)Met Asp Met His

No tag M41K 11 (f)Met Asn Lys Arg

No tag R66H 12 (f)Met Asp Met His

No tag N17,96D, M41K, R66H 13 (f)Met Asp Lys His

No tag N17D, R66H 14 (f)Met Asp Met His

No tag R66H, N96D 15 (f)Met Asn Met His

No tag M41K, R66H 16 (f)Met Asn Lys His

No tag N17,96D, M41K 17 (f)Met Asp Lys Arg

No tag N17D, M41K 18 (f)Met Asp Lys Arg

No tag M41K, N96D 19 (f)Met Asn Lys Arg

Any tag N17,96D, R66H 20 Lys/(f)Met/Absent Glu/Asp/Arg Asp Met His

No tag N17D, M41K, R66H 21 (f)Met Asp Lys His

No tag M41K, R66H, N96D 22 (f)Met Asn Lys His

6His N17D 23 Lys Asp Met Arg

6His N17,96D 24 Lys Asp Met Arg

6His N96D 25 Lys Asn Met Arg

6His N17,96D, R66H 26 Lys Asp Met His

P017240DK1 15

Position in hAlM Con'd

Tag Mutations Compound X14 X15 X16 X17 X18 is M41K 27 Lys Asn Lys Arg Asn is R66H 28 Lys Asn Met His Asn is N17,96D, M41K, R66H 29 Lys Asp Lys His Asp is N17D, R66H 30 Lys Asp Met His Asn is R66H, N96D 31 Lys Asn Met His Asp is M41K, R66H 32 Lys Asn Lys His Asn is N17,96D, M41K 33 Lys Asp Lys Arg Asp is N17D, M41K 34 Lys Asp Lys Arg Asn is M41K, N96D 35 Lys Asn Lys Arg Asp is N17D, M41K, R66H 36 Lys Asp Lys His Asn is M41K, R66H, N96D 37 Lys Asn Lys His Asp is N17D 38 Lys Asp Met Arg Asn is N17,96D 39 Lys Asp Met Arg Asp is N96D 40 Lys Asn Met Arg Asp is N17,96D, R66H 41 Lys Asp Met His Asp is M41K 42 Lys Asn Lys Arg Asn is R66H 43 Lys Asn Met His Asn is N17,96D, M41K, R66H 44 Lys Asp Lys His Asp is N17D, R66H 45 Lys Asp Met His Asn is R66H, N96D 46 Lys Asn Met His Asp is M41K, R66H 47 Lys Asn Lys His Asn is N17,96D, M41K 48 Lys Asp Lys Arg Asp is N17D, M41K 49 Lys Asp Lys Arg Asn is M41K, N96D 50 Lys Asn Lys Arg Asp is N17D, M41K, R66H 51 Lys Asp Lys His Asn is M41K, R66H, N96D 52 Lys Asn Lys His Asp

P017240DK1 16

Position in hAlM Con'd Comment

Tag Mutations Compound

Broadest claim 1 Proviso: when all X1-X14 are absent, X15 represents Asn, X16

6-His 'in sequence' mutations can be any 2 and X17 represents Arg, then X18 cannot represent A:

8-His in sequence' mutations can be any 3

No tag; in sequence' mutations can be any 4 Proviso: when X15 represents Asn, X16 represents Met,

Any tag M41K 5 and X17 represents Arg, then X18 cannot represent Asn

Any tag N17,96D 6

No tag N17D 7

No tag N17,96D 8

No tag N96D 9

No tag N17,96D, R66H 10 Preferred mutation without tag

No tag M41K 11

No tag R66H 12

No tag N17,96D, M41K, R66H 13 All mutations

No tag N17D, R66H 14

No tag R66H, N96D 15

No tag M41K, R66H 16

No tag N17,96D, M41K 17

No tag N17D, M41K 18

No tag M41K, N96D 19

Any tag N17,96D, R66H 20

No tag N17D, M41K, R66H 21

No tag M41K, R66H, N96D 22

6His N17D 23

6His N17,96D 24

6His N96D 25

6His N17,96D, R66H 26 Preferred mutation with 6His

P017240DK1 17

Position in hAlM Con'd Comment

Tag Mutations Compound

6His M41K 27

6His R66H 28

6His N17,96D, M41K, R66H 29

6His N17D, R66H 30

6His R66H, N96D 31

6His M41K, R66H 32

6His N17,96D, M41K 33

6His N17D, M41K 34

6His M41K, N96D 35

6His N17D, M41K, R66H 36

6His M41K, R66H, N96D 37

8His N17D 38

8His N17,96D 39

8His N96D 40

8His N17,96D, R66H 41 Preferred mutation with 81-lis

8His M41K 42

8His R66H 43

8His N17,96D, M41K, R66H 44

8His N17D, R66H 45

8His R66H, N96D 46

8His M41K, R66H 47

8His N17,96D, M41K 48

8His N17D, M41K 49

8His M41K, N96D 50

8His N17D, M41K, R66H 51

8His M41K, R66H, N96D 52

Alpha- 1 -microglobulin - a general background

A1 M is synthesized in the liver at a high rate, secreted into the blood stream and transported across the vessel walls to the extravascular compartment of all organs. The protein is also synthesized in other tissues (blood cells, brain, kidney, skin) but at a lower rate. Due to the small size, free A1 M is rapidly filtered from blood in the kidneys.

A1 M is a member of the lipocalin superfamily, a group of proteins from animals, plants and bacteria with a conserved three-dimensional structure but very diverse functions. Each lipocalin consists of a 160-190-amino acid chain that is folded into a β-barrel pocket with a hydrophobic interior. At least twelve human lipocalin genes are known. A1 M is a 26 kDa plasma and tissue protein that so far has been identified in mammals, birds, fish and frogs. The three-dimensional structure of A1 M determined by X-ray crystallography is shown in Figure 10. A1 M is synthesized in the liver at a high rate, se- creted into the blood stream and rapidly (T½ = 2-3 min) transported across the vessel walls to the extravascular compartment of all organs. A1 M is found both in a free, mon- omeric form and as covalent complexes with larger molecules (IgA, albumin, prothrombin) in blood and interstitial tissues. Due to the small size, free A1 M is rapidly filtered from blood in the kidneys. The major portion is then readsorbed, but significant amounts are excreted to the urine.

Antioxidants are protective factors that eliminate oxidants or prevent harmful oxidation reactions. The human organism can produce antioxidants in response to oxidative stress. Such endogenous antioxidants include the superoxide-degrading enzyme su- peroxide dismutase (SOD), the hydrogen peroxide-degrading enzymes catalase and glutathione peroxidase, and the heme-degrading enzyme heme oxygenase-1 (HO-1 ). A1 M was recently shown to be involved in protecting against oxidative tissue damage by functioning both as a scavenger of radicals and heme as well as a reductase and inhibitor of oxidation. Several recent papers demonstrate that A1 M protects cell cultures and organ explants against oxidative damage, partly by accumulating in mitochondria and protecting mitochondrial function. Indeed, infusion of human recombinant A1 M has been successfully employed for in vivo treatment of the oxidative stress-related diseases preeclampsia and hemoglobin-induced glomerular injuries in animal models. Sequence and structural properties of A 1M The full sequence of human A1 M is known. The protein consists of a polypeptide with 183 amino acid residues. Many additional A1 M cDNAs and/or proteins have been detected, isolated and/or sequenced from other mammals, birds, amphibians, and fish. The length of the peptide chain of A1 M differs slightly among species, due mainly to variations in the C-terminus. Alignment comparisons of the different deduced amino acid sequences show that the percentage of identity varies from approximately 75-80% between rodents or ferungulates and man, down to approximately 45% between fish and mammals. A free cysteine side-chain at position 34 is conserved. This group has been shown to be involved in redox reactions (see below), in complex formation with other plasma proteins and in binding to a yellow-brown chromophore. The three-dimensional structure of A1 M shows that C34 is solvent exposed and located near the opening of the lipocalin pocket (see Figure 10).

In the present context the term "a-i-microglobulin" intends to cover a-i-microglobulin as identified in SEQ ID NO: 1 (human A1 M) , SEQ ID NO: 2 (human recombinant A1 M) and A1 M from other species, including homologues, fragments or variants thereof having similar therapeutic activities. Thus, A1 M as used herein is intended to mean a protein having at least 80% sequence identity with SEQ ID NO:1 or SEQ ID NO:2. It is preferred that A1 M as used herein has at least 90% sequence identity with SEQ ID NO:1 or SEQ ID NO:2. It is even more preferred that A1 M as used herein has at least 95% such as 99% or 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:2. In a preferred aspect, the a-i-microglobulin is in accordance with SEQ ID NO: 1 or 2 as identified herein. In the sequence listing the amino acid sequences of human A1 M and human recombinant A1 M (SEQ ID NOs 1 and 2, respectively) are given. However, homo- logues, variants and fragments of A1 M having the important parts of the proteins as identified in the following are also comprised in the term A1 M as used herein. Regarding alignments/identity see the following paragraph.

Details on alignment/identity

Positions of amino acid residues herein refer to the positions in human wt A1 M as it is found in human blood plasma (SEQ ID NO:1 ). When referring to amino acid residues in recombinant A1 M, which harbors a methionine or N-formyl methionine residue N-termi- nally linked to the glycine residue that is the initial residue in wt-A1 M (SEQ ID NO: 2), or in mutated human A1 M or A1 M from other species a person skilled in the art will un- derstand how to identify residues corresponding to residues in human wt-A1 M (SEQ ID NO:1 ) even when positions are shifted due to e.g. deletions or insertions. When recombinant proteins are produced they most often start with an initial Met residue, which may be removed using e.g. a methionine aminopeptidase or another enzyme with a similar activity. The A1 M variants presented here may be with or without an initial Met residue.

Homologues of A 1M

As mentioned above homologues of A1 M can also be used in accordance with the description herein. In theory A1 M from all species can be used for the purposes described herein including the most primitive found so far, which is from fish (plaice). A1 M is also available in isolated form from human, orangutan, squirrel monkey, rat, naked mole rat, mouse, rabbit, guinea pig, cow, frog, chicken, walrus, manatee and plaice.

Considering homologues, variants and fragments of A1 M, the following has been identified as important parts of the protein for the anti-oxidative effect:

Y22 (Tyrosine, pos 22, basepairs 64-66)

C34 (Cystein, position 34, basepairs 100-102)

K69 (Lysine, pos 69, basepairs 205-207)

K92 (Lysine, pos 92, basepairs 274-276)

K1 18 (Lysine, pos 1 18, basepairs 352-354)

K130 (Lysine, pos 130, basepairs 388-390)

Y132 (Tyrosine, pos 132, basepairs 394-396)

L180 (Leucine, pos 180, basepairs 538-540)

1181 (Isoleucine, pos 181 , basepairs 541 -543)

P182 (Proline, pos 182, basepairs 544-546)

R183 (Arginine, pos 183, basepairs 547-549)

Numbering of amino acids and nucleotides throughout the document refers to SEQ ID 1 ; if other A1 M from other species, A1 M analogs or recombinant sequences thereof are employed, a person skilled in the art will know how to identify the amino acids corre- sponding to the amino acids in SEQ ID NO: 1 .

Thus, in those cases, where A1 M eg has 80% (or 90% or 95%) sequence identity with one of SEQ ID NO: 1 or 2, it is preferred that the amino acids mentioned above are present at the appropriate places in the molecule. Human A1 M is substituted with oligosaccharides in three positions, two sialylated complex-type, probably diantennary carbohydrated linked to N17 and N96 and one more simple oligosaccharide linked to T5. The carbohydrate content of A1 M proteins from different species varies greatly, though, ranging from no glycosylation at all in Xenopus leavis over a spectrum of different glycosylation patterns. However, one glycosylation site, corresponding to N96 in man, is conserved in mammals, suggesting that this specific carbohydrate may be a more important constituent of the protein than the other two oligosaccharides. A1 M is yellow-brown-coloured when purified from plasma or urine. The colour is caused by heterogeneous compounds covalently bound to various amino acid side groups mainly located at the entrance to the pocket. These modifications represent the oxidized degradation products of organic oxidants covalently trapped by A1 M in vivo, for example heme, kynurenine and tyrosyl radicals.

A1 M is also charge- and size-heterogeneous and more highly brown-coloured A1 M- molecules are more negatively charged. The probable explanation for the heterogeneity is that different side-groups are modified to a varying degree with different radicals, and that the modifications alter the net charge of the protein. Covalently linked coloured substances have been localized to C34, and K92, K1 18 and K130, the latter with molecular masses between 100 and 300 Da. The tryptophan metabolite kynurenine was found covalently attached to lysyl residues in A1 M from urine of haemodialysis patients and appears to be the source of the brown colour of the protein in this case [6]. Oxidized fragments of the synthetic radical ABTS (2,2 ^' -azino-di-(3-ethylbenzothiazoline)-6- sulfonic acid) was bound to the side-chains of Y22 and Y132.

C34 is the reactive center of A1 M. It becomes very electronegative, meaning that it has a high potential to give away electrons, by the proximity of the positively charged side- chains of K69, K92, K1 18 and K130, which induce a deprotonization of the C34 thiol group which is a prerequisite of oxidation of the sulphur atom. Preliminary data shows that C34 is one of the most electronegative groups known.

Theoretically, the amino acids that characterize the properties of A1 M (C34, Y22, K92, K1 18, K130, Y132, L180, 1181 , P182, R183), which will be described in more detail be- low, can be arranged in a similar three-dimensional configuration on another frame- work, for instance a protein with the same global folding (another lipocalin) or a completely artificial organic or inorganic molecule such as a plastic polymer, a nanoparticle or metal polymer. The three-dimensional arrangement of some of these amino acids (blue ovals, the lysines are depicted by a„+"), the A1 M-framework (barrel), the electron-flow and the radical-trapping, are illustrated in Figure 10.

Accordingly, homologues, fragments or variants comprising a structure including the re- active centre and its surroundings as depicted above, are preferred.

Modifications and changes have been made in the structure of the polypeptides of this disclosure and still resulted in a molecule having similar functional characteristics as the original polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like functional properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1 .8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (- 1.3); proline (-1 .6); his- tidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1 ); glutamate (+3.0 ± 1 ); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ± 1 ); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1 .3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids the hydrophilicity values of which are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Glni His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Val: Lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.

In the present context, the homology between two amino acid sequences or between two nucleic acid sequences is described by the parameter "identity" (see also above). Alignments of sequences and calculation of homology scores may be done using a full Smith-Waterman alignment, useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA. Alignment may be made with the FASTA package version v20u6.

Multiple alignments of protein sequences may be made using "ClustalW". Multiple alignments of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence. Alternatively different software can be used for aligning amino acid sequences and

DNA sequences. The alignment of two amino acid sequences is e.g. determined by using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the global alignment algorithm described in. The substitution matrix used is BLOSUM62, gap opening penalty is 10, and gap extension pen- alty is 0.5.

The degree of identity between an amino acid sequence; e.g. SEQ ID NO: 1 and a different amino acid sequence (e.g. SEQ ID NO: 2) is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "SEQ ID NO: 1 " or the length of the " SEQ ID NO: 2 ", whichever is the shortest. The result is expressed in percent identity. See above regarding alignment and identity.

An exact match occurs when the two sequences have identical amino acid residues in the same positions of the overlap.

If relevant, the degree of identity between two nucleotide sequences can be determined by the Wilbur-Lipman method using the LASER- GENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise align- ment parameters are Ktuple=3, gap penalty=3, and windows=20.

The percentage of identity of an amino acid sequence of a polypeptide with, or to, amino acids of SEQ ID NO: 1 may be determined by i) aligning the two amino acid sequences using the Needle program, with the BLOSUM62 substitution matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5; ii) counting the number of exact matches in the alignment; iii) dividing the number of exact matches by the length of the shortest of the two amino acid sequences, and iv) converting the result of the division of iii) into percentage. The percentage of identity to, or with, other sequences of the invention is calculated in an analogous way. By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, and wherein said alterations may occur at the amino- or carboxy-terminus positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence, or in one or more contiguous groups within the reference sequence. Conservative amino acid variants can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-me- thyl-glycine, allo-threonine, methylthreonine, hydroxy-ethylcysteine, hydroxyethylhomo- cysteine, nitro-glutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2- azaphenyl-alanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylala- nine. Several methods are known in the art for incorporating non- naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppres- sor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. In a second method, translation is carried out in Xenopus oocytes by microinjection of mu- tated mRNA and chemically aminoacylated suppressor tRNAs. Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluor- ophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions. Alternative chemical structures providing a 3-dimensional structure sufficient to support the antioxidative properties of A1 M may be provided by other technologies e.g. artificial scaffolds, amino-acid substitutions and the like. Furthermore, structures mimicking the active sites of A1 M as listed above are contemplated as having the same therapeutic or physiologic function as A1 M.

Pharmaceutical compositions and dosage

The present invention also provides a kit comprising:

i) a pharmaceutical composition comprising a contrast medium, and

ii) a pharmaceutical composition comprising A1 M, any of the SEQ ID Nos: 1-10 and 17, or any of the A1 M derived proteins mentioned herein (or a mutant, analogue, fragment or variant as defined herein). In the following a listing of the sequences are given. The invention encompass all possible variations eg such as those illustrated herein.

SEQ ID NO: 1 : wt hA1 M

SEQ ID NO: 2: rhA1 M (i.e. Met-A1 M)

SEQ ID NO: 3: Preferred mutation without "extension" - N17,96D, R66H

SEQ ID NO: 4: No extension, M41 K

SEQ ID NO: 5: Preferred mutation with 6 His, N17,96D, R66H

SEQ ID NO: 6: 6His, M41 K

SEQ ID NO: 7: preferred mutation with 8 His extension, N17,96D, R66H

SEQ ID NO: 8: 8 His, M41 K

SEQ ID NO: 9: Extension + wt hA1 M

SEQ ID NO: 10: Omnibus A1 M with possible extensions.

SEQ ID NO: 1 1-16: segments of wt hA1 M

SEQ ID NO: 17: Omnibus C-terminally truncated A1 M with possible extensions

SEQ ID NO: 18: Preferred mutation without "extension" - N17,96D, R66; C-terminally truncated.

SEQ ID NO: 19: No extension, M41 K; C-terminally truncated.

SEQ ID NO: 20: Preferred mutation with 6 His, N17,96D, R66H; C-terminally truncated. SEQ ID NO: 21 : 6His, M41 K; C-terminally truncated.

SEQ ID NO: 22: preferred mutation with 8 His extension, N 17,96D, R66H; C-terminally truncated. SEQ ID NO: 23: 8 His, M41 K; C-terminally truncated.

The kit is in the form of one package containing the above-mentioned two compositions.

The pharmaceutical composition comprising a contrast medium is typically a composition already on the market.

The pharmaceutical composition comprising A1 M (or an analogue, fragment or variant thereof as defined herein) is intended for i.v. administration. Accordingly, A1 M can be formulated in a liquid, e.g. in a solution, a dispersion, an emulsion, a suspension etc.

For parenteral use suitable solvents include water, vegetable oils, propylene glycol and organic solvents generally approved for such purposes. In general, a person skilled in the art can find guidance in "Remington's Pharmaceutical Science" edited by Gennaro et al. (Mack Publishing Company), in "Handbook of Pharmaceutical Excipients" edited by Rowe et al. (PhP Press) and in official Monographs (e.g. Ph. Eur. or USP) relating to relevant excipients for specific formulation types and to methods for preparing a specific formulation.

A1 M will be administrated in one or several doses in connection to the administration of contrast medium. Preferably, each dose will be administrated i.v. either as a single dose, as a single dose followed by slow infusion during a short time-period up to 60 minutes, or only as a slow infusion during a short time-period up to 60 minutes. The first dose may be administrated at the same time as the contrast medium, or within a period of 0-60 minutes before to 0-30 minutes after injection of the contrast medium. Additional A1 M-doses can be added, but may not be necessary, after injection of the contrast medium. Each dose contains an amount of A1 M which is related to the body- weight of the patient: 1 -15 mg A1 M/kg of the patient.

Sequence Listing Free Text SEQ ID NO: 1

<223> Wildtype human A1 M, no mutations

SEQ ID NO: 2

<223> rhA1 M, ie N-terminal Met SEQ ID NO: 3

<223> hA1M, No tag, N-terminal Met, N17,96D; R66H

SEQ ID NO: 4

<223> hA1 M, not tag, N-terminal Met, M41 K

SEQ ID NO: 5

<223> 6His, N17,96D; R66H SEQ ID NO: 6

<223> hA1M, 6His, M41K

SEQ ID NO: 7

<223> 8His, N17,96D; R66H

SEQ ID NO: 8

<223> hA1M, 8His, M41K

SEQ ID NO: 9

<223> hA1 M, 8His, no mut

SEQ ID NO: 10

<211> 193

<212> PRT

<213> Homo sapiens

<220>

<221> VARIANT

<222> 1

<223> Xaa = Met or absent <220>

<221> VARIANT

<222> 1

<223> Xaa = Met or absent <220>

<221> VARIANT

<222> 2

<223> Xaa = His or absent <220>

<221> VARIANT

<222> 2

<223> Xaa = His or absent <220>

<221> VARIANT <222> 3

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 3

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 4

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 4

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 5

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 5

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 6

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 6

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 7

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 7

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 8

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 8

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 9

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 9

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 10

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 10

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 1 1

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 1 1

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 12

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 12 <223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221> VARIANT

<222> 13

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221> VARIANT

<222>13

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221> VARIANT

<222> 14

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221> VARIANT

<222> 14

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221> VARIANT

<222> 31

<223> Xaa = Asn or Asp

<220>

<221> VARIANT

<222> 55

<223> Xaa = Met or Lys

<220>

<221> VARIANT

<222> 80

<223> Xaa = Arg or His

<220>

<221> VARIANT

<222> 110

<223> Xaa = Asn or Asp

SEQ ID NO: 11

<223> Y1

SEQ ID NO: 12

<223> Y2 SEQ ID NO : 13

<223> Y3

SEQ ID NO : 14

<223> Y4

SEQ ID NO : 15

<223> Y5 SEQ ID NO : 16

<223> Y5

SEQ ID NO : 17

<21 1 > 197

<212> PRT

<213> Homo sapiens

<220>

<221 > VARIANT

<222> 1

<223> Xaa = Met or absent

<220>

<221 > VARIANT

<222> 1

<223> Xaa = Met or absent

<220>

<221 > VARIANT

<222> 2

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 2

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 3

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 3

<223> Xaa = His or absent

<220> <221 > VARIANT

<222> 4

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 4

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 5

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 5

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 6

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 6

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 7

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 7

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 8

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 8

<223> Xaa = His or absent <220>

<221 > VARIANT

<222> 9

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 9

<223> Xaa = His or absent

<220>

<221 > VARIANT

<222> 10

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT

<222> 10

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT

<222> 1 1

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT

<222> 1 1

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT

<222> 12

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT

<222> 12

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT

<222> 13

<223> Xaa = Asp, Glu, Lys, Arg, or absent <220>

<221 > VARIANT <222> 13

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 14

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 14

<223> Xaa = Asp, Glu, Lys, Arg, or absent

<220>

<221 > VARIANT

<222> 31

<223> Xaa = Asn or Asp

<220>

<221 > VARIANT

<222> 55

<223> Xaa = Met or Lys

<220>

<221 > VARIANT

<222> 80

<223> Xaa = Arg or His

<220>

<221 > VARIANT

<222> 1 10

<223> Xaa = Asn or Asp SEQ ID NO: 18

<223> hA1 M, No tag, N-terminal Met, N17,96D; R66H; truncated SEQ ID NO: 19

<223> hA1 M, not tag, N-terminal Met, M41 K; truncated SEQ ID NO: 20

<223> 6His, N17,96D; R66H; truncated

SEQ ID NO: 21

<223> hA1 M, 6His, M41 K; truncated

SEQ ID NO: 22

<223> 8His, N17,96D; R66H; truncated SEQ ID NO: 23

<223> hA1 M, 8His, M41 K; truncated

SEQ ID NO: 24

<223> 1 .M8H5GIEGR-Mouse

SEQ ID NO: 25

<223> 2. M8H5GIEGR-Naked Mole rat SEQ ID NO: 26

<223> 3. M8H5GIEGR-Frog

SEQ ID NO: 27

<223> 4. M8H5GIEGR-Chicken

SEQ ID NO: 28

<223> 5. M8H5GIEGR-Rabbit

SEQ ID NO: 29

<223> 6. M8H5GIEGR-SQ Monkey

SEQ ID NO: 30

<223> 7. M8H5GIEGR-Walrus SEQ ID NO: 31

<223> 8. M8H5GIEGR-Manatee

SEQ ID NO: 32

<223> 9. M8H5GIEGR-Plaice

SEQ ID NO: 33

<223> 10. M8H5GIEGR-Orangutan

SEQ ID NO: 34

<223> 1 1. M8H5GIEGR-Human P35K

SEQ ID NO: 35

<223> 12. M8H5GIEGR-Human M41 K SEQ ID NO: 36

. M8H5GIEGR-Human R66H

SEQ ID NO: 37

<223> 14. M8H5GIEGR-Human T75K

SEQ ID NO: 38

<223> 15. M8H5GIEGR-Human T75Y SEQ ID NO: 39

<223> 16. M8H5GIEGR-Human M99K

SEQ ID NO: 40

<223> 17. M8H5GIEGR-Human S101Y

SEQ ID NO: 41

<223> 18. M8H5GIEGR-Human K69.92.1 18.130R SEQ ID NO: 42

<223> 19. M8H5GIEGR-Coelacanth

SEQ ID NO: 43

<223> 21. M8H5GIEGR-Human L89T

SEQ ID NO: 44

<223> 22. M8H5GIEGR-Human N1796D

SEQ ID NO: 45

<223> 23. M8H5GIEGR-Human T45K

SEQ ID NO: 46

<223> 24. M8H5GIEGR-Human A135E SEQ ID NO: 47

<223> 25. M8H5GIEGR-Human V170S

SEQ ID NO: 48

<223> 26. M8H5GIEGR-Human

SEQ ID NO: 49

<223> 27. M8H5GIEGR-Human G172Q

SEQ ID NO: 50

<223> 33. M8H4DK-Human M41 K+

SEQ ID NO: 51

<223> 34. M8H4DK-Human M41 K+N 1796D 34 SEQ ID NO: 52

<223> 35. M8H4DK-Human R66H+N1796D

SEQ ID NO: 53

<223> 36. M8H4DK-Human M41 K+R66H+N1796D

SEQ ID NO: 54

<223> 38. M8H4DK-Human R66H SEQ ID NO: 55

<223> 39.M8H4DK-Human

SEQ ID NO: 56

<223> 40. M8H-Human wt

SEQ ID NO: 57

<223> 41. M8H-Human R66H+N1796D SEQ ID NO: 58

<223> 60. M8H4DK-Human wt

SEQ ID NO: 59

<223> 1 .M8H5GIEGR-Mouse

SEQ ID NO: 60

<223> 2. M8H5GIEGR-Naked Mole

SEQ ID NO: 61

<223> 3. M8H5GIEGR-Frog

SEQ ID NO: 62

<223> 4. M8H5GIEGR-Chicken SEQ ID NO: 63

<223> 5. M8H5GIEGR-Rabbit

SEQ ID NO: 64

<223> 6. M8H5GIEGR-SQ Monkey

SEQ ID NO: 65

<223> 7. M8H5GIEGR-Walrus

SEQ ID NO: 66

<223> 8. M8H5GIEGR-Manatee

SEQ ID NO: 67

<223> 9. M8H5GIEGR-Plaice SEQ ID NO: 68

<223> 10. M8H5GIEGR-Orangutan

SEQ ID NO: 69

<223> 1 1. M8H5GIEGR-Human P35K

SEQ ID NO: 70

<223> 12. M8H5GIEGR-Human M41 K SEQ ID NO: 71

<223> 13. M8H5GIEGR-Human R66H

SEQ ID NO: 72

<223> 14. M8H5GIEGR-Human T75K

SEQ ID NO: 73

<223> 15. M8H5GIEGR-Human T75Y SEQ ID NO: 74

<223> 16. M8H5GIEGR-Human M99K

SEQ ID NO: 75

<223> 17. M8H5GIEGR-Human S101Y

SEQ ID NO: 76

<223> 18. M8H5GIEGR-Human K69.92.1 18.

SEQ ID NO: 77

<223> 19. M8H5GIEGR-Coelacanth

SEQ ID NO: 78

<223> 21. M8H5GIEGR-Human L89T SEQ ID NO: 79

<223> 22. M8H5GIEGR-Human N1796D

SEQ ID NO: 80

<223> 23. M8H5GIEGR-Human T45K

SEQ ID NO: 81

<223> 24. M8H5GIEGR-Human A135E

SEQ ID NO: 82

<223> 25. M8H5GIEGR-Human V170S

SEQ ID NO: 83

<223> 26. M8H5GIEGR-Human V148D SEQ ID NO: 84

<223> 27. M8H5GIEGR-Human G172Q

SEQ ID NO: 85

<223> 33. M8H4DK-Human M41 K+R66H

SEQ ID NO: 86

<223> 34. M8H4DK-Human M41 K+N1796D SEQ ID NO: 87

<223> 35. M8H4DK-Human R66H+N1796D

SEQ ID NO: 88

<223> 36. M8H4DK-Human M41 K+R66H+N1796D

SEQ ID NO: 89

<223> 37. M8H4DK-Human M41 K SEQ ID NO: 90

<223> 38. M8H4DK-Human R66H

SEQ ID NO: 91

<223> 39. M8H4DK-Human N1796D

SEQ ID NO: 92

<223> 40. M8H-Human wt

SEQ ID NO: 93

<223> 41. M8H-Human R66H+N1796D

SEQ ID NO: 94

<223> 42. untagged-Human R66H+N1796D SEQ ID NO: 95

<223> 61. untagged-Human wt

Abbreviations

A1 M: alpha-1-microglobulin, IB: inclusion bodies, wt: wildtype, R66H: point mutation in A1 M-gene leading to expression of His instead of Arg at position 66, N17,96D: point mutations in A1 M-gene leading to expression of Asp instead of Asn at positions 17 and 96, M8H4DK: peptide with amino acid sequence MHHHHHHHHDDDDK, M6H4DK: peptide with amino acid sequence MHHHHHHDDDDK, M8H: peptide with amino acid sequence MHHHHHHHH, M8H5GIEGR: peptide with amino acid sequence

MHHHHHHHHGGGGGIEGR, CV: column volume, SEC: size-exclusion chromatography, DLS: dynamic light scattering, DSF: differential scanning fluorimetry, Gu-HCI: guaninidine hydrochloride, ORAC: oxygen radical antioxidant capacity, SD: standard deviation, PAGE: polyacrylamide gel electrophoresis

Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined proto- cols and/or parameters unless otherwise noted. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. In this specification, unless otherwise specified, "a" or "an" means "one or more".

The terms "treatment or prophylaxis" in their various grammatical forms in relation to the present invention refer to preventing, curing, reversing, attenuating, alleviating, ameliorating, inhibiting, minimizing, suppressing, or halting (1 ) the deleterious effects of a disorder, (2) disorder progression, or (3) disorder causative agent.

The term "effective amount' in relation to the present invention refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired ther- apeutic effect in association with the required additives and diluents; i.e., a carrier, or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent a clinically significant deficit in the activity and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluents; i.e., carrier, or additive. Further, the dosage to be administered will vary depending on the active principle or principles to be used, the age, weight etc. of the patient to be treated but will generally be within the range from 0,001 to 1000 mg/kg body weight/day. Moreover, the dose depends on the administration route. The term "polypeptides" includes proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with stand- ard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Glyi G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M)i Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

"Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

The invention is further illustrated in - but not limited to - the figures.

Legend to figures

Figure 1. Amino acid sequence alignment of A 1M from 12 different species.

The amino acid sequence of human wt A1 M and 1 1 additional species investigated in project phase I were aligned using the http://www.ebi.ac.uk/Tools/msa/clustalw2/ soft- ware. The identity of the different sequences to the human sequence is presented as percent. Amino acids identical between all species in the set are marked with yellow. Additionally, amino acids believed to be important in human A1 M are marked: the unpaired cysteine residue (C34) important for reduction and antioxidant properties (All- horn et al., 2005) as well as heme binding is marked with pink (Mening and Skerra, 2012). The asparagines known to be glycosylated (N17 and 96) are marked with green (Escribano et al, 1990). The four lysines (K69, 92, 1 18 and 130) that have been found modified in urine A1 M (Akerstrom et al., 1995; Berggard et al., 1999) and are believed to be important for the reductase activity (Allhorn et al., 2005) are marked with light blue. The H123 suggested to take part in the heme-binding (Meining and Skerra, 2012) is marked with grey and finally the tyrosines (Y22 and 132) shown to be involved in radical scavenging (Akerstrom et al., 2007) are marked in dark blue.

Figure 2. SDS-PAGE of expressed proteins in phase III.

Equal amount of bacterial lysate from uninduced samples (marked 0) and samples taken one to four hours after induction (marked 1 , 2, 3, 4) were separated by SDS- PAGE. In these samples a band slightly below 25kDa is expressed at increasing amounts by time. The intensity of the bands culminates after 3 hours of induction. M8H-tagged wt-A1 M and R66K+N17,96D-A1 M are expressed with molecular weights slightly smaller than for the M8H4DK bands, but also here the expression level culminates around 3 hours. Untagged wt-A1 M and R66H+N17,96D-A1 M appear as even smaller bands, but here the intensity of the bands is stronger at 4 hours than 3 hours indicating a slightly delayed expression, in particular for wt A1 M.

Figure 3. Yield, purity and aggregation of phase III variants.

A. The yields of purified A1 M of all phase III variants were compared. All variants re- suited in good yields, with a slightly lower yield of the untagged wt-A1 M. B. The purity was investigated by separation of 10μg of all variants on SDS-PAGE. The purity was determined by densiometric analysis of the main monomeric band compared to all bands using the Image software from Bio-Rad. All histidine-tagged variants showed very high purity while the purities of the untagged variants were a little bit lower (around 90%). C. The presence of large aggregates were analysed by separating 20μg of each variant by native PAGE. The intensity of the main monomeric band and the material remaining in the application slit (very large aggregated) were determined by densiometry. Most variants showed low amounts of large aggregates with exception of M8H-wt-A1 M and the untagged variants, which show a slightly higher percentage. All variants are coded by a number given in panel A.

Figure 4. Aggregation analysis of 100μΜ and 1mM solutions of phase III variants in various buffers.

The tendency to aggregate after concentration from 100μΜ to 1 mM in different buffers was investigated by native PAGE analysis. 20μg of protein were separated in each lane. The percent large aggregates were calculated using densiometry analysis of the individual band intensities.

A. All variants were concentrated from 100μΜ to 1 mM in Tris-buffer pH 8.0 or 7.4 and separated by PAGE. The M8H4DK-wt, R66H+N1796D, M41 K, R66H, N1796D are labelled 60, 35, 37, 38 and 39 respectively, the M8H-wt and M8H-R66H+N1796D are la- beled 40 and 41 and the untagged wt and R66H+N1796D are labelled 61 and 42.

B. The variants were concentrated to 1 mM in TRIS-buffer pH 8.0 and 7.4 and in PBS pH7.4, subjected to one freeze-thaw cycle, and then separated by PAGE and analysed as described. Figure 5. Analysis of reductase and antioxidant capacity of phase III variants.

A. The reductase activity was analysed as the reduction of the ABTS radical. 0-2μΜ A1 M were added in duplicates to 56μΜ ABTS radical. The reduction was followed as decrease of absorbance at 405nm during 95seconds. The area under the curve (AUC) for each concentration was calculated and the Net AUC was calculated by subtracting the AUC of buffer only. The average Net AUC+/- SEM of the duplicates was plotted against concentration. All M8H4DK-variants have about the same activity as wt A1 M with a tendency to lower acitivty for M41 K and R66H. The M8H and untagged variants also show full activity with a small tendency of higher activity of the R66H+N1796D variants compared to wt A1 M.

B. The antioxidation ability was investigated in the ORAC assay. The activities of the A1 M-variants were compared to a Trolox standard and expressed as number of Trolox equivalents. Each assay was done in triplicates and the result of M84DK-wt was set to 100%. The antioxidation capacities of all other variants were expressed in relation. The M8H4DK-R66H+N17,96D variant showed significantly higher capacity than the

M8H4DK-wt A1 M. When the tags were shortened or removed the difference was smaller. Data presented are the combined results of two independent experiments.

Figure 6. Analysis of reductase activity and heme binding of phase III variants.

A. The ability of A1 M-variants to reduce cytochrome c was investigated by mixing dilu- tion series (0-1 ΟμΜ) of A1 M with 100μΜ cytochrome c+Ι ΟΟμΜ NADH and following the increase in absorbance at 550nm for 20 minutes. The assay was done in duplicates. The AUC was calculated for each concentration and the Net AUC was calculated by subtraction of the AUC of buffer only. Data are presented as the Net AUC +/- SEM of two independent experiments. Ovalbumin was used as a negative control. Most A1 M variants showed a slightly lower reduction capacity compared to wt at the lower concentrations. For N17,96D-A1 M and R66H+N17,96D-A1 M it is significant (at 0.3-0.6μΜ). Shortening and removal of the tag had no influence of this property.

B. The incorporation of heme into the A1 M-variants was analysed by the appearance of an absorbance peak between 410-420nm, and the magnitude of the red-shift of the peak (Karnaukhova et al., 2014; Rutardottir et al., 2016). 44μΜ protein solutions were mixed with 40μΜ heme in duplicates and incubated for 2 hours at RT. The mixtures were analysed by wavelength scan between 270-450nm. The position of the maximal peak and the 413:386 ratio were calculated. All M8H4DK-variants have about the same red-shift and ratio as wt-A1 M, while the M8H-tagged variants have significantly higher ratio. The untagged variants lack red-shift activity, confirming previous results (Karnau- khova et al., 2014).

C. The binding of A1 M to heme agarose. The specific binding of A1 M was analysed by mixing dilution series of A1 M, or the control ovalbumin, with heme-agarose or control agarose. The assay was made in duplicates. Protein quantification of the starting material and the flow-throughs from heme-agarose and control agarose incubations was de- termined using BCA. From these data the amount of protein specifically bound to heme agarose could be determined for each sample. Bound protein was plotted against added protein, and a linear correlation was seen for all variants. The slope of the line was calculated with linear regression and the average between the duplicates is shown. All variants bind heme in this method to about the same extent.

Figure 7. Rescue of K562 cells from heme-induced cell death.

K562 cells were exposed to 100μΜ heme in the presense of a dilution series of A1 M (0-1 ΟμΜ) for 1 hour. Then, cell death was monitored as release of LDH into the medium. The absorbance value from live cells was subtracted and the signal of heme-in- cubated cells without A1 M was set to 100%. The values of the A1 M incubations were calculated in relation to this. The assay was made in duplicates. The average result from three independent experiments (mean +/-SEM) is shown in the figure. No significant difference could be seen between any of the variants except for untagged

R66H+N17,96D-A1 M that has lower activity than untagged wt-A1 M.

Figure 8. Size-distribution and reductase activity of M8H4DK-R66H+ N17, 96D-A 1 M and M8H4DK-wt-A 1 M after storage at +4°C and room-temperature.

M8H4DK-Wt-A1 M and M8H4DK-R66H+N17,96D-A1 M are depicted as "wt" and "variant 35" and shown in the upper and lower panels, respectively. Aggregation was ana- lysed by SEC-FPLC. Monomeric A1 M and large aggregates were eluted around 15ml and 8ml, respectively. The small shoulder seen around 13-14ml most likely is dimeric A1 M. The percentage of large aggregates was calculated from the area under the 8ml peak compared to the total peak area. The recovery of protein (%; shown in italic) after stress exposure was calculated from the total peak areas compared to the total peak area of the starting material. The reduction activity of ABTS was analysed in 2μΜ A1 M solutions. Data are shown as average of duplicates. Freshly thawed 100μΜ A1 M solutions are shown in (A), 100μΜ A1 M exposed to five freeze-thaw cycles (B), 1 mM A1 M stored at +4°C over-night (C), 1 mM A1 M stored at +4°C for a week (D), 10ΟμΜ A1 M stored at RT for a week (E), and 1 mM A1 M stored at RT for a week (F). Data show that M8H4DK-R66H+N1796D better tolerates concentration to 1 mM (C & D) and storage at RT (E and F).

Figure 9. Size-distribution and reductase activity of 1mM M8H4DK-wt-A 1 M and M8H4DK-R66H+N17,96D-A 1M after storage at +37°C.

Wt-A1 M and R66H+N17,96D-A1 M are depicted as "ht2014" and "35" and shown in the upper and lower panels, respectively. Aggregation was analysed by SEC-FPLC. Mono- meric A1 M and large aggregates are eluted around 20min and 12min, respectively. The small shoulder seen around 18-19min most likely is dimeric A1 M. The percentage of large aggregates was calculated from the area under the 12 min peak compared to the total peak area. The recovery of protein (%; shown in italic) after stress exposure was calculated from the total peak areas compared to the total peak area of the starting material. The reduction activity of ABTS was analysed in 2μΜ A1 M solutions. Data are shown as average of duplicates. 1 mM A1 M solutions stored for 1 .5hours (B), 2.5hours (C) and 4.5hours (D) were compared to 1 mM A1 M start material (A). The M8H4DK-wt at 4.5hours was completely precipitated and could not be analysed. Data show that M8H4DH-R66H+N1796D better tolerates storage at +37°C than M8H4DK-wt.

Figure 10. Model of the structure of A 1M. The model was prepared as described (ref 29). The eight β-strands, shown as ribbons, form a slightly cone-shaped cylinder with a hydrophobic interior: the "Npocalin pocket". One side of the lipocalin pocket is open (shown by the arrow), i.e. it permits entrance of small molecules. The opposite side is closed. Two ohelices are shown as cylinders. The positions of three carbohydrate groups (T5; N17; N96) and four side-chains involved in reductase activity (C34; K92; K1 18; K130) are shown. Figure 1 1 Heme-binding analysed by migration shift/fluorescence quenching on native PAGE (A and B), and UV-absorbance spectrophotometry (C). A. Fifteen μg M8H4DK-wt A1 M (wt-A1 M) or M8H4DK-35-A1 M (35-A1 M) were incubated with different amounts of heme for 30 min at 20°C, separated by native PAGE, and the gel analysed by trypto- phan fluorescence (Flourescence) and densitometry scanning after Coommassie staining (Stain). B. The images were digitalized by using Image Lab™ Software (Bio-Rad). Heme binding, meassured as flourescence quenching (black) and migration distance (blue) were plotted against the molar ratio A1 M:heme. Mean values of duplicate experiments are shown, wt-A1 M (filled symbols), 35-A1 M (open symbols). C. A1 M and heme were mixed (32 and 19 μΜ, respectively), incubated for 2h at 20°C and scanned. The absorbance of the proteins alone at 32 μΜ are shown as comparison. The absorbance of the buffer (20 mM Tris-HCI, pH 8.0 + 0.15M NaCI) was subtracted from all scans as blank. Figure 12

Comparison of the enzymatic properties of M8H4DK-wt A1 M (wt-A1 M) and M8H4DK- 35-A1 M (35-A1 M). A. Freshly purified wt-A1 M (■) or 35-A1 M (o) at various concentrations were mixed with ABTS-radical at 56μΜ in 25mM sodium phosphate buffer pH 8.0 in microtiter plate wells, and the rate of reduction was followed by reading the absorb- ance at 405 nm during 95 seconds. The absorbance for each concentration was plotted against time and the area under the curve (AUC) between 0 and 95 s was calculated for each concentration. The net AUC was calculated by subtracting the AUC of buffer only. Mean of triplicates +/- SEM are shown. B. The ABTS-reduction rate was determined as described in A, but using wt-A1 M (■) or 35-A1 M (o) after storage for 7 days at 4°C or room-temperature and 0.1 or 1 mM. Single experiments are shown. C. The reduction of cytochrome c was investigated by mixing dilution series (0-1 ΟμΜ) of wt- A1 M (■) or 35-A1 M (o) with 100μΜ cytochrome c+100μΜ NADH and following the increase in absorbance at 550nm for 20 minutes. The assay was done in duplicates. The AUC was calculated for each concentration and the Net AUC was calculated by sub- traction of the AUC of buffer only. Data are presented as the Net AUC +/- SEM of two independent experiments. Ovalbumin was used as a negative control. D. The antioxi- dation ability was investigated in the ORAC assay. The activities of the A1 M-variants at 5μΜ were compared to a Trolox standard and expressed as number of Trolox equivalents. Each assay was done in triplicates and the result of wt-A1 M was set to 100%. Data presented are the mean of two independent experiments +/- SEM. Figure 13

K562 cells cultured at 10 ⁵ cells per well in a 96-well microtiter plate, were exposed to 100μΜ heme in the presence of a dilution series of M8H4DK-wt A1 M (wt-A1 M) or M8H4DK-35-A1 M (35-A1 M) (0-1 ΟμΜ) for 1 hour. Cell death was monitored as release of LDH into the medium. The LDH-value from live cells was subtracted and the signal of heme-incubated cells without A1 M was set to 100% and the values of the A1 M incubations were calculated in relation to this. The assay was made in duplicates. The average result from three independent experiments (mean +/-SEM) is shown. Wt-A1 M (■) or 35-A1 M (o).

Figure 14

HK-2 cells were exposed to a mixture of 200 μΜ (NH ₄ )Fe(SC>4)2, 400 μΜ hydrogen peroxide, and 2 mM ascorbate (the Fenton reaction, displayed in A and B) or 0-30 μΜ heme (displayed in C and D) with or without the simultaneous addition of 0-20 μΜ M8H4DK-wt A1 M (wt-A1 M) (displayed as■ and black columns) or M8H4DK-35-A1 M (35-A1 M) (displayed as o and white columns) for 6 hours. After incubation, cells were analyzed for cell viability using WST-1 (displayed in A and C) or mRNA expression of HO-1 and Hsp70 (displayed in B and D) as described in materials and methods. The cell viability (A and C) was normalized against control samples from untreated cells. Results are from triplicate experiments and presented as mean±SEM. The mRNA expression of HO-1 and Hsp70 (B and D) was normalized against GAPDH and is given as fold change. The fold-change values were calculated by normalizing against control samples from untreated cells. Results are from triplicate experiments and presented as mean ± SEM. Differences between the respective exposures and control conditions were analyzed using One way ANOVA with post hoc Bonferroni correction. ^* indicates statistical comparison vs. Fenton (displayed in B) or heme (displayed in D). ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 . No significant difference was observed when comparing wt- A1 M vs. 35-A1 M. Figure 15

Plasma clearance (pharmacokinetics, displayed in A) and biodistribution (displayed in B) of M8H4DK-wt A1 M (wt-A1 M) and M8H4DK-35-A1 M (35-A1 M) injected intravenously in animals. A. Wt-A1 M (■) or 35-A1 M (o) was injected (5mg/kg) in Wistar rats and blood was collected at regular intervals. A1 M-concentrations were determined by RIA using the particular A1 M-variant as standard. Each point are from three animals and presented as mean±SEM. B. Wt- or 35-A1 M, 5 mg/kg, was injected intravenously in C57BL/6NRj-mice which were sacrificed 10 and 30 min post-injection. Organs were sampled, weighed and homogenized. Concentrations of injected (human) A1 M were determined by sandwich-ELISA. Each bar are from three animals and presented as mean±SD. Wt-A1 M, 10 min (black) and 30 min (dark gray); 35-A1 M, 10 min (white) and 30 min (light gray).

Figure 16

Female C57BL/6 mice were exposed to Glycerol (2.0 ml/kg, i.m.) followed by i.v. administration of either M8H4DK-wt A1 M (wt-A1 M) (dark grey bars, n=10), M8H4DK-35- A1 M (35-A1 M) (white bars, n=10) or vehicle buffer (sham control, grey bars, n=6) 30 minutes post-glycerol injections. At 4 hours (post-glycerol administration) animals were euthanized and kidneys excised, snap frozen and subsequently analyzed for mRNA expression of HO-1 (A) and Hsp70 (B) using real-time PCR as described in materials and methods. mRNA expression were normalized against those of GAPDH and fold change values were calculated by normalizing against control samples from untreated animals (controls). Results are presented as as box plots, displaying medians and 25 ^th and 75 ^th percentiles. Statistical comparison between groups were performed by ANOVA with post hoc Bonferroni correction. ^* indicates statistical comparison vs.

Glycerol. ^* P < 0.05, ^** P < 0.01 . No significant difference was observed when comparing wt-A1 M vs. 35-A1 M.

The invention is further illustrated in the following examples. The examples are illustrative and do not limit the scope of the invention in any manner. Experimental

The experimental work was divided into three major phases:

Phase I) Expression of the 27 A1 M-variants described above followed by analysis of solubility, stability and function; Phase II) Design, expression and analysis of a few A1 M-variants with expected optimal properties based on the outcome of phase 1 ;

Phase III) Design, expression and analysis of wt-A1 M and the most successful mutated A1 M-variant equipped with or without N-terminal tags. In Phase I, four lines of reasoning were used, when selecting the positions and identities of mutated amino acid side-groups: 1 ) Animal homologues from a variety of species with different expected environmental pressure in terms of oxidative stress, temperature, oxygen pressure were expressed (N=12); 2) Single amino acid substitutions that occur frequently among the 56 sequenced A1 M-homologues at positions located in loops 1-4 or the interior surface of the hydrophobic pocket, were introduced into the human gene construct and expressed (N=3); 3) Addition or removal of favourably located lysyl or tyrosyl residues, based on the hypothesis that these may infuence pKa of the C35 thiolyl (Allhorn et al., 2005) or serve as radical-trapping sites (Berggard et al., 1999; Sala et al., 2004; Akerstrom et al. 2007) (N=5); 4) Hydrophobic^hydrophilic substitutions on the surface of the protein, with no predicted influence on function or folding (N=7).

Materials and methods

Expression

The sequences of the A1 M variants were provided to DNA2.0, Inc. (USA) which synthesized the genes and cloned them into their PJ401 Express vector (T5 promoter, kan- amycin resistance). The DNA sequence was confirmed by sequencing. The vectors were transformed into competent E. coli (BL21 Star(DE3) (Invitrogen, Life technologies corp, USA)) according to the manufacturer's instructions and four individual clones of each variants were tested for micro-expression. The clone with highest expression of each variant was prepared as a glycerol stock which was used for production expression. A1 M variant expression clones were grown in complete NYAT (15mM (NH ₄ )2S04,

84mM K ₂ HP0 ₄ , 23mM NaH ₂ P0 ₄ xH ₂ 0, 2.2mM (NH ₄ ) ₂ Hcitrate, 1 % (w/v) glucose, 2mM MgS0 ₄ , 9μΜ CaCI ₂ x2H ₂ 0, 85μΜ FeCI ₃ x6 H ₂ 0, 1.3μΜ ZnS0 ₄ x7 H ₂ 0, 1 .3μΜ CuS0 ₄ x5 H20, 1 .8μΜ MnS0 ₄ x H20, 1.5μΜ CoCI ₂ x6 H ₂ 0, 108μΜ EDTA, 50μg/ml kanamycin) to an OD600 of 1 .5. Then protein expression was induced by addition of 1 mM IPTG. The production went on for 4 hours. Samples for SDS-PAGE analysis were taken before induction, and 1 , 2, 3 and 4 hours after induction.

Purification

Bacteria from the cultures were collected by centrifugation at 5000 rpm, 15 minutes. The bacterial pellets were lysed by five freeze-thaw cycles, diluted 3 times in 20 mM Tris-HCI pH 8.0 and sonicated. The inclusion bodies (IB ^' s) were collected by centrifu- gation at 6000 rpm, 30 minutes, and washed by three more cycles of resuspension and centrifugation. For extraction, the IB ^' s were resuspended in 6M guanidine-hydrochlo- ride, 20 mM Tris-HCI pH 8.0 (6M Gu-HCI) and incubated with stirring overnight at +4°C. The extract was clarified by high-speed centrifugation at 26000 g, 60minut.es. The supernatant was saved for further clarification, while the pellet went through another cycle of extraction. The supernatants of the extractions were combined and further clarified by depth filter filtration (K700P filter laid on top of KS50P filter, Pall Corp., USA). A1 M in the clarified extract was purified, using a Ni-agarose resin (Sigma-Aldrich, USA). Briefly, the resin was packed into a 10-ml disposable chromatography column (Bio- Rad, USA) and equilibrated in 6M Gu-HCI. The A1 M extract was applied onto the column using free-flow and the flow-through collected. The column was washed with five column volumes of 6M Gu-HCI and then eluted with four volumes of 6M Gu-HCI +0.5 M imidazole. Starting material, flow-through and eluted fractions were precipitated with ethanol, resolved in 1xSDS-PAGE loading buffer and separated by SDS-PAGE. The purification procedure was repeated if the flow through contained significant amounts of A1 M. The protein content of the extract was determined by absorbance 280nm.

The Ni-agarose eluates were diluted to an approximate A280 of 5.0 and cooled to +4°C before refolding. The eluate was then mixed with 2/3 volumes of 0.275 M L-cystein in 20 mM Tris-HCI pH 9.5 + 0.1 M NaCI. Then 16.7 volumes refolding buffer were quickly added. The final buffer concentration was: (0.2 mg/ml A1 M, 0.1 M Tris, 0.6 M NaCI, 0.45 M L-arginine, 2 mM EDTA, 10 mM L-cystein and 1 mM L-cystein, pH 9.5). The mixture was then stirred for 1 hour at +4°C, and the solution concentrated to the initial volume of the A1 M solution using Centricon plus 70, 10K ultrafiltration devices (Merck Millipore; USA). After concentration, the A1 M solution was diafiltrated to 20mM Tris- HCI pH 8.0 using the same devices, by 10 consecutive 2.5x dilution/concentration cycles. After diafiltration the solution was clarified by centrifugation at 15000g for 15 minutes and then run through a 0.2μηη filter.

The refolded A1 M was immediately applied to a 5ml Bio-Scale Mini UNOsphere Q Cartridge (Bio-Rad), equilibrated with 20mM Tris-HCI pH 8.0. The column was run on an ΑΚΤΑ purifier 10 instrument (GE Healthcare, USA) according to Bio-Rad's instruction for the cartridges. After sample application the column was washed with five column volumes (CV) of 20mM Tris-HCI pH 8.0 before elution with a 20 CV linear gradient from 0-0.35M NaCI. Finally, the column was washed with three CVs of 1 M NaCI. The flow- though and selected fractions collected during the linear gradient were analysed by SDS-PAGE. Flow-through with remaining A1 M was immediately re-run on a new column and the A1 M containing fractions were pooled, concentrated to 100μΜ, sterile filtered and frozen at -20°C in aliqoutes.

Gel electrophoresis

SDS-PAGE was run according to Laemmli (Laemmli, 1970) using standard protocols. Proteins were separated on stain-free 4-20% TGX gels (Bio-Rad) at 300V for 17 minutes. Native PAGE was run witout SDS and without reducing agents on stain-free 4-20% TGX gels at 200V for 40 minutes. The gels were analysed on a Chemidoc MP instrument (Bio-Rad).

Circular Dichroism

The circular dichroism spectra were recorded on a Jasco-J180 spectrofluorimeter in- strument (JASCO Inc., Japan) of 10 μΜ solutions in 20 mM Tris-HCI pH 8.0 + 0.15 M NaCI in a 2-mm cuvette. The solutions were scanned at +22°C between 190-260 nm. Three runs were overlayed for each sample. The percentage of a-helix and β-sheet structure was calculated using the http://k2d3.oqic.ca software. SEC-FPLC

Proteins were analysed by size exclusion on an AKTA purifier 10 instrument using a 24-ml Superose 12 10/30 GL column (GE Healthcare). The column was equilibrated with 20 mM Tris-HCI pH 8.0 + 0.15 M NaCI using a flow-rate of 1 ml/min. 100-200 μg of protein were loaded onto the column in a volume of 100 μΙ and eluted with 20 mM Tris- HCI pH 8.0 + 0.15 M NaCI using a flow-rate of 0.75 ml/min. Typically, monomeric A1 M was eluted after 15 ml/20 min, dimeric after 13-14 ml/18-19 min, and large aggregates were eluted after 8 ml/12 min. The percentage of large aggregates was calculated from the area under the 8-ml peak compared to the total peak area. The percentage of total protein retrieved on the column after stress treatments (see below) was calculated by comparing the total peak areas of treated vs. non-treated samples.

RP-HPLC

Reversed phase HPLC was run on an Agilent 1260 Infinity Binary LC system using an Aeris Wildpore 3.6 μΜ XP-C8 column (Phenomenex Inc., USA). The column was run at +25°C using a flow rate of 1 ml/min and equilibrated with a mixture of 70% H2O+0.1 % TFA and 30% Acetonitrile+0.1 % TFA. 10 μΙ (=10 μg of protein) were loaded and eluted with a linear gradient of 30-50% acetonitrile over 20 minutes. The column was regenerated by washing with 95% acetonitrile for 10 minutes.

Dynamic light scattering

Dynamic light scattering (DLS) analysis of non-stressed and shearing-stressed samples was done using the service of SARomics Biostructure AB, Lund. A1 M samples, diluted to 10μΜ in 10mM Tris-HCI pH 8.0 + 0.125 M NaCI, were analysed on a Malvern APS instrument at +20°C. Samples were prepared in duplicates and each sample was monitored three times.

Differential scanning fluorimetry

Thermostability of the A1 M variants was analysed by differential scanning fluorimetry (DSF) using the service of SARomics Biostructure AB, Lund. A1 M diluted to 4.4 μΜ in 10 mM HEPES pH 8.0 + 0.125 M NaCI was mixed with SYPRO orange (1000x dilution of SYPRO orange in total). The analysis was made in duplicates and the average melting temperature (T _m ) was calculated.

Introduction of stress by shearing forces

10 μ I of 100-μΜ A1 M solutions were exposed to shearing force stress by 80 pipettings with a multiple channel pipett using 0-10 μΙ pipett tips. The stress-treatment was performed in duplicates. After pipetting, the duplicates were combined and diluted 10 times before analysis with DLS as described.

High concentration-induced stress

Solubility and stability of A1 M was analysed at high concentration. 500 μΙ of 100 μΜ A1 M solutions were concentrated tenfold to 50μΙ using Amicon Ultra-0.5, 10K devices (Merck Millipore) by centrifugation at 14000g for 10 min. After concentration the volumes were corrected to exactly 50μΙ using the respective flowthroughs. Concentrated and non-concentrated samples (10 μg) were compared side-by-side on native PAGE. The influence of different buffers were examined by diafiltration of the samples before concentration. This was done by five cycles 10-time dilution/concentration in Amicon Ultra-15, 10K devices.

Quantification of free thiol groups

The free thiol groups of the A1 M variants were determined with the "Thiol and sulfide quantification kit" from Molecular probes. The assay was performed in 96-well plates according to the kit manual. A volume of 3μΙ standard or A1 M (1 ΟΟμΜ) was mixed with 3μΙ cystamine work solution, 10ΟμΙ papain-SSCH, 10ΟμΙ L-BAPNA. The assay was read as absorbance 405nm. ABTS reduction assay

The assay is a modification of (Akerstrom et al 2007). 7mM of 2,2 Azino-bis (3-etylben- zothiazoline-6-sulfonic acid) di-ammonium salt (Sigma-Aldrich) was oxidized with 2.45mM K2S2O8 overnight, and then diluted to 56μΜ in 25mM sodium phosphate buffer pH 8.0. 10ΟμΙ of the ABTS working solution was added per well in a 96-well plate. A time-point zero measurement was done at A405 using a Perkin Elmer Plate reader. 2μΙ of an A1 M solution (0-100 μΜ) were quickly added by a multiple channel pipett. The kinetics of the decrease in absorbance at 405nm was quickly followed for 95s. For practical reasons only 8wells were analysed at the time. The A1 M dilution series was run in duplicate or triplicates. If the number of samples to be analysed required several plates, new ABTS stock was diluted into working stock for each plate and an wt-A1 M reference sample was included in all plates. The absorbance for each concentration was plotted against time and the area under the curve (AUC) was calculated for each concentration. The net AUC was calculated by subtracting the AUC of buffer only. Oxygen radical antioxidant capacity (ORAC) assay

The commercial kit OxySelect™ Oxygen radical antioxidant capacity (ORAC) activity assay (Cell Biolabs, Inc. USA) is based on the oxidation and destruction of a fluorescent probe by peroxyl radicals. When an antioxidant is present this destruction is inhibited. As standard the water soluble vitamin E derivate, Trolox is used. Performance, analysis and calculations followed the kit manual and an A1 M concentrations of 2.5- 5μΜ fitted nicely in to the standard curve. Ovalbumin (Sigma-Aldrich) was used as a negative protein control.

Cytochrome c reduction assay

The assay is modified from (Allhorn el al. 2005). A working solution was made by mixing 100μΜ cytochrome c (Sigma-Aldrich) and 100μΜ NADH in 10mM Tris-HCI pH 8.0 + 0.125M NaCI. 1 1 μΙ of an A1 M solution (0-100 μΜ) were added to a 96-well plate in duplicates. 100μΙ of the cytochrome c working solution was quickly added per well using a multichannel pipett. The kinetics of the increase in absorbance at 550nm was followed for 20 minutes. One plate was analysed at the time. No biases over time could be observed. If several plates were to be analysed the same working solution was used for all without any observable artefacts caused by this procedure. After measurements the results were analysed as described for the ABTS assay.

The red-shift assay

The incorporation of free heme in A1 M yields a red-shift of the Soret band absorbance peak (Karnaukhova et al., 2014; Rutardottir et al., 2016), and was evaluated in the A1 M-mutants as the A413/A386 ratio. 44μΜ A1 M in 20mM Tris-HCI pH 8.0 + 0.25M NaCI was mixed with 40μΜ free heme (Applichem). The incubations were done in duplicates and incubated for 2 hours at room temperature. The red-shift was analysed in four times diluted samples by wavelength scan in a Beckman Spectrophotometer. The average maximal peak of the duplicates as well as the average ratio between the absorb- ances at 413 and 386nm was calculated. Ovalbumin and buffer only were used as negative controls. Specific binding of A IM to heme-agarose

Binding of A1 M to heme-agarose was analysed by the method of (Larsson et al., 2004), modified as described (Rutardottir et al., 2016). Heme-agarose (Sigma-Aldrich) and control Sepharose 4B (Sigma-Aldrich) were equilibrated with 20mM Tris-HCI pH 8.0 + 0.25M NaCI and prepared as 50% slurries. 75μΙ of an A1 M dilution series (0- 13.3μΜ) were added to duplicate 96-well plates (one plate for heme agarose and one for control Sepharose). 20μΙ of heme-agarose och control Sepharose slurry were added to the wells by careful pipetting to assure transfer of similar amounts, and incubated for 30 minutes at RT during rotational stirring. The mixtures were carefully transferred to an AcroPrep Advance 96-well filter plate, 1 .2μηη Supor membrane (Pall Corp). The plates were centrifuged for 2min at 1000g collecting the flow-through in a low-binding microplate. 25μΙ of each flow-through, as well as the non-incubated starting material, were assayed for protein content using Pierce BCA Protein Assay kit (Thermo Scientific Inc., USA). Protein amounts were recalculated as amount bound (added minus amount in flow-through) for both the heme- and control Sepharose-incubated samples. After subtraction of the amount bound to the control Sepharose, the amount specifically bound to heme agarose was plotted against the added amount. The slope of the curve was calculated with linear regression. The SD of the duplicates were used to evaluate the significance of the differences between the A1 M variants.

Heme binding of M8H4DK-wt A 1M (Wt-A 1M) and M8H4DK-35-A 1 M (35-A 1M) Heme binding was analysed as previously described by native PAGE (Karnaukhova et al., 2014) and UV-spectrophotometry (Ruttarsdottir et al., 2016). Briefly, for native PAGE, A1 M and various concentrations of heme were incubated in Tris-buffer, pH 8.0 for 30 min at room temperature, separated by native PAGE on stain-free 12% Criterion TGX gels at 200V for 40 minutes. The gel bands were analysed on a Chemidoc MP instrument (Bio-Rad) for tryptophan fluorescence using the Stain-free setting, stained with Coommassie Brilliant Blue, destained and imaged again on the Chemidoc using the Coommassie setting. Both sets of bands were then quantified using Image Lab™ Software (Bio-Rad). Heme binding was then estimated as the amount of quenching of the tryptophan fluorescence relative to total protein amounts after Coommassie staining. Absorbance spectra were measured on a Beckman (Beckman Instruments, Fuller- ton, CA) DU 800 spectrophotometer using a scan rate of 600 nm/min in the UV-VIS region between 250 and 700 nm at 22 C. Concentrations of A1 M and heme were 32 and 19 μΜ, respectively, in 20 mM Tris-HCI, pH 8.0, 0.15 M NaCI. Heme was added from a stock solution of 10 mM in DMSO. Protein solutions were scanned 2 h after mixing.

Plasma clearance and biodist bution

For the plasma clearance studies, each A1 M-variant was injected intravenously (i.v) in six male Wistar rats (5.0 mg/kg; stock-solutions in 20 mM Tris-HCI, pH 8.0) and blood samples were taken in EDTA-tubes at 1 , 5, 15, 30 min, and 1 , 3, 6, 16 and 24 h post- injection, using different sampling intervals in groups of three rats to avoid over-sampling. Plasma was aspirated after centrifugation 140xg for 10 min, and the concentration of A1 M was determined by radioimmunoassay (RIA) as described (Gram et al. 2015) using the specific A1 M-variant as standard. For the biodistribution studies, the A1 M-variants were injected i.v. in C57BL/6NRj mice (5.0 mg/kg; stock-solutions in 20 mM Tris-HCI, pH 8.0). The mice were sacrificed 10 min post-injection (n=3), and after 30 min (n=3). Organs were sampled, weighed and homogenized in 5:1 (vohweight) Cell Extraction Buffer (Invitrogen, cat. No. FNN001 1 ), containing 50 μΙ/ml complete Mini, EDTA-free proteinase inhibitor cocktail tablets (Roche, cat. no. 1 1836170001 ). A1 M- concentrations were determined by an in-house sandwich ELISA. Briefly, 96-well microliter plates were coated overnight at 4°C with mouse monoclonal anti-A1 M (clone 35.14, 5 μg/ml in PBS), washed, and then incubated with A1 M-standards (human urinary A1 M, purified as described at our laboratory (Akerstrom et al., 1995) or homogenised tissue samples, diluted in incubation buffer (PBS + 0.05% Tween 20 + 0.5% bo- vine serum albumin), for 60 min at RT. After washing, the wells were incubated with horseradish peroxidase-conjugated mouse monoclonal anti-A1 M (clone 57.10, 5 ng/ml in incubation buffer) for 60 min at RT. The plates were washed and developed by incubating with SureBlue TMB Microwell Peroxidase Substrate (KPL) in the dark for 20 min, and finally stopped with 1 M sulfuric acid. Absorbance was read at 450 nm in a Wallac 1420 Multilabel Counter. The two mouse monoclonal antibodies were raised by

AgriSera AB (Vannas, Sweden) against human urinary A1 M. The ELISA was specific for human A1 M, did not cross-react with endogenous mouse plasma A1 M, and reacted with human urinary A1 M, M8H4DK-wt A1 M (wt-A1 M) and M8H4DK-35-A1 M (35-A1 M) equally well. Rhabdomvolvsis induced kidney damage

This study was approved by the ethical committee for animal studies in Malmo-Lund, no. M21-15. Female C57BL/6 mice with a body weight of 20.5 ± 0.7 g were obtained from Taconic (Denmark), housed in Type III cages with wire lids, at a constant room temperature with 12 hour light dark cycles. Temperature (20±0.5°C) and relative moist (50±5%) was maintained throughout the studies. All animals had free access to food (RM1 (E) SQC, SDS, England), tap water and cage enrichment. After overnight water deprivation (15 hours) animals were weighed and anaesthetized using isoflurane and then allocated to the following four groups: 1 ) Control (n=6), received no intramuscular (i.m.) or intravenous (i.v.) administration; 2) Glycerol (n=10), received 50% sterile glyc- erol (Teknova, Hollister, CA, USA) i.m. (2.0 ml/kg body weight, single dose, divided into both hind limbs); 3) Glycerol + M8H4DK-wt A1 M (wt-A1 M) (n=10), received wt-A1 M i.v. (7 mg/kg body weight, single dose) 30 minutes after glycerol i.m. (2.0 ml/kg body weight) administration; and 4) Glycerol + M8H4DK-35-A1 M (35-A1 M) (n=10), received 35-A1 M i.v. (7 mg/kg body weight, single dose) 30 minutes after glycerol i.m. (2.0 ml/kg body weight) administration. Following i.m. administration animals were placed on a heat pad during awakening and then put back to their cages and supplied with free access to food and water. After 4 hours (post-glycerol injection) animals were anaesthetized using isoflurane and kidney collected for RNA extraction followed by mRNA evaluation as described below.

RNA isolation and real-time PCR

Total RNA was isolated from HK-2 cells, using Direct-zol™ RNA MiniPrep supplied by Zymo Research (Irvine, CA, USA), or mouse kidneys, using NucleoSpin RNA/Protein (Machery-Nagel, Duren, Germany) followed by RNeasy® Mini Kit (QIAGEN, German- town, MD, USA). The OD ratio (optical density at 260 nm/280 nm) of RNA was always higher than 1 .9. Reverse transcription was performed according to the manufacturer on 1.0 μ9 total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad, CA, USA). RT ² qPCR Primer Assay (human (HK-2 cells) and mouse (kidneys) primers from QIAGEN) were used to quantify the mRNA expression of heme oxygenase 1 (HO-1 ) and heat shock protein 70 (Hsp70). Data were normalized to glyceraldehyde-3-phosphate

dehydrogenase (human (HK-2 cells) and mouse (kidney) GAPDH, RT ² qPCR Primer Assay from QIAGEN). Data are presented as as columns, displaying mean ± SEM, for in vitro data and box plots, displaying medians and 25 ^th and 75 ^th percentiles, for in vivo data. The fold change values were calculated by normalizing against control samples from untreated cells or animals (controls). Expression was analyzed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad). Amplification was performed as described by the manufacturer (Bio-Rad) for 40 cycles in an iCycler Thermal Cycler (Bio-Rad) and data analyzed using iCycler iQ Optical System Software (Bio-Rad).

Rescue of K562 cells from heme induced cell death

A1 M was previously shown to inhibit heme-induced cell-death of human erythroid K562 cells (Olsson et al., 2008). The cells were cultured in DMEM with glutamax + 10% FCS and antibiotics (Gibco, Life Technologies Corp., USA) according to the instructions at ATCC. Cells were washed and resuspended in DMEM without phenol red and FCS but supplemented with glutamax I and antibiotics (Gibco). Cells were seeded into 96-well plates, 10 ⁵ cells per well, and exposed to 100μΜ heme in the presence of a 0-10 μΜ A1 M dilution series. As a positive control for cell death, 10μΙ of lysis solution from the LDH detection kit (see below) was added. The cells were incubated in a 37°C C02-in- cubator for 1 hour. The plates were quickly centrifuged at 350g for 4 minutes before 50μΙ of the medium was transferred to a 96-well microplate for analysis of LDH release using the cytoTox 96® Non-Radio. Cytotoxicity Assay (Promega Biotech AB, Sweden) according to the manufacturer's instructions. Heme-induced cells typically gave 7 times higher signal compared to live cells and 70% of the signal of completely lysed cells. The average signal of cells incubated without heme- or A1 M-addition was subtracted from all and the signal of heme only-incubated cells was set to 100%. All other signals were related to this value. This procedure enabled comparison of several independent experiments.

Protection of HK-2 cells

Human kidney cortex proximal tubule epithelial cells (HK-2, ATCC® CRL-2190, ATCC, Teddington, UK) were cultured in keratinocyte serum free medium (K-SFM) supplemented with bovine pituitary extract (BPE, 0.05 mg/ml) and epidermal growth factor (5 ng/ml_)(all from Invitrogen, Paisley, UK). When cells reached approximately 80-90% confluence, heme (0-30 μΜ, from a freshly prepared 10 mM stock solution) or a mixture of (NH4)Fe(S04)2, hydrogen peroxide, and ascorbate (0-200 μΜ, the Fenton reaction), with or without the simultaneous addition of A1 M (0-20 μΜ, M8H4DK-wt A1 M (wt-A1 M) and M8H4DK-35-A1 M (35-A1 M)), were added and cells were incubated for 6 hours. After incubation, cells were analyzed for cell viability using WST-1 (the measured metabolic activity of cells, e.g. measurment of cellular cleavage of the WST- 1 stable tetrazolium salt to the soluble formazan dye, is a direct correlate to the number of viable cells)(Roche Diagnostics GmbH, Mannheim, Germany) according to the instructions from the manufacturer. The cell viability was normalized against control samples from untreated cells. Parallel cultures were harvested using Qiazol™ Lysis reagent (for RNA extraction, QIAGEN, Germantown, MD, USA). Total RNA was extracted from cells to evaluate mRNA expression as described below. Statistics

Comparisons between unrelated groups were performed by ANOVA with post hoc Bonferroni correction. P-values <0.05 were considered significant.

Results and Discussion

Project overview

The purpose of this investigation is to identify more stable and soluble variants of A1 M with preserved functional properties. The project was performed in three phases. In phase I, A1 M of different species and point mutations of human A1 M were screened to identify individual amino acids that had a positive effect on stability without compromising function. In phase II, amino acids shown to have positive effect in phase I, were combined to find even better combinations. Finally in phase III, data from phase I and II were confirmed and the effect of different N-terminal tags was investigated. In all three phases, the proteins were expressed in the same E coli system, using the same vector and purification protocol. All variants were expressed, purified and analysed in parallel, using similar protocols and procedure, within each phase. The analysis panel of each phase is summarized in Table 1. Amino acid and DNA sequence of all constructs can be found below. Table 1. Analyses performed in the different phases of the A1 M variant project

^* Only performed on human wt and the M8H4DK-R66H+N1796D variant.

Amino acid sequence of all constructs: 60. M8H4DK-Human wt (rhA1 M) (SEQ ID NO: 9)

MH JDDKGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIM-

DRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK-

SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

1.M8H5GIEGR-Mouse (SEQ ID NO: 24)

MHHHHHHHHGGGGGIEGRDPASTLPDIQVQENFSESRI-

YGKWYNLAVGSTCPWLSRIKDKMSVQTLVLQEGATETEISMTSTRWRRGVCEEIT- GAYQKTDIDGKFLYHKSKWNITLESYWHTNYDEYAIFLTKKSSHHHGLTI- TAKLYGREPQLRDSLLQEFKDVALNVGISENSIIFMPDRGECVPGDREVEPTSIAR

2. M8H5GIEGR-Naked Mole rat (SEQ ID NO: 25)

MHHHHHHHHGGGGGIEGRNPVPMPPDNIQVQENFDESRI- YGKWFNLATGSTCPWLKRIKDRLSVSTMVLGKGTTE-

TQISTTHTHWRQGVCQETSGVYKKTDTAGKFLYHKSKWNVTMESYV-

VHTNYDEYAIILTKKFSHHHGPTITAKLYGREPRLRDSLLQEFREMALGVGIPEDSI FT-

MANRGECVPGDQAPESTPAPR 3. M8H5GIEGR-Frog (SEQ ID NO: 26)

MHHI ! ) I ) I ) I ) I ) "GGGGGIEGRCSPIQPEDNIQIQENFDLQRIYGKWYDIAIGSTCK- WLKHHKEKFNMGTLELSDGETDGEVRIVNTRMRHGTCSQIVGSYQKTETPGKFDYF- NARWGTTIQNYIVFTNYNEYVIMQMRKKKGSETTTTVKLYGRSPDLRPT- LVDEFRQFALAQGIPEDSIVMLPNNGECSPGEIEVRPRR

4. M8H5GIEGR-Chicken (SEQ ID NO: 27)

MHHHHHHHHGGGGGIEGRTPVGDQDEDIQVQENFEPERMYGKWYDVAVGTTCK- WMKNYKEKFSMGTLVLGPGPSADQISTISTRLRQGDCKRVSGEYQKTDTPGKYTYYN PKWDVSIKSYVLRTNYEEYAVILMKKTSNFGPTTTLKLYGRSPELREEL- TEAFQQLALEMGIPADSVFILANKGECVPQETATAPER

5. M8H5GIEGR-Rabbit (SEQ ID NO: 28)

MHh 3GGGIEGRDPVPTLPDDIQVQENFELSRI- YGKWYNLAVGSTCPWLKRIKDRMAVSTLVLGEGTSETEISMTSTHWRRGVCEE- ISGAYEKTDTDGKFLYHKAKWNLTMESYWHTNYDEYAIFLTKKFSRRHGPTI- TAKLYGREPQLRESLLQEFREVALGVGIPENSIFTMIDRGECVPGQQEPKPAPVLR

6. M8H5GIEGR-SQ Monkey (SEQ ID NO: 29)

MHHHHHHHHGGGGGIEGRSPVPTPPEGIQVQENFNLSRIYGKWYNLAIGSTCPWLK- KIMDRLKVSTLVLEEGATEAEISMTSTRWRKGFCEQTSWAY- EKTDTDGKFLYHEPKWNVTMESYVAHTNYEEYAIFLTKKFSRHHGPTI- TAKLYGREPQLRESLLQDFRVVAQGVGIPEDSIFTMANRGECVPGEQEPQPILHRR

7. M8H5GIEGR-Walrus (SEQ ID NO: 30)

MHHHHHHHHGGGGGIEGRSPVLTPPDAIQVQENFDISRIYGKWFH-

VAMGSTCPWLKKFMDRMSMSTLVLGEGATDGEISMTSTRWRRGTCEEISGAY-

EKTSTNGKFLYHNPKWNITMESYWHTDYDEYAIFLTKKFSRHHGPTI-

TAKLYGRQPQLRESLLEEFRELALGVGIPEDSIFTMANKGECVPGEQEPEPSPHMR

8. M8H5GIEGR-Manatee (SEQ ID NO: 31 )

MHHHHHHHHGGGGGIEGRSPVKTPLNDIQVQENFDLPRIYGKWFNIAIG- STCQWLKRLKAGPTMSTLVLGEGATDTEISTTSTRWRKGFCEEISGAYEKTD- TAGKFLYHGSKWNVTLESYVVHTNYDEYAIFLTKKFSRYGLTI- TAKLYGRQPQVRESLLEEFREFALGVGIPEDSIFTTADKGECVPGEQEPEPTAALR

9. M8H5GIEGR-Plaice (SEQ ID NO: 32)

MHH HHHHHHGGGGGIEGRLPVLPEP- LYPTQENFDLTRFVGTWHDVALTSSCPHMQRN- RADAAIGKLVLEKDTGNKLKVTRTRLRHGTCVEMSGEYELTSTPGRIFYHIDRW-

DADVDAYWHTNYDEYAIIIMSKQKTSGENSTSLKLYSRTMSVRDTVLDDFKTLVRHQ GMSDDTIIIKQNKGDCIPGEQVEEAPSQPEPKR

10. M8H5GIEGR-Orangutan (SEQ ID NO: 33)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIM-

DRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNIT MESYVVHTNYDEYAIFLTKKFSRRHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGI- PEDSIFTMADRGECVPGEQEPEPILIPR

1 1. M8H5GIEGR-Human P35K (SEQ ID NO: 34)

HHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCKWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

12. M8H5GIEGR-Human M41 K (SEQ ID NO: 35)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIKDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI- TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

13. M8H5GIEGR-Human R66H (SEQ ID NO: 36)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK-

KIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHK-

SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

14. M8H5GIEGR-Human T75K (SEQ ID NO: 37)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK-

KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEEKSGAYEKTDTDGKFLYHK-

SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

15. M8H5GIEGR-Human T75Y (SEQ ID NO: 38)

MHHh 3GGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK-

KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEEYSGAYEKTDTDGKFLYHK-

SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

16. M8H5GIEGR-Human M99K (SEQ ID NO: 39)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITKESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

17. M8H5GIEGR-Human S101Y (SEQ ID NO: 40)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMEYYWHTNYDEYAIFLTKKFSRHHGPTI- TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

18. M8H5GIEGR-Human K69.92.1 18.130R (SEQ ID NO: 41 ) MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRRGVCEETSGAY- EKTDTDGKFLYHRSKWNITMESYVVHTNYDEYAIFLTKRFSRHHGPTITAR- LYGRAPQLRETLLQDFRVVAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

19. M8H5GIEGR-Coelacanth (SEQ ID NO: 42)

MHHHHHHHHGGGGGIEGRGSPLRDEDIQVQENFDLPRIYGKWYEIAI- ASTCPWVKNHKDKMFMGTMVLQEGEQSDRISTTSTRIRDGTCSQITGYYTLTTTPGK- FAYHNSKWNLDVNSYWHTNYDEYSIVMMQKYKSS-

NSTTTVRLYGRTQELRDSLHAEFKKFALDQGIDEDSIYILPKRDECVPGEPKAESLM AR

21. M8H5GIEGR-Human L89T (SEQ ID NO: 43)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFTYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

22. M8H5GIEGR-Human N1796D (SEQ ID NO: 44)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLK-

KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK-

SKWDITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

23. M8H5GIEGR-Human T45K (SEQ ID NO: 45)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK-

KIMDRMKVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK-

SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

24. M8H5GIEGR-Human A135E (SEQ ID NO: 46)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGREPQLRETLLQDFRVVAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

25. M8H5GIEGR-Human V170S (SEQ ID NO: 47)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI- TAKLYGRAPQLRETLLQDFRVVAQGVGIPEDSIFTMADRGECSPGEQEPEPILIPR

26. M8H5GIEGR-Human V148D (SEQ ID NO: 48)

MHHHHHHHHGGGGGIEGRGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK- KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRDVAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR 27. M8H5GIEGR-Human G172Q (SEQ ID NO: 49)

MHh GGGG' , GPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLK-

KIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPQEQEPEPILIPR

33. M8H4DK-Human M41 K+R66H (SEQ ID NO: 50)

IVIH 5DDKGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKI K- DRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR 34. M8H4DK-Human M41 K+N1796D (SEQ ID NO: 51 )

3DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIK- DRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWDITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

35. M8H4DK-Human R66H+N1796D (SEQ ID NO: 52)

3DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM- DRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHK- SKWDITMESYWHTNYDEYAIFLTKKFSRHHGPTI- TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

36. M8H4DK-Human M41 K+R66H+N1796D (SEQ ID NO: 53)

)DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIK- DRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHK- SKWDITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

37. M8H4DK-Human M41 K (SEQ ID NO: 8)

3DDKGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIK- DRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

38. M8H4DK-Human R66H (SEQ ID NO: 54)

)DDKGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIM- DRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR 39.M8H4DK-Human N1796D (SEQ ID NO: 55)

Mh 3DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM-

DRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK-

SKWDITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR 40. M8H-Human wt (SEQ ID NO: 56)

MH GPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIM- DRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK- SKWNITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

41. M8H-Human R66H+N1796D (SEQ ID NO: 57)

MH GPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM- DRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHK- SKWDITMESYWHTNYDEYAIFLTKKFSRHHGPTI-

TAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

42. untagged-Human R66H+N1796D (SEQ ID NO: 3)

i GPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGA- TEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHKSKWDITMESYV- VHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDSIFT- MADRGECVPGEQEPEPILIPR 61 untagged-Human wt (SEQ ID NO: 2)

MGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGA- TEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYV- VHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDSIFT- MADRGECVPGEQEPEPILIPR

DNA sequence of all constructs:

60. M8H4DK-Human wt (SEQ ID NO: 58)

ATGCATCACCATCACCATCACCATCAC* iCGAIGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGG- CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

1.M8H5GIEGR-Mouse (SEQ ID NO: 59)

A 1 UA 1 UAUUA I UAUUA I UAUUA I LAUbb I bbAbbAbbbbu 1 A 1 UbAbbb-

CCGCGACCCTGCGTCAACACTGCCAGATATCCAGGTTCAGGAGAACTTCAGTGAG TCCCGGATCTATGGAAAATGGTACAACCTGGCGGTGGGATCCACCTGCCCGTGG- CTGAGCCGCATTAAGGACAAGATGAGCGTGAGCACGCTGGTGCTGCAGGAGGGG GCGACAGAAACAGAGATCAGCATGACCAGTACTCGATGGCGGAGAGGTGTCTGT- GAGGA-

GATCACTGGGGCGTACCAGAAGACGGACATCGATGGAAAGTTCCTCTACCACAAA TCCAAATGGAACATAACCTTGGAATCCTATGT- GGTCCACACCAACTATGACGAATATGC-

CATTTTCCTTACCAAGAAGTCCAGCCACCACCACGGGCTCACCATCACTGCCAAG

CTCTATGGTCGGGAGCCACAGCTGAGGGACAGCCTTCTGCAGGAGTTCAAG-

GATGTGG-

CCCTGAATGTGGGCATCTCTGAGAACTCCATCATTTTTATGCCTGACAGAGGGGA ATGTGTCCCTGGGGATCGGGAGGTGGAGCCCACATCAATTGCCAGATGA

2. M8H5GIEGR-Naked Mole rat (SEQ ID NO: 60)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCAATCCTGTGCCGATGCCGCCAGACAACATCCAAGTGCAGGA- GAACTTTGATGAATCCCGGATCTATGGGAAATGGTTCAACCTGGCTACGGG- CTCCACGTGCCCGTGGCTGAAGAGGATCAAAGACAGGCTGAGTGT- GAGCACAATGGTGCTGGGCAAGGGGACCACGGA-

GACACAGATCAGCACAACCCACACCCACTGGCGGCAAGGGGTGTGCCAGGA- GACCTCAGGGGTTTACAAGAAAACAGACACGG- CTGGGAAGTTCCTCTACCACAAGTCCAAATGGAATGTAACCATGGAGTCCTATGT- GGTCCACACCAACTATGATGAGTATGC-

CATCATTCTAACTAAGAAGTTCAGCCACCACCATGGACCGAC- CATTACTGCCAAGCTCTATGGGAGAGAGCCGCGGCTGAGA- GACAGCCTCCTGCAGGAATTCAGGGAGATGGCCCTGGGCGTAGG- CATCCCCGAGGATTCCATCTTCACAATGGCCAACAGAGGGGAATGT- GTCCCTGGTGACCAGGCACCAGAGTCCACCCCAGCCCCGAGGTGA

3. M8H5GIEGR-Frog (SEQ ID NO: 61 )

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCTGCAGCCCAATCCAGCCAGAGGACAATATCCAGATCCAGGA- GAACTTTGATCTCCAGAGGATTTATGGCAAATGGTACGACATTGCCATCGG- CTCCACCTGCAAATGG-

CTGAAGCACCACAAGGAAAAGTTCAACATGGGGACACTGGAGCTTAGCGATGGG- GAGACCGACGGGGAGGTGCGGATTGTGAACACAAG-

GATGAGGCACGGAACCTGCTCTCAGATTGTTGGGTCCTATCAGAAGACAGA-

GACCCCAGGGAAGTTCGACTATTTCAACGCACGGTGGGGAACCACGATCCAAAA

CTACATTGTCTTCACTAACTACAATGAG-

TATGTCATCATGCAGATGAGGAAGAAGAAGGGATCGGA- GACCACCACGACCGTCAAGCTGTATGGGCGGAGCCCA-

GACTTGCGTCCGACCCTCGTTGATGAATTCAGGCAGTTTGCCTTGGCTCAGGG-

CATTCCTGAAGACTCCATCGTGATGCTACCTAACAATGGTGAGT-

GCTCTCCAGGGGAAATAGAAGTGAGACCACGGAGATGA

4. M8H5GIEGR-Chicken (SEQ ID NO: 62)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCACGCCTGTTGGGGACCAGGATGAGGACATTCAAGTGCAAGAGAATTTTGA GCCTGAGCGGATGTATGGGAAATGGTATGACGTAGCTGTTGG- CACCACCTGCAAGT-

GGATGAAGAACTACAAGGAGAAGTTCAGCATGGGCACACTGGTGCTGGGCCCCG GCCCCAGCGCTGACCAGATCAGTACCATCAGCACCAGGCTGCGG- CAAGGTGACTGCAAACGT-

GTCTCAGGAGAGTACCAGAAAACTGACACCCCTGGCAAATACACCTACTATAACC CCAAGTGGGATGTGTCTATCAAGTCCTACGT-

GCTTCGCACCAACTATGAAGAATACGCAGTCATTCTGATGAAGAAGACAAGTAATT TTGGCCCAACCACCACACTGAAGCTGTATGGGAGAAGCCCAGAGCTGCGGGAA- GAGCTCACCGAGGCTTTCCAGCAGCTGGCTCTGGAGATGGGCATCCCTGCAGAT TCCGTCTTCATCCTGGCCAACAAAGGTGAATGTGTCCCACAGGA- GACTGCCACTGCCCCTGAGAGGTGA

5. M8H5GIEGR-Rabbit (SEQ ID NO: 63)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGACCCCGTGCCCACCCTGCCGGACGACATCCAAGTGCAGGA- GAACTTCGAGCTCTCTCGGATCTACGGGAAATGGTACAACCTGGCTGT- GGGGTCCACCTGCCCGTGGCTGAAGAGGATCAAGGACAGGATGGCCGT- GAGCACGCTGGTGCTGGGAGAGGGGACGAGCGAGACGGA- GATCAGCATGACCAGCACGCACTGGCGGAGGGGCGTCTGTGAGGA- GATCTCCGGGGCCTATGA-

GAAAACGGACACTGACGGGAAGTTCCTGTACCACAAAGCCAAATGGAACTTAAC- CATGGAGTCCTACGTGGTGCACACCAACTACGATGAGTATGC- CATTTTTCTCACCAAGAAATTCAGCCGCCGCCACGGCCCCAC- CATCACCGCCAAGCTCTATGGGCGGGAGCCGCAGCTGAGGGA-

GAGCCTCCTGCAGGAGTTCAGGGAGGTGGCTCTCGGGGTGGGGATCCCCGA-

GAACTCCATCTTCACCATGATCGACAGAGGGGAATGTGTGCCCGGG-

CAGCAGGAACCAAAGCCTGCCCCCGTGTTGAGATGA

6. M8H5GIEGR-SQ Monkey (SEQ ID NO: 64)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG-

CCGCAGCCCAGTGCCGACGCCGCCCGAAGGCATTCAAGT-

GCAGGAAAACTTCAATCTCTCTCGGATCTACGGCAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTAAAGAAGATCATGGACAGGTTGAAAGT- GAGCACGCTGGTGCTGGAAGAGGGCGCCACGGAGGCGGA- GATCAGCATGACCAGCACTCGCTGGCGGAAAGGTTTCTGTGAGCA- GACCTCTTGGGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACGAACCCAAATGGAACGTAAC- CATGGAGTCCTATGTGGCCCACACCAACTATGAGGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTATGGGCGG-

GAGCCACAGCTGAGGGAAAGCCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGATTCCATCTTCACCATGG- CTAACCGAGGTGAATGCGTCCCTGGG-

7. M8H5GIEGR-Walrus (SEQ ID NO: 65) ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCAGTCCCGTGCTGACGCCGCCTGACGCCATCCAAGTGCAAGA- GAACTTCGACATCTCTCGGATCTACGGGAAGTGGTTTCATGTGGCCATGGG- CTCCACCTGCCCGTGGCTGAAGAAGTTCATGGACAG- GATGTCCATGAGCACGCTGGTGCTGGGCGAGGGGGCGACGGATGGGGA- GATCAGCATGACCAGCACACGTTGGCGGAGAGGCACCTGTGAGGA- GATCTCTGGGGCTTATGAGAAAACCAGCACTAACGGAAAGTTCCTCTATCA- TAATCCCAAATGGAACATCACCATGGAGTCCTATGTGGTCCACACCGACTAT- GATGAGTACGCCATCTTTCTGACCAAGAAATTCAGCCGCCACCATGGGCCCAC- CATTACTGCCAAGCTCTATGGGCGACAGCCGCAGCTTCGAGAAAGCCTGCTG- GAGGAGTTCAGGGAGCTTGCCTTGGGTGTGGG- CATCCCCGAGGACTCCATCTTCACCATGGCCAACAAAGGTGAGTGT- GTCCCTGGGGAGCAGGAACCAGAGCCCTCTCCACACATGAGGTGA

8. M8H5GIEGR-Manatee (SEQ ID NO: 66)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCAGCCCAGTGAAAACACCACTCAACGACATCCAAGTGCAGGA- GAACTTTGACCTCCCTCGGATCTACGGGAAATGGTTCAACATAGCCATTGG- CTCCACCTGCCAATGGCTGAAGAGGTTGAAGGCCGGGCCGAC- CATGAGCACCCTGGTCCTGGGAGAGGGAGCTACAGACACAGA- GATCAGCACAACCAGCACTCGTTGGCGGAAAGGCTTCTGTGAGGA- GATCTCTGGGGCATATGAGAAAACAGACACAGCTGGGAAGTTCCTTTATCACG- GATCCAAATGGAATGTAACCTTGGAGTCCTATGTGGTCCACACCAACTAT- GATGAGTACGCCATTTTTCTGACCAAGAAATTCAGCCGCTATGGACTCAC- CATTACTGCTAAGCTCTATGGGCGGCAGCCTCAGGTGAGGGAGAGCCTCCTG- GAGGAGTTCAGGGAATTTGCCCTGGGTGTGGGCATCCCTGAG- GATTCCATCTTCACCACGGCCGACAAAGGTGAGTGTGTCCCTGGAGAGCAG- GAGCCAGAACCCACCGCAGCCCTGAGATGA

9. M8H5GIEGR-Plaice (SEQ ID NO: 67)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCCTCCCTGT-

GCTCCCTGAACCTCTTTACCCGACACAGGAGAACTTTGATCTGACCCGGTTTGTG GGGACATGG-

CACGATGTTGCCTTGACGAGCAGCTGCCCCCATATGCAGCGTAACAGGGCG- GATGCAGCCATTGGTAAACTGGTTCTGGAGAAAGACACTGGAAACAAACTCAAGG TGACACGAACTAGACTCAGACATGGAACATGTGTGGAGATGTCTGGA- GAATATGAGT-

TAACCAGCACACCAGGACGAATCTTCTACCATATTGACAGGTGGGATGCAGACGT GGACGCCTACGTGGTTCACACCAACTACGACGAGTACGCAATTATAA- TAATGAGCAAACAGAAAACATCGGGGGAGAACAGCACCTCACTCAAGCTGTACAG TCGGACGATGTCTGTGAGAGACACTGTGCTGGATGACTTCAAAACTCTGGTCA- GACATCAGGGAATGAGTGACGACACCATTATCATCAAGCAGAACAAAGGTGACTG TATTCCTGGAGAGCAGGTGGAAGAAGCACCATCTCAGCCA- GAGCCCAAGCGGTGA

10. M8H5GIEGR-Orangutan (SEQ ID NO: 68)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCGACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACCCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACATCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCGTCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG-

CGCCGCAGCTGAGGGAAACCCTCCTGCAGGACTTCAGAGT-

GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG-

CTGACCGAGGTGAATGTGTCCCTGGGGAACAGGAACCAGAGCCCATCT- 1 1. M8H5GIEGR-Human P35K (SEQ ID NO:69)

ATGCATCACCATCACCATCACCATCACf AGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCAAATGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA-

GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

12. M8H5GIEGR-Human M41 K (SEQ ID NO: 70

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCGGATC TATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATC

GACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACAAATCCAAATGG AACATAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCACCATTACTGCCAAG CTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCG AGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGA- GATGA

13. M8H5GIEGR-Human R66H (SEQ ID NO: 71 )

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCATTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

14. M8H5GIEGR-Human T75K (SEQ ID NO: 72)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT-

GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGAAATCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT-

GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG-

CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT-

TAATCCCGAGATGA 15. M8H5GIEGR-Human T75Y (SEQ ID NO: 73)

ATGCATCACCATCACCATCACCATCACl 3AGGAGGGGGTATCGAGGG- CCGCGGCCCTGT-

GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCGGATC TATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGGC GCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGT- GAG-

GAGTATTCTGGAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACAA ATCCAAATGGAACATAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAG- TATGC-

CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCACCATTACTGCCAAG CTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCG AGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGA- GATGA

16. M8H5GIEGR-Human M99K (SEQ ID NO: 74)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAACCAAAGAGTCCTATGT- GGTCCACACCAACTATGATGAGTATGCCATTTTCCTGACCAAGAAATTCAGCCGC- CATCATGGACCCACCATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT-

GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG-

CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT-

TAATCCCGAGATGA

17. M8H5GIEGR-Human S101Y (SEQ ID NO: 75)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAACCATGGAG TATGT- GGTCCACACCAACTATGATGAGTATGCCATTTTCCTGACCAAGAAATTCAGCCGC- CATCATGGACCCACCATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

18. M8H5GIEGR-Human K69.92.1 18.130R (SEQ ID NO: 76)

ATGCATCACCATCACCATCACCATCACf AGGAGGGGGTATCGAGGG- CCGCGGCCCTGT-

GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCGGATC

TATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG-

CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGGC GCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGGCGGCGT- GGTGTCTGTGAGGA-

GACGTCTGGAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACCGTT CCAAATGGAACATAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAG- TATGC-

CATTTTCCTGACCAAGCGTTTCAGCCGCCATCATGGACCCACCATTACTGCC CTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCG AGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGA- GATGA

19. M8H5GIEGR-Coelacanth (SEQ ID NO: 77)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG-

CCGCGGAAGTCCCCTTCGAGATGAAGACATCCAAGTGCAGGA- GAACTTTGACCTTCCCAG-

GATTTATGGAAAATGGTACGAAATTGCAATCGCTTCGACCTGTCCCTGGGTGAAG

AATCACAAGGATAAGATGTTCATGGGAACTATGGTGCTACAAGAGGGAGAGCA-

GAGTGACCGGATCAGTACCACCTCCACCCGAATCAGG-

GATGGAACCTGCTCACAGATCACTGGATATTACACGT- TAACCACAACACCTGGGAAGTTCGCTTATCACAATTCTAAATGGAACTTGGATGTC

AACAGTTATGTTGTTCACACTAACTATGACGAATACTCGATTGT-

GAT-

GATGCAGAAATACAAAAGCTCTAACTCTACCACTACAGTCCGACTCTATGGAAGAA CTCAAGAGCTACGAGACAGCTTGCATGCCGAGTTCAAAAAGTTTGCTCTG- GATCAGGGAATAGATGAGGACTCCATTTACATTCTGCCAAAAAGAGATGAATGT- GTACCTGGTGAACCTAAAGCAGAATCTCTCATGGCACGTTGA

21. M8H5GIEGR-Human L89T (SEQ ID NO: 78)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT-

GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACTGATGGGAAGTT-

TACCTATCACAAATCCAAATGGAACATAACCATGGAGTCCTATGT- GGTCCACACCAACTATGATGAGTATGCCATTTTCCTGACCAAGAAATTCAGCCGC- CATCATGGACCCACCATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA 22. M8H5GIEGR-Human N1796D (SEQ ID NO: 79)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGT-

GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCGATATCTCTCGGATC TATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGGC GCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGT- GAGGA- GACGTCTGGAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACAAAT CCAAATGGGATATAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAG- TATGC-

CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCACCATTACTGCCAAG CTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCG AGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGA- GATGA

23. M8H5GIEGR-Human T45K (SEQ ID NO: 80)

A I bL A I UAL-OA I UAUUA I UAUUA I UAb I bbAubAbbbbb I A I LbAbbb- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGAAAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG-

CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

24. M8H5GIEGR-Human A135E (SEQ ID NO: 81 )

ATGnATnACCATCACnATnACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGG-

CGGGAACCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

25. M8H5GIEGR-Human V170S (SEQ ID NO: 82) ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA-

GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTTCTCCTGGGGAGCAGGAACCAGAGCCCATCT-

26. M8H5GIEGR-Human V148D (SEQ ID NO: 83)

ATGCATCACCATCACCATCACCATCACGGTGGAGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC-

CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC-

CATTACTGCCAAGCTCTACGGGCGGG-

CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGA-

GATGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT-

TAATCCCGAGATGA

27. M8H5GIEGR-Human G172Q (SEQ ID NO: 84)

ATGCATCACCATCACCATCACCATCACi 3AGGAGGGGGTATCGAGGG- CCGCGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT-

GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT-

GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTCAGGAGCAGGAACCAGAGCCCATCT-

33. M8H4DK-Human M41 K+R66H (SEQ ID NO: 85) ATGCATCACCATCACCATCACCATCAC< vCGATGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCAAAGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCATTGG-

CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG-

CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

34. M8H4DK-Human M41 K+N1796D (SEQ ID NO: 86)

ATGCATCACCATCACCATCACCATCA GATGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCGATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCAAAGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGG-

CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGGATATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG-

CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

35. M8H4DK-Human R66H+N1796D (SEQ ID NO: 87)

ATGCATCACCATCACCATCACCATCAC* vCGATGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCGATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCATTGG-

CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGGATATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG-

CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

36. M8H4DK-Human M41 K+R66H+N1796D (SEQ ID NO: 88)

ATGCATCACCATCACCATCACCATCAO vCGATGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCGATATCTCTCGGATC TATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCAAA-

GACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA GATCAGCATGACCAGCACTCATTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACAAATCCAAATGG GATATAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCACCATTACTGCCAAG CTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCG AGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGA- GATGA

37. M8H4DK-Human M41 K (SEQ ID NO: 89)

ATGCATCACCATCACCATCACCATCAC* vCGATGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCAAAGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGG- CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

38. M8H4DK-Human R66H (SEQ ID NO: 90)

ATGCATCACCATCACCATCACCATCAC< GATGACAAGGGCCCTGT- GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCATTGG- CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

39. M8H4DK-Human N1796D (SEQ ID NO: 91 )

ATGCATCACCATCACCATCACCATCAC< vCGATGACAAGGGCCCTGT-

GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCGATATCTCTCGGATC TATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGGC GCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGT- GAGGA-

GACGTCTGGAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACAAAT CCAAATGGGATATAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAG- TATGC-

CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCACCATTACTGCCAAG CTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCG AGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGA- GATGA

40. M8H-Human wt (SEQ ID NO: 92)

ATGCATCACCATCACCATCACCATCACGGCCCTGT-

GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGG- CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT-

GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG-

CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT-

TAATCCCGAGATGA

41 . M8H-Human R66H+N 1796D (SEQ ID NO: 93)

ATGCATCACCATCACCATCACCATCACGGCCCTGT-

GCCAACGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCGATATCTCTCG- GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGG- CTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG- CGCTACAGAGGCGGAGATCAGCATGACCAGCACTCATTGG- CGGAAAGGTGTCTGTGAGGAGACGTCTGGAGCTTATGAGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGGATATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT-

GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG-

CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT-

TAATCCCGAGATGA

42. untagged-Human R66H+N 1796D (SEQ I D NO: 94)

GGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCGATATCTCTCG-

GATCTATGGGAAGTGGTACAACCTGGCCATCGGTTCCACCTGCCCCTGGCTGAA

GAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTGGGAGAGGG-

CGCTACAGAGGCG- GAGATCAGCATGACCAGCACTCATTGGCGGAAAGGTGTCTGTGAGGAGACGTCT G GAG CTTATG AGAAAACAGATACT- GATGGGAAGTTTCTCTATCACAAATCCAAATGGGATA- TAACCATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGCCATTTTCCTG ACCAAGAAATTCAGCCGCCATCATGGACCCACCATTACTGCCAAGCTCTACGGG- CGGG-

CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGTGGTTGCCCAGGGTG TGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCGAGGTGAATGT- GTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGAGATGA

61. untagged-Human wt (SEQ ID NO: 95)

ATGGGCCCTGTGCCAACGCCGCCCGACAACATCCAAGT- GCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCTGG- CCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGT- GAGCACGCTGGTGCTGGGAGAGGGCGCTACAGAGGCGGA- GATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGACGTCTG- GAGCTTATGAGAAAACAGATACT-

GATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC- CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGC- CATTTTCCTGACCAAGAAATTCAGCCGCCATCATGGACCCAC- CATTACTGCCAAGCTCTACGGGCGGG- CGCCGCAGCTGAGGGAAACTCTCCTGCAGGACTTCAGAGT- GGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGG- CTGACCGAGGTGAATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCT- TAATCCCGAGATGA

Rationale of A 1M variant constructions

Human wt-A1 M, 1 1 A1 M-homologues from various species, and 15 A1 M-variants with point mutations were constructed, expressed, purified and analysed in phase I, i.e. a total of 27 A1 M-variants (Table 2). The 1 1 A1 M-homologues were selected as follows. A1 M is well conserved between species. A1 M sequences of different species were searched for in data bases (www.ncbi.nlm.nih.qov and www.uniprot.org). AMBP sequences from 67 different species were found. The sequences were investigated for presence of A1 M-specific functional groups (K69, K92, K1 18, K130, Y22; Y132, H122 and H123), lipocalin motifs (SCR1 , 2, 3) (see refs in Introduction) and predicted carbohydrate binding sites (Escribano et al., 1990). Five were classified as non-A1 M and dismissed because they lacked cystein 34, three were dismissed because they lacked cystein 169, four because they were incomplete, and two because they had long, ques- tionable, inserts. The amino acid sequences of the remaining 53 homologues were aligned and their suggested 3D structure were modelled by projecting their amino acid sequences on the crystal structure of human A1 M (Meining and Skerra, 2012) to identify the location of individual amino acid side chains in the lipocalin loops or on the inside or outside of the pocket. The result of this was used for construction of point muta- tions affecting A1 M-function as described below. The rationale for selection of the final 1 1 homologues (see Table 2) was a combination of wide-spread evolutionary representation, methodological feasibility, potentially increased environmental oxidative stress in the living habitat of the species, presence or absence of A1 M-specific functional groups, and lack of glycosylation. The 15 A1 M-variants with point mutations were selected as follows. Eight of the variants were mutated in strategic amino acids for functional properties and seven variants in exposed positions to improve stability/solubility (Table 2). The eight functional mutations were selected as they 1 ) occurred in other species in positions in the 3D-structural model (see above) that could potentially influence the function of A1 M and/or 2) theoretically would lower the pKa of cystein 34 and/or 3) theoretically would provide an extra radical trapping site. Predicted data for all variants was calculated using the http://www.alphalyse.com/qpmaw lite.html tool (Ta- ble 3).

P017240DK1 80

Table 2. Variants expressed and ana ysed in A1 M variants project phase I

P017240DK1 81

P017240DK1 82

• Numbering corresponds to the sequences mentioned above

Table 3. Predicted data for phase I variants

Phase I

The variants were expressed in parallel shake-flasks. There was a variation in expression levels and for a few A1 M-variants expression had to be repeated several times to obtain reasonable protein amounts. Good expression and relatively large amounts after purification (>10mg/L culture) were obtained for mouse, squirrel monkey, walrus, orangutan, M41 K, R66H, T75Y, M99K, N 17,96D, T45K and V148D. The expression and amount of purified protein was similar to previous yields for human wt A1 M

(12mg/L) expressed and purified under similar conditions. Variants with lower expression levels resulting in lower yields (1-10mg/L) were naked mole rat, frog, rabbit, manatee, P35K, T75K, S101Y, L89T, V170S and G172Q. Chicken, plaice and coelacanth A1 M displayed suboptimal expression levels resulting in a purification yield of <1 mg/L culture. The quadruple human mutant K69,92,1 18,130R expressed well, but was impossible to refold successfully. Also T75K-A1 M showed increased precipitation tendencies during purification. All variants were purified to >99% purity according to SDS-PAGE except chicken and coelacanth A1 M, which were purified to 95% purity. According to SDS-PAGE all variants contained a maximum of 0.5% covalent dimers with no major differences between the variants. Circular dichroism revealed about 2% alpha helix and 40% β-sheet structure for all variants without major differences, the expected values for A1 M (Kwasek et al., 2007). After purification, all variants were analysed as summarized in Table 4.

Firstly, stability and solubility was analysed. The aggregation tendency and thermostability of freshly thawed, unstressed 0.1 mM protein solutions were analysed by dynamic light scattering (DLS), PAGE (aggregation) and differential scanning fluorimetry (DSF) (thermostability). In addition, the tendency to aggregate in response to shearing forces or concentration to 1 mM was investigated. Human wt A1 M performed relatively well in these assays (Table 4), but some variants performed even better in the stability/solubility properties: chicken A1 M, manatee A1 M and N17,96D-A1 M. The chicken and manatee A1 M differed from human wt A1 M in too many positions to enable assumptions on individual amino acids beneficial for human A1 M and was not used in further studies. When functional properties were investigated, M41 K and R66H were found to have the same (R66H) or improved (M41 K) functions. As these variants also had higher thermostability and only slightly higher tendency to aggregate than most other variants, they were selected for further investigation. Hence, N17,96D-A1 M, M41 K-A1 M and R66H- A1 M were continued to phase II of the project. P017240DK1 85 Table 4. Solubility, Stability and functional properties of phase I variants.

P017240DK1 86

P017240DK1 87

Properties regarded to be better for variant compared to human wt A1 M are marked in dark blue with white, bold text. Properties regarded to be inferior of variant compared to human wt A1 M are marked in red. All other properties are regarded as similar to human wt A1 M.

In PAGE, variants with a significantly higher percentage of large aggregates are judged as inferior to human wt A1 M.

In DLS, an average radius » 3nm is regarded as indication of aggregates as well as difficulties in recording of measurements (seen as lower 5 number of measurements used for calculations).

In DSF a T _m >3 S.D of human wt A1 M is regarded as higher or lower.

Free thiols of human wt A1 M is typically in the range 0.6-0.8 anything significantly below this is regarded as inferior.

In heme binding assay, human wt A1 M binds to =80% in this assay. Binding of other variants below 75% and above 85% is regarded as different.

10 In heme reduction assay a Soret band peak maximum <400nm is regarded as inferior of human wt A1 M and a shift≥409nm is regarded as better than human wt A1 M.

In ABTS assay, a T-test p-value of <0.2 is regarded to indicate an inferior ABTS reduction capacity.

Phase II

The aim of phase II of the project was to combine the beneficial mutations (M41 K, R66H and N17,96D) to investigate if even more stable variants with similar or improved function could be constructed. In addition, the same N-terminal tag (M8H4DK) which is used for human wt A1 M was introduced (Table 5). Thus, the new combined M8H4DK- variants were compared to M8H4DK-human wt.

Table 5. Variants expressed in phase I with predicted data

All four new variants (M8H4DK-M41 K+R66H, M8H4DK-M41 K+N17,96D, M8H4DK- R66H+N17,96D and M8H4DK-M41 K+R66H+N17,96D) expressed very well and yields of 29, 13, 37 and 15 mg pure protein/L were achieved. The four new variants were purified to >99% purity according to SDS-PAGE and showed the same or lower percentage of covalent dimers except M8H4DK-M41 K+R66H that showed approximately 2% covalent dimers.

Solubilization/stability properties were analysed (Table 6a). Data revealed that the combination of M41 K and R66H was suboptimal for thermostability. The N17,96D mutation, on the other hand, was generally beneficial for solubilization and stability, and the combination with R66H (i.e. the M8H4DK-R66H+N17,96D-A1 M) yielded even better solubilization and stability. Functional properties were analysed (Table 6b). Data showed that the M41 K mutation had increased heme reduction capacity, but decreased heme binding. When the M41 K mutation was combined with N17,96D, however, it showed less potential to rescue cells from heme-induced cell death. The R66H+N17,96D combination yielded similar results compared to wt-A1 M in all functional aspects investigated. Therefore, the M8H4DK-tagged R66H+N 17,96D A1 M is a promising candidate after phase II, while the M41 K-mutation is less useful.

P017240DK1 90

Table 6a. Solubility, Stability and functional properties of phase II variants.

Properties regarded to be better for variant compared to human wt A1 M are marked in dark blue with white, bold text. Properties regarded to be inferior of variant compared to human wt A1 M are marked in red. All other properties are regarded as similar to human wt A1 M.

In PAGE, variants with a significantly higher percentage of large aggregates are judged as inferior to human wt A1 M.

In DLS an average radius » 3nm is regarded as indication of aggregates as well as difficulties in recording of measurements (seen as lower number of measurements used for calculations).

In DSF a T _m >3 S.D of human wt A1 M is regarded as higher or lower.

P017240DK1 91

Table 6b. Functional properties of phase II variants.

Human wt A1 M binds to =80% in the heme-agarose binding assay. Binding of other variants below 75% and above 85% is regarded as different.

In heme-binding absorbance assay, a redshift to <400nm is regarded as inferior of human wt A1 M and a shift≥417nm is regarded as better than human wt A1 M.

In ABTS assay, a T-test p-value of <0.2 is regarded to indicate an inferior ABTS reduction capacity.

Phase III

The first aim of phase III was to compare M8H4DK-tagged R66H+N17,96D-A1 M with wt-A1 M and promising M8H4DK-tagged single mutations of phase I and II, when expressed in parallel under identical conditions. The second aim was to investigate the influence of the tag on stability, solubility and function. The N-terminal tag of human wt A1 M (M8H4DK) consist of 8 histidines tag followed by an enterokinase cleavage site. The cleavage site enables cleavage of the histidine tag after expression and purification. Therefore, wt-A1 M and R66H+N17,96D-A1 M were expressed with N-terminal M8H4DK- and M8H-tags and without a tag. The third aim was to compare in more de- tail the properties of M8H4DK-wt A1 M (Wt-A1 M) and M8H4DK-R66H+N17,96D-A1 M (35-A1 M). All expressed variants of phase III with predictive data are shown in Table 7.

Table 7. Variants expressed in phase II with predicted data

The parallel expression and purification allowed more accurate comparison of expression levels and purification yields. It was found that all A1 M variants expressing bacteria grow with the same rate (not shown) and the same relatively good yield (Figure 2). The only difference was a slower expression of the untagged variants (#60 and #42) compared to the others. From the 2x750ml expressions it was possible to purify 20- 60mg of each variant (Figure 3a). All histidine-tagged variants were purified to >99% purity, while the untagged variants (#61 and #42) showed a somewhat lower purity (around 90%) (Figure 3b). All displayed a low amounts of covalent dimers. PAGE analysis showed less than 1 -2% large aggregates of all variants except M8H-wt (40) and untagged wt-A1 M (#61 ) (Figure 3c). Thus, all single and multiple M8H4DK-tagged, M8H-tagged and untagged A1 M variants could be produced.

The aggregation and thermostability of 100μΜ solutions were analysed by reversed- phase HPLC, dynamic light scattering and differential scanning fluorimetry (Table 8). The M8H4DK-tagged variants were eluted in RP-HPLC at approximately 12 min, with 87-90% of the protein in the main peak. The only exception was M8H4DK-M41 K that appeared at a retentation time of 9.8 minutes and only 80% of the protein in the main peak. The data once more indicate that the M41 K mutation yields unexpected protein properties of A1 M, and is suboptimal. Changing the tag to M8H or completely removing the tag, resulted in increased retentation times to 12.2 and 12.9 min, respectively, indi- eating more hydrophobic molecules, as expected. Less protein appeared in the main peak for M8H-wt and both untagged variants, possibly reflecting the slightly lower purity seen in SDS-PAGE of these variants. DLS-data indicated an average radius around 3 of all variants, and the possibility to collect data from all 6 recordings. All variants had the same or higher thermostability as M8H4DK-wt A1 M. Higher stability was seen for all variants carrying the N17,96D mutation.

Table 8. Aggregation and thermostability of non-stressed phase III variants.

The variants of phase I I I were exposed to shearing forces by pipetting 80 times in a narrow pipett tip. The shearing-stress induced aggregation was the analysed with DLS. In general, all variants with the N 17,96D mutation showed high tolerance towards shearing stress (Table 9), with the exception of M8H-tagged R66H+N 1796D variant (#41 ). Also, the tolerance towards stress induced by concentration to 1 mM in different buffers was investigated. All variants were concentrated to 1 mM in 20mM Tris-HCI + 0.125M NaCI pH 8.0 or 7.4, and were immediately analysed by PAGE (Figure 4a, Table 10). The 0.1 mM and 1 mM samples were also analysed with PAGE after a single freeze-thaw cycle in the Tris-buffers or PBS (Figure 4b, Table 10). Among the

M8H4DK-tagged variants all the N 17,96D-carrying mutations displayed an increased tolerance to concentration, freeze-thawing, decreased pH and buffer-change (to PBS) compared to wt A1 M. The addition of the R66H mutation improved the stability and solubility even further. The R66H mutation alone had approximately the same tolerance as wt A1 M, while the M41 K mutation had significantly lower tolerance. Shortening or removal of the N-terminal tag had a negative effect on the tolerance, which was more pronounced for wt A1 M compared to corresponding R66H+N 17,96D variants.

Table 9. Shearing stability of phase I I I variants.

P017240DK1 95

Table 10. Concentration and freeze-thaw stability of phase III variants in different buffers.

The functional activities of all phase III variants were investigated in an extended program to cover all possible aspects of known A1 M functions. Non-heme related reduction capacity was investigated on the synthetic ABTS radical and in the commercially available oxygen radical antioxidant capacity (ORAC) assay (Figure 5). M8H4DK- tagged N17,96D-A1 M and R66H+N 17,96D-A1 M showed a similar reduction capacity as wt A1 M, while M8H4DK-tagged M41 K-A1 M and R66H-A1 M showed a slightly lower ABTS reduction capacity (Figure 5A). Shortening or removal of the tag had little effect, but the R66H-N17,96D variants show a tendency to higher reduction capacity than wt A1 M when M8H-tagged and untagged. The performance of M8H4DK-tagged wt A1 M in the ORAC assay was set to 100% and the other variants were compared in relation to this. All M8H4DK- variants showed the same results in ORAC as M8H4DK-wt except M8H4DK-R66H+N17,96D which had significantly higher capacity (Figure 5b). Also the M8H-tagged and untagged R66H+N17,96D-A1 M showed a slightly higher ORAC ca- pacity than wt-A1 M. The reduction capacity was also investigated in a cytochrome c reduction assay (Figure 6). Most variants showed a slightly lower reduction capacity at the lower concentrations, compared to wt-A1 M, and for N17,96D and R66H+N17,96D this was significant (at 0.3-0.6μΜ) (Figure 6A). The importance of this is not understood. Shortening and removal of the tag had no influence of this property.

Heme-binding was investigated in a free heme incorporation assay and in a heme-agarose binding assay (Figure 6). The incorporation and coordination of free heme is seen as the appearance of an absorbance peak around 410-415 nm, the so-called Soret band (Karnaukhova et al., 2014; Rutardottir et al, 2016). Heme normally has an ab- sorbance peak around 380-390nm and this can be seen when it is dissolved alone or mixed and incubated with a non-binding control protein like ovalbumin. However, when heme is mixed with wt A1 M, an absorbance peak is formed between 410-415nm, yielding a so-called red-shift (i.e. a shift of the peak towards higher wave-lengths), and an increased Abs(413/386) ratio. It was shown that the degree of red-shift and the Abs(413/386) ratio are related to the heme-binding function of A1 M (Rutardottir et al, 2016). These properties were therefore compared for all variants in phase III (Figure 6b). Among the M8H4DK-tagged variants all showed about the same red-shift as wt A1 M except the M41 K variants which had a stronger shift. Interestingly, shortening the tag to M8H increased the red-shift, while removal of the tag decreased the red-shift. Clearly, the tag had a strong influence of this property. The binding of heme to A1 M was investigated as binding of a dilution series of A1 M to heme-agarose in comparison to binding to a control agarose. Typically, a control protein like ovalbumin shows no binding to heme-agarose above the background binding to the control agarose (Figure 6C). Typically, 70-80% of added M8H4DK-wt A1 M (35-A1 M) was bound to the heme agarose, whereas no-binding was seen to control agarose. The binding was compared for all variants in phase III. All showed similar degree of binding to the heme-agarose (66-72%), while ovalbumin showed no binding.

Heme binding of M8H4DK-wt A1 M (wt-A1 M) and M8H4DK-35-A1 M (35-A1 M) was further analysed (Figure 1 1 ) using a combination of migration shift and fluorescence anal- ysis (Karnaukhova et al. 2014). As a result of heme-incorporation, the migration of wt- A1 M and 35-A1 M in native PAGE analysis was slower at heme:protein ratio < 1 , and faster at heme:protein ratio > 1 , showing the same dependence upon heme-concentra- tion (Fig. 1 1A and B). At high heme concentrations, both variants showed a tendency towards oligomerization, supporting previous findings for wt-A1 M (Karanaukhova et al. 2014). Likewise, heme-incorporation induced quenching of tryptophan fluorescence in both wt-A1 M and 35-A1 M, with similar kinetics (Fig. 1 1 B). The coordination of heme in A1 M was previously shown to induce formation of a UV-absorbance peak at 415 nm (Ruttarsdottir et al., 2016; karnaukhova et al., 2014). Similar UV-absorbance patterns of wt-A1 M- and 35-A1 M/heme complexes were seen (Fig. 1 1 C), with only a small red- shift of the 35-A1 M peak (415^417 nm).

The rate of reduction of ABTS-radicals of wt-A1 M and 35-A1 M was investigated [10] and was similar for wt-A1 M and 35-A1 M using unstressed, freshly prepared proteins (Fig. 12A). After storage for 7 days, wt-A1 M displayed a slower reduction rate after concentration to 1 mM and at 22°C (Fig. 12B), suggesting a loss of protein activity in addition to the aggregation shown at these conditions (Table 1 1 ). The cytochrome c- reduction (Allhorn et al., 2005) was slightly slower for 35-A1 M (Fig. 12C), whereas the antioxidation capacity measured by the ORAC assay was somewhat higher for 35-A1 M (Fig. 12D). Table 1 1 . Physicochemical properties of M8H4DK-wt A1 M (wt-A1 M) and M8H4DK-35- A1 M (35-A1 M).

Conditions ¹ Concentration Method Parameter wt-A1 M 35-A1 M

HPLC Elution time (min) 12.1 12.1

Monomer (%) 87 89

DLS Radius (nm) 2.9 3.2

PAGE Aggregates (%) 1 .5 0.4

SEC Monomer (%) 87 93

Stressed

Heat 0.1 mM DSF T _m (°C) 46.6 51 .6

High cone 1 mM PAGE Aggregates (%) 1 .1 0.4 pH 7.4 ³ 1 mM PAGE Aggregates (%) 1 .2 0.4

Freeze-thaw ² 1 mM PAGE Aggregates (%) 2.0 0.4

Freeze-thaw/PBS 1 mM PAGE Aggregates (%) 13.2 7.6

Prolonged storage ⁴

4°C - 1 day 1 mM SEC Recovery ⁵ (%) 100 100

Aggregates (%) 17.9 3.9

4°C - 7 days 1 mM SEC Recovery (%) 100 100

Aggregates (%) 12.1 3.7

RT - 7 days 1 mM SEC Recovery (%) 44 100

Aggregates (%) 0.2 0.1

37°C - 1.5 h 1 mM SEC Recovery (%) 67 100

Aggregates (%) 53 28

37°C - 4.5 h 1 mM SEC Recovery (%) 0 44

Aggregates (%) ND ⁶ 15

Footnotes:

1. Room temperature unless otherwise stated.

2. 20 mM Tris-HCI, 0.125 M NaCI, pH 8.0

3. 20 mM Tris-HCI, 0.125 M NaCI, pH 7.4

4. PBS 5. Calculated after centrifugation 10,000xg, from total peak area compared to starting material.

6. Not determined A biologic assay where the ability of the A1 M variants to rescue K562 cells from heme- induced cell death was investigated (Figure 7). The degree of cell death, according to lactate dehydrogenase release, was set to 100% for cells incubated with heme without A1 M. All M8H4DK-tagged variants, including wt-A1 M, showed almost 100% rescue at 10μΜ (Figure 7), with no significant differences between the variants. The M8H-tagged variants showed almost the same rescuing potential, while a slightly lower potential was observed for the untagged variants.

Cell protection capacity of M8H4DK-wt A1 M (35-A1 M) and M8H4DK-35-A1 M M-A1 M) The cell protection properties of the two A1 M-variants were tested in K562 cells and a human kidney proximal tubule epithelial cell line (HK-2), exposed to free heme and free iron. Fig. 12 shows that both A1 M-variants completely inhibited the cell-death, measured by extracellular release of the cytosolic marker LDH, of K562-cells exposed to 0.1 mM heme. The dose-response curves of wt-A1 M and 35-A1 M overlaps almost completely, suggesting similar cell-protection capacities.

Protection of HK-2 cells are shown in Fig. 14. First, cell damage was induced by the Fenton reaction, a mixture of free iron, ascorbate and hydrogen peroxide which generates hydroxyl radicals. Cell viability, measured by the WST-1 assay, was restored by both A1 M-variants, following overlapping dose-response curves (Fig. 14A). The upreg- ulation of heme oxygenase-1 (HO-1 ), a well-documented biomarker of oxidative stress- response (Alam et al., 1999), was significantly suppressed by wt-A1 M and 35-A1 M to similar degrees (Fig. 14B). Cell damage of HK-2 cells was also induced by incubation with heme, and could be inhibited by both A1 M-variants (Fig. 14C and D). Again, cell viability measured by the WST-1 assay was restored by both proteins to a similar de- gree, using two heme concentrations, 10 and 30 μΜ (Fig. 14C). The upregulation of HO-1 and another cellular stress response gene, Hsp70, was inhibited to a similar degree by both A1 M-variants (Fig. 14D).

In vivo distribution of M8H4DK-wt A 1M (wt-A 1M) and M8H4DK-35-1M (35- A 1M) in mice Intravenously injected A1 M was previously shown to be rapidly cleared from blood and predominantly localized to kidneys and liver in rats and mice (Larsson et al., 2001 ; Ahlstedt et al., 2015). We compared the clearance rates in blood and distribution in organs of wt-A1 M and 35-A1 M (Fig. 15). Similar turnover rates were seen during the first 60 min, whereas 35-A1 M was cleared more rapidly after 1 h. The distribution of A1 M in the investigated organs after 10 and 30 min did not show any significant differences between the two proteins. Both wt-A1 M and 35-A1 M were found predominantly in kidneys, and smaller amounts were seen in heart, liver, lung, skin and spleen, while neglible amounts were found in brain.

In vivo protection of kidneys bv M8H4DK-wt A1 M (wt-A1 M) and M8H4DK-35-A1 M (35- A1 IV0

Previously, A1 M has shown in vivo therapeutic effects in animal models where heme- and oxidative stress-related kidney injuries are induced (Wester-Rosenlof et al., 2014; Naav et al., 2015; Sverrison et al., 2014). Here, we investigated the in vivo protective effects of the two A1 M-variants in a glycerol-injection rhabdomyolysis mouse model where acute kidney injuries (AKI) develops as a result of muscle rupture with release of myoglobin, free heme, radicals and other tissue components. The glycerol-injection resulted in a massive upregulation of the HO-1 and Hsp70 genes, two biomarkers of cel- lular stress (Fig. 16A and B), and most of the upregulation was inhibited by simultanous injection of wt-A1 M or 35-A1 M. No significant difference between the two A1 M-variants were seen at the applied doses (7 mg/kg animal weight).

In summary, the investigations in phase III showed that the N17,96D-containing A1 M- mutations significantly added stability to the A1 M molecule, which was further improved when the R66H mutation was added. Furthermore, M8H4DK-tagged A1 M variants were more stable than variants with a shorter tag or no tag. Therefore, the tag does not only serve as a purification tool but also provide A1 M with increased stability and stability and does not interfere with function. The M8H4DK-R66H+N1796D (35-A1 M) mole- cule show the same or better functional properties, possibly with the exception of the cytochrome c reduction. In conclusion, stability/solubility and functional studies suggest that M8H4DK-R66H+N17,96D (35-A1 M) has improved molecular properties compared to M8H4DK-wt A1 M (wt-A1 M), and both Wt-A1 M and 35-A1 M show in vivo protective effects on kidneys using a rhabdomyolysis glycerol-injection model of acute kidney in- jury. Further stability studies on M8H4DK-R66H+N1796D vs M8H4DK-wt.

To further compare M8H4DK-R66H+N17,96D-A1 M and wt-A1 M, aggregation and function were tested with SEC-FPLC and the ABTS reduction assay after freeze-thaw cycles and storage at +4°C and room temperature (Figure 8). Samples (100μΜ) of wt- A1 M and R66H+N17,96D-A1 M both tolerated 5x freeze-thaw cycles very well. As previously shown wt-A1 M showed more aggregation after concentration to 1 mM than R66H+N17,96D-A1 M, and similar results were seen after storage for 1 week compared to overnight. The increased aggregation of wt-A1 M did not affect the ABTS reduction activity of 2μΜ-83ΓηρΙβ8. Ocular inspections show completely clear solutions of both variants after freeze-thaw, concentration and storage for 1 week at +4°C. To stress the A1 M solutions further, 100μΜ and 1 mM solutions were stored in room temperature for 1 week. Ocular inspection of the 100μΜ solutions showed a slight cloudiness of wt- A1 M, but the R66H+N17,96D-A1 M solution remained clear. SEC-FPLC showed a decreased area under the curve of wt-A1 M to 80% of the starting material, indicating loss of wt-A1 M, although no increase in large aggregates was seen. The loss was most likely caused by precipitation of protein and removal by the filtration before the FPLC- run. For R66H+N17,96D-A1 M, some increased aggregation could be seen, but no loss of protein after the storage at room-temperature for one week at 100 μΜ. In the ABTS assay, R66H+N17,96D-A1 M still showed full activity, while a small decrease was seen for wt-A1 M. Ocular inspection of 1 mM solutions stored for 1 week showed cloudiness of both variants, but the wt-A1 M solution appeared thicker. When the solutions were diluted 10 times for loading onto the SEC, the precipitate of the R66H+N17,96D-A1 M, but not wt-A1 M, was resolved. In the SEC, very little aggreagates were seen, but only 44% of the wt-A1 M starting material remained. In contrast, 100% of R66H+N17,96D- A1 M remained. In the ABTS assay wt-A1 M had seriously decreased activity while the activity of R66H+N17,96D-A1 M stayed the same. This study shows that M8H4DK- R66H+N17,96D-A1 M can tolerate storage at high concentrations at room temperature, whereas wt-A1 M cannot. The stability of 1 mM solutions at 37°C was investigated. The samples were incubated for 1.5, 2.5 and 4.5 h and then ocularly inspected, analysed by SEC-FPLC and the ABTS reduction assay (Figure 9). After 1 .5 h incubation, both solutions were still clear when ocularly inspected, but the SEC-FPLC revealed more aggregates in the wt-A1 M solution (53%) compared to the R66H+N 17, 96D-A1 M solution (28%). Only 67% of the starting material was eluted on the SEC of wt-A1 M, while 100% was eluted of

R66H+N17,96D-A1 M, estimated by the area under the curve. Both variants showed full activity in the ABTS assay. After 2.5 h of incubation the wt-A1 M solution had become cloudy with precipitates impossible to resolve. 40% of the starting material was found on the SEC and the ABTS activity was significantly decreased. The R66H+N17,96D- A1 M solution was still clear and showed full activity in the ABTS assay, but large ag- gregates had increased from 28 to 38% in the SEC-FPLC. After 4.5 h, the wt-A1 M had developed into a thick substance, impossible to pipett. Therefore, it was not further analysed. The R66H+N17,96D-A1 M had become cloudy, but was still possible to pipette. Some precipitation remained after dilution. The amount of protein eluted on the SEC was 44% of the starting material and a decreased activity in the ABTS assay was seen. The study suggest that following incubation at +37°C, A1 M first forms resolvable aggregates, then it forms irreversible aggregates and loss of activity in the ABTS assay. Complete irreversible precipitation of wt-A1 M was seen after 2.5 hours of incubation, and partial irreversible precipitation was seen only after 4.5 hours for M8H4DK- R66H+N17,96D-A1 M. Hence, M8H4DK-R66H+N17,96D-A1 M is more resistant to stor- age at +37°C than M8H4DK-wt-A1 M.

References

Ahlstedt J, Tran TA, Strand F, Holmqvist B, Strand S-E, Gram M, and Akerstrom B. Bi- odistribution and pharmacokinetics of recombinant a-i-microglobulin and its potential use in radioprotection of kidneys. Am J Nucl Med Mol Imaging 5(4): 333-347, 2015. Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, and Cook JL. Nrf2, a

Cap'n'Collar transcription factor, regulates induction of the heme-oxygenase gene. J Biol Chem 274: 26071-26078, 1999.

Allhorn M, Berggard T, Nordberg J, Olsson ML, Akerstrom B. Processing of the lipocalin a-i-microglobulin by hemoglobin induces heme-binding and heme-degradation properties. Blood 99; 2002: 1894-1901 . Allhorn M, Klapyta A, Akerstrom B. Redox properties of the lipocalin a-i-microglobulin : reduction of cytochrome c, hemoglobin, and free iron. Free Radic Biol Med 38; 2005: 557-567.

Anderson UD, Rutardottir S, Centlow M, Olsson MG, Kristensen, K, Isberg PE, Thilaga- natan B, Akerstrom B, Hansson SR. Fetal hemoglobin and a-i-microglobulin as first and early second trimester predictive biomarkers of preeclampsia. Am J Obst Gynecol 204; 201 1 : 520.e1 -520.e5

Berggard T, Cohen A, Persson P, Lindqvist A, Cedervall T, Silow M, Th0gersen IB, Jonsson J-A, Enghild JJ, Akerstrom B. CH-Microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound. Protein Sci. 8; 1999: 261 1 -2620.

Berggard T, Thelin N, Falkenberg C, Enghild JJ, Akerstrom B. Prothrombin, albumin and immunoglobulin A form covalent complexes with i-microglobulin in human plasma. Eur J Biochem 245; 1997: 676-683.

Bratt T, Olsson H, Sjoberg EM, Jergil B, Akerstrom B. Cleavage of the a-i-microglobulin -bikunin precursor is localized to the Golgi apparatus of rat liver cells. Biochim Biophys Acta 1 157; 1993: 147-154.

Centlow M, Carninci P, Nemeth K, Mezey E, Brownstein M, Hansson SR. Placental expression profiling in preeclampsia: local overproduction of hemoglobin may drive pathological changes. Fertility and sterility 90; 2008: 1834-1843.

Ekstrom B., Berggard I. Human a-i-microglobulin: Purification procedure, chemical and physicochemical properties. J Biol Chem 252(22); 1977: 8048-8057.

Escribano J, Lopez-Otin C, Hjerpe A, Grubb A, Mendez E. Location and characteriza- tion of the three carbohydrate prosthetic groups of human protein HC. FEBS Lett 266(1 -2); 1990: 167-170.

Flower DR. The lipocalin family. Biochem J 318; 1996: 1-14. Gram M, Anderson UD, Johansson ME, Edstrom-Hagerwall A, Larsson I, Jalmby M, Hansson SR, and Akerstrom B. The human endogenous protection system against cell-free hemoglobin and heme is overwhelmed in preeclampsia and provides potential biomarkers and clinical indicators. PloS One 10(9): e01381 1 1 , 2015. Hansson SR, Gram M and Akerstrom B. Fetal hemoglobin in preeclampsia: a new etiological tool for prediction/diagnosis and a potential target for therapy. Curr Opin Obstet Gynecol. 25(6); 2013: 448-455. Karnaukhova E, Rutardottir S, Majabi M, Wester Rosenlof L, Alayash Al, Akerstrom B. Characterization of heme binding to recombinant ai-microglobulin. Front Physiol 5; 2014: 465. doi: 10.3389/fphys.2014.00465.

Kaumeyer JF, Polazzi JO, Kotick MP. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes ai-microglobulin (protein HC). Nucleic Acids Res 14(20); 1986: 7839-7850.

Kwasek A, Osmark P, Allhorn M, Lindqvist A, Akerstrom B, Wasylewski Z. Production of recombinant human ai-microglobulin and mutated forms involved in chromophore formation. Prot. Expr. Purif. 53; 2007: 145-152.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227; 1970: 680-685. Larsson J, Wingardh K, Berggard T, Davies JR, Logdberg L, Strand S-E, Akerstrom B. Distribution of ¹²⁵ l-labelled a-i-microglobulin in rats after intravenous injection. J Lab Clin Med 137: 165-175, 2001

Lindqvist A, Bratt T, Altieri M, Kastern W, Akerstrom B. Rat a-i-microglobulin: co-ex- pression in liver with the light chain of inter-alpha-trypsin inhibitor. Biochim Biophys Acta 1 130; 1992: 63-67.

May K, Rosenlof L, Olsson MG, Centlow M, Morgelin M, Larsson I, Cederlund M, Rutardottir S, Schneider H, Siegmund W, Akerstrom B, Hansson SR. Perfusion of human placenta with haemoglobin introduces preeclampsia-like injuries that are prevented by ai-microglobulin. Placenta 32(4); 201 1 : 323-332.

Meining W, Skerra A. The crystal structure of human a-i-microglobulin reveals a potential haem-binding site. Biochem J. 445(2); 2012: 175-82. Naav A, Erlandsson L, Axelsson J, Larsson I, Johansson M, Wester Rosenlof L, Morgelin M, Casslen V, Gram M, Akerstrom B, Hansson SR. A1 M ameliorates preeclampsia-like symptoms in placenta and kidney induced by cell-free fetal hemoglobin in rabbit. PloS One, 10(5); 2015: e0125499.

Olsson MG, Allhorn M, Larsson J, Cederlund M, Lundqvist, K, Schmidtchen A, S0ren- sen OE, Morgelin M, Akerstrom B. Up-regulation of AI M/a-i-microglobulin in skin by heme and reactive oxygen species gives protection from oxidative damage. PLoS One 6(1 1 ); 201 1 : Θ27505.

Olsson MG, Centlow M, Rutardottir S, Stenfors I, Larsson J, Hosseini-Maaf B, Olsson ML, Hansson SR, Akerstrom B. Increased levels of free hemoglobin, oxidation markers, and the antioxidative heme scavenger ai-microglobulin in preeclampsia. Free Rad. Biol. Med. 48; 2010: 284-291 .

Olsson MG, Olofsson T, Tapper H, Akerstrom B. The lipocalin ai-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species. Free Rad Res. 42; 2008: 725-736. Olsson MG, Rosenlof LW, Kotarsky H, Olofsson T, Leanderson T, Morgelin M, Fellman V, Akerstrom B. The radical-binding lipocalin A1 M binds to a Complex I subunit and protects mitochondrial structure and function. Antiox Redox Signal. 18(16); 2013: 2017- 2028. Rutardottir S, Karnaukhova E, Nantasenamat C, Songtawee N, Prachayasittikul V, Ra- jabi M, Wester Rosenlof L, Alayash Al, Akerstrom B. Studies of two heme binding sites in AI M/a-i-microglobulin using site-directed mutagenesis and molecular simulation. Bio- chim Biophys Acta 1864(1 ); 2016: 29-41. Sala A, Campagnoli M, Perani E, Romano A, Labo S, Monzani E, Minchiotti L, Galliano M. Human ai-microglobulin is covalently bound to kynurenine-derived chromophores. J Biol Chem 279(49); 2004: 51033-51044. Sverrisson K, Axelsson J, Rippe A, Gram M, Akerstrom B, Hansson SR. Rippe B. Extracellular fetal hemoglobin (HbF) induces increases in glomerular permeability. Inhibition with ai-microglobulin (A1 M) and Tempol. Am J Physiol Renal Physiol 306(4); 2014: F442-448.

Wester-Rosenlof L, Casslen V, Axelsson J, Edstrom-Hagerwall A, Gram M, Holmquist M, Johansson ME, Larsson I, Ley D, Marsal K, Morgelin M, Rippe B, Rutardottir S, Shohani B, Akerstrom B, Hansson SR. A1 M/ai-microglobulin protects from heme-in- duced placental and renal damage in a pregnant sheep model of preeclampsia. PLoS One 9(1 ); 2014: e86353.

Akerstrom B, Borregaard N, Flower D, Salier JP. Lipocalins, an introduction. In

Lipocalins. Eds. Akerstrom B, Borregaard N, Flower D, Salier JP, Landes Bioscience, Georgetown TX.

Akerstrom B, Gram M. A1 M, an extravascular tissue cleaning and house-keeping protein. Free Rad Biol Med 74; 2014: 274-282.

Akerstrom B, Maghzal GJ, Winterbourn CC, Kettle AJ. The lipocalin ai-microglobulin has radical scavenging activity. J Biol Chem 282; 2007: 31493-31503.

Akerstrom B, Bratt T, Enghild JJ. Formation of the ai-microglobulin chromophore in mammalian and insect cells: a novel post-translational mechanism? FEBS Lett 362, 50-54, 1995.