PRODUCTS AND METHODS AFFECTING CELL AND TISSUE GROWTH

Background of the Invention

Field of the Invention

The present invention relates to new products, and methods, which can be used in regulating cell and tissue growth. In particular, the present invention relates to new products, and methods, which can be used for improving cell or tissue growth.

Description of Related Art

By tumour is meant a swelling caused by uncontrolled growth of cells. In cancer the tumours are malignant, which means that the cells in the tumour are aggressive, invasive and sometimes metastatic. On the other hand, there are conditions, where improved growth of cells or tissues would be of advantage. For example, in wound healing improved growth of cells and tissues would be of advantage. Furthermore, in in vitro cell and tissue cultures, regulation and in particular improved growth would be of advantage.

In many diseased conditions associated with cell apoptosis, such like tissue degenerative disorders, may benefit the treatments increasing cell growth. Not only the diseases discussed below but also apoptosis plays an important role in the differentiation, development and homeostatic maintenance of many tissues. There is a growing evidence that apoptosis and necrosis are not always separable phenomena and that necrosis can be one outcome of an initially apoptotic process. For example, in the focal ischemia model of stroke the center of the infarct area dies by necrosis. Recent studies suggest the same situation is true in several muscular dystrophies. Markers of apoptosis and necrosis coexist in the muscle of Duchenne muscular dystrophy (DMD) patients Diseases associated with apoptosis and/or necrosis are thus of interest.

There is thus a need for new methods and products for regulating cell and tissue growth both in vitro and in vivo, and in particular for methods and products improving cell and tissue growth.

Summary of the Invention

It is an aim of the present invention to eliminate at least some problems of the prior art.

One object of the invention is to provide products and methods for regulating the growth of cells and tissues both in vitro and in vivo.

It is also an object of the present invention to improve the growth of cells and tissues in cell and tissue culture systems.

It is also an object of the present invention to improve growth of cells and tissues in cell and tissue growth systems and in human or animal body.

The present invention is based on the finding that lysyl hydroxylase enzyme having glycosyltransferase activity can affect cell growth. More specifically, it is the glycosyltransferase activity of lysyl hydroxylase 3 (LH3) enzyme, which has been shown to affect cell growth. Glycosyltransferase activity of lysyl hydroxylase 3 (LH3) enzyme or other lysylhydroxylase having glycosyltransferase activity can thus be used in particular to improve cell or tissue growth; it can also be used to maintain integrity.

Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. Galactosyltransferase (GT) and glucosyltransferase (GGT) activities are called also glycosyltransferase activities. Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme being able to modify lysyl residues in collagenous sequences, i.e. in collagens and proteins having collagenous domain in their structure. LH3 is producing hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues in the proteins mentioned above. LH3 is located in cells in endoplasmic reticulum, but in addition to that it is found also in extracellular space and in serum.

In some species lysyl hydroxylase activity is encoded by three genes. In these cases, isoform 3, LH3, has glycosyltransferase activity, but the other iso forms do not have this activity. In species, where lysyl hydroxylase activity is encoded by one gene, there is only one form of lysyl hydroxylase, LH, and it has also glycosyltransferase activity.

The present invention is based on studies where it has been shown that LH3 is bound to cell surface of cultured cells, and it is able to modify extracellular proteins in their native conformation. Furthermore, it has been indicated that extracellular LH3 glycosyltransferase activity (not lysyl hydroxylase activity) is able to increase cell proliferation, and on the other hand, it has been indicated that a lack of glycosyltransferase activities of LH3 in extracellular space is causing cell growth arrest and finally a cell death. The most dramatic effects were seen in transformed cells (cancer cells).

Since lysyl hydroxylase enzyme, LH, from species having one lysyl hydroxylase encoding gene, has glycosyltransferase activity, it can be used in the present invention in addition to LH3 isoform enzyme.

According one aspect of the present invention it is possible to prevent cell or tissue growth by inhibiting, preventing, decreasing or removing the glycosyltransferase activity of lysyl hydroxylase 3 (LH3) enzyme in cell or tissue systems.

According another aspect of the present invention it is possible to improve cell or tissue growth in cell cultures or other growth systems or in human or animal body by raising glycosyltransferase activity in the cell or tissue culture systems or other growth systems or in the human or animal body. In particular, it is possible to increase glycosyltransferase activity in the environment of cells or tissues, on the surface of cells or tissues or in the extracellular space surrounding cells or tissues.

More specifically, the method for regulating the growth of cells or tissues in vitro according to the present invention is mainly characterized by what is stated in claim 1.

The method for improving the growth of cells or tissues in vivo according to the present invention is mainly characterized by what is stated in claim 3.

Use of a product according to the present invention for improving cell or tissue growth in vitro, is mainly characterized by what is stated in claim 5.

A product for improving cell or tissue growth in vivo, is mainly characterized by what is stated in claim 6.

Use of an agent according to the present invention for preparing a product for improving cell or tissue growth in vivo is mainly characterized by what is stated in claim 7.

Considerable advantages are obtained by the present invention.

The possibility for regulating cell or tissue growth in cell or tissue cultures or in human or animal body is of great advantage. In particular in cases, where there are difficulties in culturing certain cells, for example nerve cells, it is of advantage, if the cell growth can be improved. In some cases the improvement of the growth of cells in the human or animal body is of advantage. Also agents used in this invention are not toxic or harmful to the treated patients.

Brief Description of the Drawings

Fig. 1 Overexpression of LH3 cDNAs in HT- 1080 cells. Cells were transfected with different human LH3 cDNAs in a pcDNA3 vector with a 6 ^χ His-tag at the N-terminal after the signal peptide. His-tag antibodies were used to detect the overexpressed LH3 and its mutated variants by Western blot analysis in HT- 1080 cell lysates after Nickel purification (Wang et al. 2002a). The molecular weight of the LH3 bands corresponds to about 85 kDa. Panel A: the transfected cells were kept under constant selection pressure with G418 at 750 μg/ml. Panel B: the LH3 protein produced from the single stably transfected clones.

Fig. 2 The effect of LH3 siRNAs on HT- 1080 cells. A. GT and GGT activities in untransfected (white), vector transfected (pRNA U6.1/Neo) (grey), and LH3 siRNA (black) expressing HT- 1080 cells after 10 days and 20 days of the treatment. The untreated cells were taken as 100 %. B. Light microscopic view at 10 days after trans fection of HT- 1080 cells transfected either with empty pRNA U6.1/Neo vector (control) or with LH3 siRNA-expressing constructs. The vector transfected cells grew normally with no obvious morphological change, whereas the LH3 siRNA transfected cells showed growth retardation, an abnormal rounded shape (a sign of cell detachment), and finally cell death.

Fig. 3 The binding of the LH3 N-terminal fragment and its glycosyltransferase-deficient counterpart on HT- 1080 cell surfaces. A. Western blot analysis after incubation with the hLH3Nl antibody: lanes 1-3 are cell pellets from untreated cells, DXD fragment treated cells, and wild type LH3 N-terminal fragment treated cells, respectively, demonstrating that both fragments (30 kDa) bound to cell membranes; lanes 4-6 are medium samples purified on a Nickel column from the untreated cells, DXD fragment treated cells, and LH3 N-terminal fragment treated cells, respectively, showing the same amount of the fragments (30 kDa) used in all experiments. Lanes 7-9 are cell lysates purified on a Nickel column from untreated cells, DXD fragment treated cells, and LH3 N-terminal fragment treated cells, indicating trace amount of the fragments (30 kDa) taken into the cells. B. Immunofluorescence staining of non-permeabilized cells by the affinity-purified PLOD3 antibody (panels A, D, G), Wheat germ agglutinin (cell surface marker, panels B, E, H), and Hoechst 33258 (nuclei marker). Panels C, F and I show the overlapping of the LH3 staining with the WGA staining on the cell surface (indicated by arrowheads). Untreated cells (panel C) are used as a background control, showing a small amount of endogenous LH3 on the cell surface. The cells treated with the DXD fragment (panel F) or the LH3N fragment (panel I) show much more overlap of the LH3 with the WGA, indicating that both fragments are bound to the cell surface.

Fig. 4 Visualization of the permeabilized cell bodies by type IV collagen staining after incubation of HT- 1080 cells with media containing the LH3 N-terminal fragment with (LH3N fragment) or without (DXD fragment) glycosyltransferase activities. The cells were incubated for 24 h, 48 h, and 72 h with the fragments before the staining. The morphological change of the cells treated with DXD fragment occurred within 24h and remained the same during the treatment whereas no change was observed in untreated cells or cells treated with the active LH3N fragment.

Fig. 5 The effect of the glycosyltransferase activity of LH3 on HT- 1080 cell proliferation. The cells were grown under the conditions described in experimental procedures. Student's t-test (one-tailed) was used for the statistical analysis, p<0.05 was taken as a significant change. The changes of cell numbers deviating significantly from control cells are indicated by stars. A. Extracellular effect in the presence of the wild type LH3 N-terminal fragment or its glycosyltransferase-deficient form (DXD fragment) in cell medium. Untreated cells were used as controls. Significant inhibition of cell proliferation was

observed with the DXD fragment treated cells. B. Intracellular effect by stably overexpressing full length LH-defϊcient LH3 (clone H 14-3). Remarkable acceleration of cell growth was seen, compared to the pcDNA3 vector (clone 9) transfected cells, when the glycosyltransferase activities of LH3 (not LH activity of LH3) increased in HT- 1080 cells (clone H14-3).

Fig. 6 Cytoskeleton protein staining of HT- 1080 cells after treatment of the LH3 N- terminal fragment with (LH3N fragment) or without (DXD fragment) glycosyltransferase activities. A. Filamentous actin was visualized by Alexa Fluor 568 phalloidin. The filopodia indicated by arrowheads were well-developed in the control and the LH3N fragment treated cells, whereas the filopodia protrusions at the cell periphery were severely disrupted in the DXD fragment treated cells. B. The overall architecture of the microtubule network was not considerably altered. However, brighter perinuclear tubulin staining around the centrosome, with much less staining extending towards the filopodia, was observed in the DXD treated cells than in the untreated controls and in the LH3N fragment treated cells (indicated by arrowheads). C. No obvious difference of vimentin staining was seen in the treated cells compared to the controls.

Fig. 7 The effect of the LH3 N-terminal fragment and the DXD fragment on 293 and CCD- 19LU cells. The cells were incubated with the fragments for 48 h. A. Permeabilized 293 cells were stained with type IV collagen antibody. Cell morphology changes and growth arrest were observed in the DXD fragment treated cells. B. Permeabilized CCD- 19LU cells were stained with procollagen I antibody. No obvious change was observed.

Fig. 8 Immunofluorescence staining of His-tag proteins in permeabilized HT- 1080 cells overexpressing full length LH3 in native or mutated forms. Human LH3 and mutant LH3s were overexpressed using a pcDNA3 expression vector. The cells were constantly under selection pressure with 750 μg/ml G418 for one month, then stained by His-tag antibody. A. pcDNA3 control. B. Full-length human LH3 transfected cells. C. Glycosyltransferase- deficient LH3 transfected cells. D. LH-deficient LH3 transfected cells. A change in cell morphology was observed when the glycosyltransferase-deficient LH3 was overexpressed in the cells (panel C). Some nonspecific staining was seen, as in panel A with His-tag antibodies.

Fig. 9 to 16 Amino acid and nucleotide sequences

Fig. 17A and 17B Normal lung fibroblasts (Fig. 17 A. CCD- 19LU cell), the expression of endogenous LH3 was significantly reduced after manipulation of the LH3 N fragment (Fig. 17A. bottom panel), whereas in fibrosarcoma cells (Fig. 17 B. HT- 1080 cell) no reduction was observed (Fig. 17 B. bottom panel) suggesting a defect regulation mechanism in controlling of the expression level of the endogenous LH3 in tumour cells compared to the normal ones.

The sequences of the following LH3 and LH are presented in the Figures. All these sequences are also publicly available in data banks.

HumanLH3: Accession no. NP OO 1075, amino acid sequence, signal peptide amino acids

1 - 24 (the mature protein starts from the 25 ^th amino acid), D-rich region 187-191;

Rat LH3: Accession no. CAD23628, amino acid sequence, signal peptide amino acids 1 - 27 (the mature protein starts from the 28 ^th amino acid), D-rich region 190-194;

Caenorhabditis elegans LH: Accession no. Q20679, amino acid sequence, the predicted signal peptide amino acids 1 - 16 (the mature protein starts from the 17 ^th amino acid), D- rich region 175-179;

Zebrafish Zebrafish (Danio rerio) LH3 (diwanka): Accession no. NP 001037808, amino acid sequence, the predicted signal peptide amino acids 1 - 20 (the mature protein starts from the 21 ^st amino acid), D-rich region 176-180;

Xenopus laevis (African clawed frog) procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3

(plod): Accession no. NP 001080446, the predicted signal peptide amino acids 1 - 20 (the mature protein starts from the 21 ^st amino acid); the proposed signal peptide amino acids 1 - 20 (the mature protein starts from the 21 ^st amino acid), D-rich region 181-185;

Takifugu rubripes (Fugu rubripes) LH3: Accession no. NP OO 1093075, amino acid sequence, the predicted signal peptide amino acids 1 - 21 (the mature protein starts from the 22 ^nd amino acid), D-rich region 179-183.

The nucleotide sequence of the full-length human LH3 (coding sequence) Accession no. NM OO 1084;

The nucleotide sequence of the glycosyltransferase-deficient human LH3 ( remains 0%

GGT/GT activity);

The nucleotide sequence of the human LH3 mutant (C ₁₄₄ I) ( remains<15% GGT/GT activity):

Detailed Description of Preferred Embodiments

Definitions

By "a nucleic acid sequence encoding lysyl hydroxylase enzyme having glycosyltransferase activity" is meant here a nucleic acid sequence encoding lysyl hydroxylase enzyme isoform 3 (LH3), or encoding lysyl hydroxylase (LH) (in species where only one isoform of LH is available). Correspondingly by "a lysyl hydroxylase enzyme having glycosyltransferase activity" is meant here lysyl hydroxylase enzyme isoform 3 (LH3), or lysyl hydroxylase (LH) (in species where only one isoform of LH is available).

LH3 or LH or the nucleic acid sequence encoding LH3 or LH may originate from any source, for example from eukaryote, from mammalian, from insect origin or even from nematodes and metazoa. The enzyme may originate for example from human, bovine, porcine, monkey, dog, horse, chicken, murine, mouse, nematode, zebrafϊsh, fly or platypus origin. Suitable sources are organisms having collagen or protein having collagenous domain or collagen-type protein, or it may be an organism not having the mentioned collagen proteins, but still producing lysyl hydroxylase, which has glycosyltransferase activity. The nucleotide sequence may be synthetic or at least partly synthetic. Within the scope of the invention are also nucleotide sequences encoding lysyl hydroxylases isolated from new organism groups provided that the nucleotide sequence encodes an enzyme having also glycosyltransferase activity. Nucleotide sequences, which are currently publicly available are for example the following:

Human LH3, Rat LH3, C. elegans LH, Zebrafϊsh (Danio rerio) LH3 (diwanka), Xenopus laevis (African clawed frog) procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (plod), Takifugu rubripes (Fugu rubripes) LH3.

The nucleotide and amino acid sequences and fragments and mutants thereof of human LH3, mouse LH3 and C. elegans are described for example in WO 01/92505 (the whole content of which is incorporated herein by reference).

In addition, nucleotide and amino acid sequences are available also from: chimpanzee, ape, canis familiaris (dog), equus caballus (horse), monodelphia domestica , gallus gallus, macaca mulatta (monkey), drosophila, ornithorhynchus anatinus (platypus).

The present invention comprises an LH3 or LH enzyme or parts thereof the full-length of said protein comprising an amino acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28, or an amino acid sequence selected from the group comprising a sequence having at least 80 % identity, preferably at least 85 % identity, more preferably at least 90 % identity, still more preferably at least 95 %, most preferably at least 98% identity to the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. Preferably, the identity is measured compared to the mature protein sequence without signal peptide.

In Table 1 has been presented the amino acid and nucleotide sequence accession numbers of the currently available amino acid and nucleotide sequences of LH3 and LH enzymes.

The present invention comprises proteins or fragments or portions of the proteins comprising the amino acid sequence (or part thereof) or being encoded by the nucleotide sequence (or part thereof) as listed in Table 1. The present invention comprises also proteins or fragments or portions of the proteins the full-length of said protein having at least 80 % identity, preferably at least 85 % identity, more preferably at least 90 % identity, still more preferably at least 95 %, most preferably at least 98% identity to the amino acid sequence selected from the group of sequences having the accession numbers NP 001075, XP 887254, XP 536856, XP 858413, Q9R0E1, Q5R6K5, CAD23628, NP_001037808, NP_001080446, NP_001093075, Q20679, NP_648451, NP_729687, XP 001653115, Q5UQC3 and EDP31117. Preferably, the identity is measured compared to the mature protein sequences without signal peptide.

In Table 2 has been presented the amino acid and nucleotide sequence accession numbers of amino acid sequences and nucleic acid sequences of proteins similar to LH3 or LH, but which are not called LH3 or LH.

The present invention comprises proteins or fragments or portions of the proteins comprising the amino acid sequence (or part thereof) or being encoded by the nucleotide sequence (or part thereof) as listed in Table 2. The present invention comprises also proteins or fragments or portions of the proteins the full-length of said protein having at least 80 % identity, preferably at least 85 % identity, more preferably at least 90 % identity, still more preferably at least 95 %, most preferably at least 98% identity to the amino acid sequence selected from the group of sequences having the accession numbers XP 001516384, XP 001601697, XP 001371229 and XP OOl 142249. Preferably, the identity is measured compared to the mature protein sequences without signal peptide.

In Table 3 has been presented the amino acid and nucleotide sequence accession numbers of amino acid sequences and nucleic acid sequences of proteins which are not called LH3 or LH, but which have DYnD motif or Cysteine in a corresponding position as Cysteine is human LH3 amino acid sequence. In human LH3 Cysteine is in position 144.

Hence, the present invention comprises also proteins or fragments or portions of the proteins comprising the amino acid sequence (or part thereof) or being encoded by the nucleotide sequence (or part thereof) as listed in Table 3. The present invention comprises also proteins or fragments or portions of the proteins the full-length of said protein having at least 80 % identity, preferably at least 85 % identity, more preferably at least 90 % identity, still more preferably at least 95 %, most preferably at least 98% identity to the amino acid sequence selected from the group of sequences having the accession numbers XP 001633491, XP 001504506, and CAF89795, XP 321614, XP 973819, XP 001678516, XP 001353930, XP OOl 181677. Preferably, the identity is measured compared to the mature protein sequences without signal peptide.

The enzyme may be full length LH3 or LH, or a fragment of LH3 or LH, in particular an N-terminal fragment of LH 3 or LH having glycosyltransferase activity. The enzyme or fragment of enzyme may be with or without signal peptide. For example antibodies are raised against the mature protein without signal peptide.

By the term "identity" is here meant the identity between two amino acid sequences compared to each other from the first amino acid encoded by the corresponding gene to the last amino acid. Preferably the identity is measured by comparing the amino acid sequences without sequences of the signal peptide. The identity of the full-length sequences is measured by using Needleman-Wunsch global alignment program at EMBOSS (European Molecular Biology Open Software Suite; Rice et al, 2000) program package, version 2.9.0, with the following parameters: EMBLOSUM62, Gap penalty 10.0, Extend penalty 0.5.

An "isolated" or "purified" polypeptide, protein or enzyme is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language "substantially free" means

preparation of the protein or enzyme having less than about 30%, less than about 20%, less than about 10% and more preferably less than about 5% (by dry weight), of contaminating proteins or chemicals or chemical precursors or culture medium, if the protein is recombinantly produced.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence or without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention are predicted to be particularly unamenable to alteration.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A predicted nonessential amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the coding sequence. The resultant mutants can be screened for biological activity to identify mutants that have the desired activity. Following mutagenesis the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

Biologically active fragments or portions of the protein or enzyme of the present invention include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein.

In particular by a fragment or portion of LH3 or LH enzyme is meant here a fragment or portion of said enzyme having biological or antigenic activity.

By biological activity is meant here in particular that the fragment or portion of enzyme (still) has glycosyltransferase activity. However, the fragment or portion may or may not have lysylhydroxylase activity.

The fragment of LH3 or LH is any fragment lacking at least one amino acid compared to the full-length polypeptide. Preferably the fragment is an amino -terminal fragment comprising an amino acid sequence of less than 600 amino acids, less than 500, 310 or less than 310 amino acids, 300 or less than 300 amino acids or 290 or less than 290 amino acids calculated from the first amino acid. In addition the fragment may lack the signal peptide.

An amino -terminal fragment from for example human LH3 with GGT/GT activities (Heikkinen et al., 2000, Wang et al., 2002a and 2002b) can be constructed to correspond nucleotides encoding amino acids 25-290 (GenBank™/EBI Bank with accession number AF046889; SEQ ID NO:9) .

The lysyl hydroxylase enzyme having glycosyltransferase activity may comprise amino acid sequence SEQ ID NO: 9 or a sequence having at least 80%, preferably at least 85%, more preferably at least 90 %, more preferably at least 95%, most preferably at least 98% identity to SEQ ID NO: 9. Corresponding N-terminal fragments can be prepared from other LH3 or LH sequences.

By "a nucleic acid sequence encoding lysyl hydroxylase enzyme being essentially deficient of glycosyltransferase activity " is meant here a nucleic acid sequence encoding lysyl hydroxylase enzyme isoform 3 (LH3), or encoding lysyl hydroxylase (LH) (in species where only one isoform of LH is available), but being genetically modified in such a manner that it does not encode any essential amount of glycosyltransferase activity. This means that the modified nucleic acid sequence encodes less than 20 %, less than 15 %, preferably less than 10 %, more preferably less than 5 %, still more preferably less than 2 % glycosyltransferase activity compared to wild-type (natural or non-modified) form of the enzyme. The nucleic acid sequence may or may not encode lysyl hydroxylase activity.

By "LH3 or LH enzyme being essentially deficient of glycosyltransferase activity" is meant here full length LH3 or LH, or a fragment of LH3 or LH, in particular an N-terminal fragment of LH3 or LH, which has been made essentially deficient of glycosyltransferase

activity. This means that the modified protein has less than 20 %, less than 15 %, preferably less than 10 %, more preferably less than 5 %, still more preferably less than 2 % glycosyltransferase activity compared to wild-type (natural or non-modified) form of the enzyme.

The enzymes, biologically active or antigenic fragments of the enzymes, antibodies against the enzymes or against the fragments of the enzymes, Fab fragments or inhibitors of the enzymes or fragment of enzymes, or nucleotide sequences encoding the enzymes or fragments thereof are useful, as reagents or targets in assays applicable to treatment and diagnosis of disorders, which can be affected by glycosyltransferase activity. The presence or absence or level of the glycosyltransferase activity can be used in assays for diagnosis.

If the fragment or portion or enzyme is essentially deficient of glycosyltransferase activity it means that the fragment or portion or enzyme is made essentially deficient of glycosyltransferase activity by one or other method (e.g. genetically or chemically modified, by using antibodies or inhibitors).

Assys for LH, GT and GGT activity are described in Kivirikko and Myllyla, 1982 (LH activity) and Myllyla et al. 1975 (GT and GGT activity). For a person skilled in the art it is easy to study, whether an enzyme or fragment or portion of enzyme has LH, GT or GGT activity.

The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include scFV and dcFV fragments, Fab and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as papain or pepsin, respectively.

Specifically, glycosyltransferase inactivated LH3 or LH or a fragment of these, in particular N-terminal fragment of LH3 or LH can be prepared by genetically modifying a nucleic acid sequence encoding LH3 or LH.

LH3 or LH enzyme typically comprises a short Aspartic Acid rich conserved motif DYnD responsible of glycosyltransferase activity. In the motif Y typically comprises 1, 2 or 3

amino acids, which vary in different species. For example in human LH3 the motif is DDDDD, whereas in insect Drosophila LH the motif is DTADD. When at least one Aspartic Acid in the D-rich motif is genetically changed not to be Aspartic Acid (D), the enzyme loses partly or completely its glycosyltransferase activity.

According to one embodiment of the invention the enzyme comprises a conserved motif DYnD motif, wherein D is Aspartic Acid, n is 1 , 2 or 3 and Yn means D or any amino acid, same or different amino acid compared to each other; or DYi Y ₂ Y ₃ D motif, wherein Yi is any amino acid, preferably D, T, K or N. Also I, M, A, E or S are possible; Y ₂ is any amino acid, preferably D or A. Also E is possible; Y ₃ is any amino acid, preferably D. Also S is possible.

Thus, for example motifs DYi D, DYi Y ₂ D and DYi Y ₂ Y ₃ D are possible, or DYi DDD Or DYi Y ₂ DD.

The motif is responsible of glycosyltransferase activity. If at least one of the Aspartic Acids in the motif is genetically changed not to be Aspartic Acid, the protein or enzyme loses partly or completely its glycosyltransferase activity.

In human LH3 the D-rich motif DDDDD is in the sequence SEQ ID NO:1 (human LH3) at position 187-191, whereas in LH3 or LH from other species D-rich region may be in a corresponding, although slightly different position. In the Figures the D-rich region is marked as darker area and the location can be seen in different sequences.

LH3 enzyme being essentially deficient of glycosyltransferase activity can be constructed for example from human LH3 coding sequence, which may cover the nucleotides 214- 2447 (GenBank™/EBI Bank with accession number AF046889, Valtavaara et al, 1998 (SEQ ID NO: 11). Histidines can be inserted at the N-terminus after the signal peptide to help the purification of the enzyme. The glycosyltransferase-defϊcient mutant can be constructed for example by mutating a short conserved DDDDD motif in the sequence at position 187-191 to ADAAA (Wang et al., 2002b; SEQ ID NO:7). This mutation leads to an enzyme without any GGT/GT activity. An alternative fragment with DDDDDi 87-191 ADADD mutations, is called here DXD fragment (SEQ ID NO:10). It has 98% of the

glycosyltransferase activities eliminated. Since DDDDD motif is critical for GGT/GT activity of human LH3, different mutations of the sequence i.e. ADAAA, ADADD, ADADA or AAAAA may lead to inactivation of glycosyltransferase activity as well.

According to one embodiment of the invention the enzyme comprises an amino acid sequence, where Cysteine is in position 144 in SEQ ID NO:1 or in a corresponding position in another LH3 or LH sequence, said Cysteine being responsible of glycosyltransferase activity.

According to another embodiment of the invention the enzyme comprises an amino acid sequence, where Cysteine in position 144 in SEQ ID NO:1 or in a corresponding position in another LH3 or LH sequence is genetically changed to Isoleucine or to some other amino acid. In the Figures the location of Cysteine and Isoleucine is marked as darker area and can be seen in different sequences.

By "DXD like fragment" is here meant glycosyltransferase inactivated fragment of LH3 or N-terminal fragment of LH3 having prepared by another method as the DXD fragment but having at least 80 %, preferably at least 85%, more preferably at least 90%, still more preferably at least 95 %, most preferably at least 98 % of the glycosyltransferase activities eliminated. For example amino acid Cysteine at position 144 can be mutated not to be

Cysteine. It can be mutated to for example Isoleucine as described in Wang et al., JBC 277, 18568, (2002a) (SEQ ID NO:8). Said mutated sequence has less than 15 % GGT/GT acitivity left. In a corresponding manner a "DYnD like sequence or fragment" can be prepared by changing DYnD in human LH3 or in a corresponding position in another LH3 or LH, not to be DYnD.

Useful variations of the above enzymes or fragments of enzymes are enzymes or fragments that are sufficiently or substantially identical to the amino acid sequences shown here. The enzyme may comprise an amino acid sequence selected from the group comprising SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 10 or may comprise an enzyme having at least 80%, preferably at least 85 %, more preferably at least 90 % identity, still more preferably at least 95 %, most preferably at least 98% identity to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10.

By " antibodies against LH3 or LH enzyme or against a fragment, such as against the N- terminal part of LH3 or LH enzyme or " "Fab fragments" of the antibodies can be prepared by conventional methods well known to a person skilled in the art.

It may be of advantage, if the enzyme used to regulate the cells or tissues of one species originates from the same species. However, LH3 or LH from different species can also be used to regulate cells or tissues both in vitro and in vivo. In particular, in cases, where antibodies are raised against lysyl hydroxylase enzyme from one species, the antibodies can be used to inhibit lysylhydroxylase enzyme or the glycosyltransferase activity of said enzyme from other species. For example, if antibodies are to be used to inhibit human LH3 or the glycosyltransferase activity of said enzyme, antibodies raised against a bovine or mouse LH3 enzyme can be used. This is because the antibodies cross-react with human LH3.

A method for preventing cell or tissue growth (described in more detail in our copending PCT Application titled "Products and Methods for Preventing Tumour Growth"), comprises that the glycosyltransferase activity in the cell or tissue, or surface of cell, or in the extracellular space surrounding a cell is inhibited, prevented or removed. This can be achieved by targeting to cell or tissue one of the following agents: - LH3 or LH enzyme or a fragment of these being essentially deficient of glycosyltransferase activity;

- a nucleic acid sequence encoding LH3 or LH enzyme or a fragment of these being essentially deficient of glycosyltransferase activity;

- antibodies against LH3 or LH enzyme or against a fragment of LH3 or LH being responsible of glycosyltransferase activity;

- Fab fragments of LH3 or LH enzyme antibodies,

- inhibitors of LH3 or LH glycosyltransferase activity; and

- inhibitors of transcription or translation of LH3 or LH glycosyltransferase activity.

As will appear from the copending application, by "inhibitor" is meant inhibitors capable of preventing sugar transfer to hydroxylysine residues. The inhibitor may be a cofactor/co- substrate analogue or derivative, such as a modification of UDP-glucose, or the inhibitor

may be UDP, analogue of UDP, such as chemically modified UDP, derivative of UDP or a heavy metal displacing manganese needed for the reaction.

Transcription of LH3 or LH glycosyltransferase activity can be regulated and inhibited by using short oligonucleotides, which bind to the promoter region or translation by using short oligonucleotides, which bind to mRNA. Inhibition by using binding of oligonucletotides to mRNA, called inhibition by RNAi, is exemplified here in the examples.

The present invention can be applied in particular for preventing different types of tumours and tumour cells. In particular, the present invention can be applied to tumours, the cells of which produce collagenous proteins, since these cells have collagenous protein sequences on the cell surface, which functions as an attachment site for lysylhydroxylase enzymes. Before the decision to treat cancer by preventing a certain type of tumour, the tumour cells can be studied for their response to treatment by decreasing the glycosyltransferase activity in the tumour or in the extracellular space of tumour. If decreased or removed glycosyltransferase activity prevents the cell growth, the cells become apoptotic, which can be seen in the cell morphology.

The modified enzyme can be targeted according to the disclosure to cells or to tumour, the growth of which is to be prevented. A suitable amount for preventing cell or tumour growth is 0.1 to 50 μg/ml, preferably 0.1 to 20 μg/ml, typically the amount is 0.1-10 μg/ml, preferably 1 - 6 μg/ml, more preferably 2 - 4 μg/ml, most preferably 3 μg/ml into the medium or into tumour volume, if fragments of the enzyme are used. If full-length enzyme is used the amounts used should be 0.5 to 10 times, preferably 1 to 5 times, advantageously 2 to 2.5 times the amount of fragments used. In tissues the amounts of enzymes or fragments may be 5, 10, 20, 30, 50, 100 or 150 times the above mentioned amounts, since the transfer of the enzyme or fragment is slower.

Antibodies and Fab fragments can be used in the same molar amounts as the enzymes or their fragments above.

To improve the growth of cells or tissues in a cell or tissue culture can be achieved by incubating the cells with LH or LH3 enzyme or fragment of these enzymes, in particular an N-terminal fragment of LH or LH3 having GT/GGT activity. Fragments of the enzyme are preferably used 0.1 to 50 μg/ml, preferably 0.1 to 20 μg/ml, typically the amount is 0.1 - 10 μg/ml, preferably 1 - 6 μg/ml, more preferably 2 - 4 μg/ml, most preferably 3 μg/ml into the medium or into tumour volume, if fragments of the enzyme are used. If full-length enzyme is used the amounts used should be 0.5 to 10 times, preferably 1 to 5 times, advantageously 2 to 2.5 times the amount of fragments used. In tissues the amounts of enzymes or fragments may be 5, 10, 20, 30, 50, 100 or 150 times the above mentioned amounts, since the transfer of the enzyme or fragment is slower.

Another method to improve cell growth would be to introduce to the cells or tissues a nucleic acid sequence encoding LH or LH3 or a fragment thereof having GT/GGT activity.

By the term "transform" is here meant any method by which a nucleic acid sequence is introduced into a cell or tissue, such as transformation, transfection, electroporation etc.

The enzyme addition to cell culture is repeated preferably after 8 hours, preferably 12 hours, more preferably after 16 hours, most preferably after 24 hours incubation. In case of a culture transformed by a nucleic acid sequence encoding LH, LH3 or a fragment thereof the improvement in growth can be seen after a culturing period, typically one day culturing period.

The enzymes or fragments of enzymes of the invention can be used in various type of growth systems. For example, the enzymes or fragments of enzymes can be attached to a solid or semisolid or liquid material, which can be used to culture cells, such as a microtiter plates (solid material) or a gel system for culturing cells, such as Matrigel™ Basement Membrane Matrix.

The enzymes or fragments of the enzymes of the invention can be used in various type of growth or culture systems as a growth factor.

The invention includes also vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term "vector" refers to a

nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

The recombinant expression vectors of the invention can be designed for expression of the proteins of the invention in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed for example in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.

When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

The present invention can be used also for improving the growth of cells or tissues in vivo, which comprises that glycosyltransferase activity in the environment of cells or tissues, on or in surface of cells or tissues or in the extracellular space surrounding cells or tissues is increased. This comprises that the cells or tissues are transformed to produce LH or LH3 enzyme or a fragment of LH or LH3 enzyme having glycosyltransferase activity or the amount of LH or LH3 enzyme or a fragment of LH or LH3 enzyme having glycosyltransferase activity or glycosyltransferase activity is raised in cell or tissue culture system or in the environment of cells or tissues, on surface of cells or tissues or in the extracellular space surrounding cells or tissues.

By the term "transform" is here meant any method by which a nucleic acid sequence is introduced into a cell, tissue or tumour, such as transformation, transduction, transfection, electroporation etc.

The use of enzymes or fragments of enzymes of the invention may be of particular advantage in tissue engineering both in vitro and in vivo. For example, neuronal cell growth, growth of skin cells or bone cells may be improved in vitro and in vivo. Also the

enzymes or fragments of enzymes of the invention may be used for example in amending neuronal damages, amending bones or wound healing.

The nucleic acid and polypeptides, fragments thereof, as well as antibodies of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, antibody, Fab fragment, inhibitor and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

One likely method of administering the therapy are formed by Gene therapy and Cellular therapy. Both methods cover a multitude of possibilities including RNAi and similar techniques, cellular therapy for example combining the possibility to include other factors. To a person skilled in the art of these therapies, the methods of gene therapy and cellular therapy are well-known.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous

preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of

the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.05 to 22 mg/kg or 0.1 to 20 mg/kg body weight, and even more preferably about 0,2 to 10 mg/kg, 0, 5 to 9 mg/kg, 1 to 8 mg/kg, 2 to 7 mg/kg, or 3 to 6 mg/kg body weight. The protein or polypeptide can be

administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The person skilled in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Lower dosages and less frequent administration may thus be possible.

As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, relieve, alter, alleviate, ameliorate, remedy, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, antibodies, peptides, ribozymes and antisense oligonucleotides.

In particular, the treatment is directed towards degenerative disorders as examples of states, where oncrease the cell number may be a benefit. Neurogenenerative disorders, such as Parkinson disease, Alzheimer disease and other dementing disorders, muscle degenerations, retina degenerations, spinal cord traumas.

Thus, according to one embodiment, the present invention comprises a method to treat degenerative disorders including retinal degeneration, skeletal muscular atrophy, spmal muscular atrophy (SMA), spmobulbar muscular atrophy (SBMA), amyolateral sclerosis (ALS), muscular dystrophy, Parkinson's disease, Alzheimer's Disease, cardiac injury, cardiac disease and liver disease, and it can be used for the reduction of cell apoptosis during transplantation of cells.

The cells treated can be selected from neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, precursor cells, muscle precursor cells, bone precursor cells, cartilage precursor cells, myoblast, cardiomyoblast, neural precursor cells glial precursor cells, neuronal precursor cells, hepatoblasts, neurons, oligodendrocytes, astrocytes, Schwann cells, skeletal muscle cells, cardiomyocytes, and pancreatic cells and hepatocytes.

Cancerous disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, for example malignant tumour growth, or may be categorized as non-pathologic, in other words a deviation from normal but not associated with a disease state, for example cell proliferation associated with wound repair and heal. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of stage of invasiveness or histopathologic type. The term "cancer" includes malignancies of the various organ systems, such as those affecting breast, lymphoid, thyroid, lung, genitourinary tract and gastrointestinal, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non- small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, fibrocarcinomas and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of sarcomatous and carcinomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumour cells form recognizable glandular structures. The term "sarcoma" is known in the art and refers to malignant tumours of mesenchymal derivation.

Collagens constitute a highly specialized family of extracellular matrix proteins, which are universally distributed in the animal body. In addition to the maintenance of the architecture of tissues, they have regulatory functions important for cell behaviour

(Reichenberger and Olsen, 1996, Vogel et al, 1997, Shrivastava et al, 1997). To date, more than 40 vertebrate collagen genes have been described, the products of which combine to form at least 29 distinct homo- and/or heterotrimeric molecules (Kadler et al., 2007, Sόderhall et al., 2007). The biosynthesis of collagen is a multi-step process characterized by a large number of co- and post-translational modifications, many of which are unique to collagens and collagen-like proteins. These modifications, which typically occur within the rough endoplasmic reticulum of the cell, include hydroxylation of lysyl residues catalyzed by lysyl hydroxylase (EC 1.14.11.4, LH). Hydroxylysyl residues participate in the formation of collagen cross-links, which stabilize the extracellular matrix. They also serve as attachment sites for sugar units. The carbohydrates linked to hydroxylysyl residues are either a monosaccharide galactose or a disaccharide glucosylgalactose (Kivirikko et al., 1992, Kielty et al., 1993, Kadler, 1994, Prockop and Kivirikko, 1995, Ayad et al. 1998, Kivirikko and Myllyla, 1979). The formation of hydroxylysyl-linked carbohydrate units is catalyzed by two specific enzyme activities, hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) and galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT). The extent of hydroxylation and glycosylation varies considerably among different collagen types and within the same collagen type from various sources or even in the same tissue under different physiological and pathological conditions (Kivirikko and Pihlajaniemi, 1998). The function of the hydroxylysyl-linked carbohydrate units remains poorly understood.

Three LH isoforms, LHl, LH2a/2b and LH3, originating from three different genes, have been characterized so far from human, mouse, rat, and zebrafish (Valtavaara et al., 1997, Valtavaara et al., 1998, Passoja et al., 1998, Ruotsalainen et al., 1999, Yeowell and Walker, 1999, Mercer et al., 2003, Schneider and Granato 2006 and 2007, Myllyla et al. 2007). We have demonstrated that LH3 and C. elegans LH, the only ortholog for lysyl hydroxylase in the nematode, are multifunctional enzymes possessing LH, GT, and GGT activities in vitro (Myllyla et al., 2007, Heikkinen, et al. 2000., Wang et al., 2002a, Wang et al., 2002b). The amino acids important for the catalytic activity of glycosyltransferases associated with LH3 are localized at the amino -terminal part of the molecule, being separate from the active site of LH (Wang et al., 2002a, Wang et al., 2002b). Our previous results demonstrate that LH3 resides not only in the ER, but also in the extracellular space (SaIo et al., 2006a and 2006b) such as in serum, and on cell surfaces associated with collagenous proteins. Furthermore, transgenic mouse studies show that a knock-out of the

LH3 gene causes lethality at an early embryonic stage (Ruotsalainen et al., 2006, Rautavuoma et al., 2004) and that the dramatic reduction of GGT activity, not LH activity, of LH3 disrupts the formation of basement membranes during mouse embryogenesis (Ruotsalainen et al., 2006). The absence of the multifunctional LH in C. elegans results in retention of type IV collagen within the cells and in failure to complete embryogenesis (Norman and Moerman, 2000). Schneider and Granato showed recently (Schneider and Granato, 2006) that the glycosyltransferase domain of myotomal diwanka (LH3 in zebrafish) plays a critical role in growth cone migration. It acts through myotomal type XVIII collagen, a ligand for neural-receptor protein tyrosine phosphatases that guide motor axons.

The present invention is based on studies, where we explored the role of LH3, in particular its glycosyltransferase activities in the extracellular space of cultured HT- 1080 cells. The data from the cells provide direct evidence that LH3 is able to hydroxylate lysyl residues and further glycosylate hydroxylysyl residues in these cells. HT- 1080 cells synthesize mainly type IV collagen (Alitalo et al., 1980, Wang et al., 2000), in which hydroxylysyl residues are highly glycosylated, about 75% in αl(IV) (Kivirikko and Myllyla, 1979). The increase of hydroxylysine and glycosylated hydroxylysines caused by overexpression of LH3 is quite small, which is probably explained by the fact that type IV collagen in HT- 1080 cells has already been highly hydroxylated and glycosylated, nearly reaching the saturating limit of modifications in collagens. Importantly, knock down of the LH3 gene by the LH3 siRNA resulted in a great reduction of GGT and GT activities in HT- 1080 cells compared to the control. Thus the results suggest that LH3 functions in vivo, not only as lysyl hydroxylase but also as both glycosyltransferases (GGT and GT). The finding is in agreement with our recent data (Sipila et al., 2007) showing that LH3 knock-out cells produce type I, IV and VI collagens that lack all the hydroxylysine linked disaccharides, revealing that other cellular galactosyltransferases and glucosyltransferases are not able to compensate the function of LH3 in ER.

The present disclosure of the extracellular role of LH3, especially the glycosyltransferase portion of the molecule, revealed it as important to cell growth and survival. The overexpression of a glycosyltransferase-deficient mutant, but not the full length LH3 or the LH-deficient mutant, and knock down of the LH3 gene by siRNA resulted in abnormal cell morphology, growth retardation, and cell death, clearly indicating that LH3, but not its

lysyl hydroxylase activity, is necessary for HT- 1080 cell survival. Furthermore, our data indicate that morphology changes and cell lethality occur very quickly after adding glycosyltransferase-deficient LH3 N-terminal fragment to the extracellular space. Similar results were observed in transformed embryonic kidney cells. It is remarkable to notice that the cell morphology of the LH3 siRNA expressing cells differed from those treated extracellularly with the glycosyltransferase-deficient LH3 fragment, suggesting different mechanisms may be applied by the cells under different treatment conditions. It is also possible that in the LH3 siRNA transfection, the GT/GGT activities were reduced too much at a very short time that there is no chance for the cells to show the stretched shape (a sign of cells under certain stress) as seen in the DXD treated cells or those overexpressing the glycosyltransferase-deficient mutant intracellularly.

The cells recovered if the glycosyltransferase-deficient fragment was removed from the medium, revealing that the phenomenon is reversible. The addition of the glycosyltransferase-deficient fragment to the extracellular space of some other cell lines has only a mild effect, if any, on cell behavior, suggesting cell membrane structure, probably receptor composition, may affect the phenomenon. Interestingly, the most remarkable changes were observed in transformed cells, this may make LH3 a potential target to cancer. The morphology change occurred in one day in HT- 1080 cells, if the glycosyltransferase-deficient LH3 fragment was added to the extracellular space, whereas additional days were required, if the full-length mutant was overexpressed intracellularly. The delay in the overexpressed situation can be explained by the fact, that some time is needed to synthesize and secrete the mutated LH3 into the cell medium.

The presence of carbohydrates linked to hydroxylysyl residues is a unique feature of collagens and the proteins with collagenous sequences. However the function of the sugar moieties and the factors which determine the level of glycosylation remain elusive. It was reported that the urinary excretion levels of hydroxylysine glycosides, especially galactosylhydroxylysine, in osteoporotic patients were indirectly related to fractures in bone in osteoporosis (Yoshihara et al, 1994). Studies on fibrillar collagens have indicated that the hydroxylysyl- linked sugar units in collagens may play a role in the control of organization of the fibrils (Brinckmann et al., 1999, Notbohm et al., 1999). Our recent analysis (Sipila et al. 2007) from LH3 knock- out cells revealed that hydroxylysyl glycosylation is required for secretion of type IV collagen thus explaining the lack of type

IV collagen in knock-out embryos. In addition, our data indicated that unglycosylated type VI collagen cannot form tetramers inside the cell. Hydroxylysyl glycosylation therefore affects type VI collagen oligomerization and secretion. In addition, some specific hydroxylysine glycosylations are needed for the correct distribution of type VI collagen and probably for the formation or interactions of microfibrils (Sipila et al. , 2007). It has also been reported that melanoma cell CD44 interaction with the αl(IV) 1263-1277 region from basement membrane collagen is regulated by ligand glycosylation. A marked reduction in cell adhesion and spreading was observed due to the presence of the single galactose residue in the αl(IV) sequence, suggesting significant biological consequences of even subtle changes in collagen carbohydrate content (Lauer-Fields et al. 2003). The metastatic process involves a coordinated series of tumour cell behaviors, including adhesion to and migration on extracellular matrix components and invasion of the basement membranes. Such interactions may be mediated by a great variety of cell surface biomolecules, including integrins. The CCsβi integrin from two tumor cell types (melanoma and ovarian carcinoma) has been shown to bind directly to a glycosylated region within type IV collagen (Lauer-Fields et al., 2003, Miles et al., 1995).

Currently nine members in the family of collagenous transmembrane proteins have been characterized (Franzke et al., 2005). They probably all act as membrane-bound receptors. Therefore the hydroxylysine- linked carbohydrates in their collagenous sequences may have a regulatory role in their function as well as in their association with ligands. However, no data has been reported so far concerning the glycosylation of hydroxylysine residues in these molecules. Our recent data indicate that LH3 is localized to the ER as well as to the extracellular space including the cell surface. Cell surface localization was confirmed by the LH3 N-terminal fragments used in this study. Furthermore, we have indicated that LH3 is able to modify lysyl residues of extracellular proteins in their native, nondenatured state, implying that LH3 may have a widespread regulatory function in tissues (SaIo et al., 2006a). The multifunctionality and intra- as well as extracellular localization of LH3 in vivo may enable the molecule to play roles on cell surfaces or in the extracellular space not only in modulating the post-translational modifications of lysyl residues in a dynamic way but also in regulating cell behavior via cell-matrix interactions or receptor activation. It is also possible that the LH3 glycosyltransferase moiety is functioning as a coreceptor, controlling receptor clustering and higher order oligomerization, similar to the kinase domain for epidermal growth factor receptors

(Clayton et al, 2007). It is known that receptor chains have to be arranged in the correct orientation with respect to each other for signaling to occur. The possible role of LH3 with receptor activation or receptor clustering followed by a signaling pathway is further supported by our results obtained from cytoskeletal stainings. Our data indicate a lack of fϊlopodies in HT- 1080 cells, and the actin and tubulin remodeling after binding of the DXD fragment to the cell membrane, when compared with untreated HT- 1080 cells or the cells treated with LH3 N-terminal fragment. It should be noticed, that HT- 1080 cells are producing LH3 and secreting it into the extracellular space, a big part of this endogenous LH3 is bound to cell surface as shown earlier (SaIo et al., 2006a). By adding LH3 N- terminal fragment in the cell medium we just overstimulate the in vivo condition, whereas by adding the DXD fragment we change the in vivo condition by dramatically reducing LH3 glycosyltransferase activities on the cell membrane. The binding of the added fragments is probably associated with endocytosis, because a band corresponding to the size of added fragments were observed in cell lysate when immunostained by antibody against purified LH3 N-fragment. Intriguingly, both fragments behaved similarly in the staining. Precise regulation of the cytoskeleton is required for diverse cellular processes such as changes in cell shape, proliferation, adhesion, migration and polarity, and changes in the organization, distribution and dynamics of cytoskeletal proteins mediate these processes. It is known that several extracellular factors trigger signaling cascades that modulate the cytoskeleton. In addition, it is known that endocytosis regulates many cellular signaling processes by controlling the number of functional receptors available at the cell surface. Conversely, some signaling processes regulate the endocytic pathway.

Partners for the LH3 glycosyltransferase moiety on the cell surface are not known, but our data clearly indicate that binding to the cell surface occur after the addition of the LH3 N- terminal fragment or its glycosyltransferase-deficient counterpart to the cell medium. The addition of these fragments to the cell medium affects cell behavior, leading to arrest of cell growth and further to cell lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of cell proliferation if the fragment contains LH3 glycosyltransferase activities. These findings indicate that cell growth is dependent on the LH3 glycosyltransferase activities in the extracellular space. More studies are required to analyze the exact mechanisms or possible signaling events behind the phenomena. The results are highly significant because they demonstrate for the first time that LH3 dependent glycosylation in the extracellular space influences cell behavior. These results

also propose LH3 as a potential target for medical applications, such as cancer therapy. This therapeutic targeting could be achieved similarly as shown here, by generating DXD fragment of LH3, or by generating antibodies against LH3 or developing inhibitory compounds and targeting them to tumor tissues to prevent LH3 glycosyltransferase activities.

Lysyl hydroxylase (LH) isoform 3, called here LH3, is according to this disclosure found intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT- 1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multifunctionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-defϊcient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The arrest effect is reversible, the cells recovering after removal of the glycosyltransferase- deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.

In this study we have used HT- 1080 cells to obtain more information about LH3, especially its extracellular role in cells. We demonstrate that treatment of cells with a mutated 30 kDa LH3 N-terminal fragment (DXD fragment) lacking glycosyltransferase activity in medium dramatically affects cell behaviour, slowing the growth rate, and changing cell morphology, whereas treatment with the glycosyltransferase active fragment stimulates cell growth. The phenomenon is rapid, cell-type specific and reversible, demonstrating for the first time that LH3 -dependent glycosyltransferase activities in the

extracellular space are important for cell growth and proliferation. The requirement of LH3 for cell viability was also demonstrated by the targeted disruption of LH3 by siRNA in cells, and the importance of the glycosyltransferase activity of LH3 for cell viability was confirmed by overexpression of the full-length LH3 in native and mutated forms in HT- 1080 cells.

Examples

The following cell lines were used: human fibrosarcoma cells (HT- 1080, ATCC CCL- 121), African green monkey kidney cells (Cos7, ATCC CRL- 1651), human embryonic kidney cells (293, ATCC CRL-1573), human lung fibroblasts (CCD-19LU, ECACC 90112708), locally established human skin fibroblasts (MR and NAF), and human osteosarcoma cells (MG-63, ATCC CRL- 1427). The cells were grown in MEM or DMEM (GIBCO) plus 10% fetal calf serum (Promocell) at 37 ⁰ C in a humidified atmosphere of 95% air and 5% CO ₂ .

Overexpression of full-length LH3 cDNA and its mutants in HT- 1080 cells

The human LH3 coding sequence covering the nucleotides 214-2447 [GenBank™/EBI Bank with accession number AF046889, Valtavaara et al., 1998] was subcloned into

BamHI and Xhol sites of a pcDNA3 vector (Invitrogen). Six histidines were inserted at the N-terminus after the signal peptide. The glycosyltransferase-deficient mutant was constructed by mutating a short conserved DDDDD motif in the sequence at position 187- 191 to ADAAA (Wang et al., 2002b). The LH-deficient mutant was made by mutating the aspartate at position 669 to alanine (Heikkinen et al., 2002). Nucleofector (Amaxa) was used for delivery of LH3 and the mutated cDNAs directly into the cell nuclei. 2x10 ⁵ - 2x10 ⁶ HT- 1080 cells were plated at least one day before the transfection. To establish the stably transfected clones, the cells were kept under constant selection pressure with G418 at 750 μg/ml and those originated from the single clones were finally picked up and transferred to new culture dishes.

Hydroxylation and glycosylation analysis

HT- 1080 cells stably transfected with the pcDNA3 vector (clone 9), full-length LH3 cDNA (clone HO- 12), and the LH-defϊcient mutant (clone H 14-3) were grown under standard conditions for 48 h. The collagenous proteins in the cells and media were collected and digested by highly purified collagenase (Sigma) as described elsewhere (SaIo et al, 2006a). After being hydro lysed in 2M NaOH, the amount of galactosylhydroxylysine (GH) and glucosylgalactosylhydroxylysine (GGH) as fluorescent dansyl derivatives were measured by slightly modifying the HPLC method presented by Moro et al. (1984). GH and GGH standards were kindly obtained from Professor R. Tenni, Italy. The amount of hydroxy Iy sine in collagenous proteins was measured in the culture medium of cells using an amino acid analyzer, after hydro lysing the samples at 110 °C overnight in 6 M HCl.

Gene silencing by RNA interference

A Silencer™ siRNA cocktail kit (RNase III) was purchased from Ambion. Two 500 bp- long target sequences, LH3 siRNAs-1 and LH3 siRNAs-2, corresponding to 378-878 nt and 1111-1611 nt (54-55% identity with the corresponding nucleotide sequence of LHl and 52-63% identity with that of LH2) located at the 5' and medial regions of human LH3 cDNA (Valtavaara et al., 1998), were selected as templates. Long dsRNAs were generated by in vitro transcription. The dsRNAs were then cleaved by RNase III into a mixture or cocktail of siRNA molecules capable of inducing the RNAi effect very specifically in mammalian cells. In order to observe the long term effect of LH3 siRNAs on cell behavior, nucleotides corresponding to 380-398 nt, 630-648 nt, and 1125-1243 nt (37-73% identity with the corresponding nucleotides of the other LH iso forms) of the human LH3 sequence (Valtavaara et al. 1998 and Myllyla et al., 2007) were synthesized and cloned into pRNA U6.1/Neo (Genscript) and pSilencer 2.1-U6 neo (Ambion), respectively. Empty pRNA U6.1/Neo vector and pSilencer 2.1-U6 neo negative control were used as controls. RNAi expression vectors were introduced into HT- 1080 and Cos7 cells by Nucleofector transfection (Amaxa). G418 was used for selection (750 μg/ml for the first 3 days and 375 μg/ml for the rest of the selection process).

LH3 N-terminal fragment with or without glycosyltransferase activities

A 30 kDa His-tagged amino -terminal fragment of human LH3 with GGT/GT activities (Heikkinen et al., 2000, Wang et al., 2002a and 2002b) corresponding to nucleotides encoding amino acids 25-290, was cloned into a pET-15b vector (Novagen). The fragment, produced in an E. coli system, was purified with Ni-NTA agarose (Qiagen) (Wang et al., 2002a). In order to avoid any cytotoxic effect from the elution buffer (30 mM MES, 300 mM imidazole, 400 mM NaCl, 10% glycerol, pH 6.5), the eluate was equilibrated in culture medium by using Amicon Ultra centrifugal filter devices (10,000 MWCO, Millipore). The same fragment with DDDDD187-191 ADADD mutations, designated the DXD fragment having 98% of the glycosyltransferase activities eliminated (Wang et al., 2002b), was constructed and purified in the same way. The purity of both fragments after equilibration in culture medium was checked by 10% SDS-PAGE, the results indicating one 30 kDa band in the gel. The GGT activity assay revealed that one μg of the LH3 N- terminal fragment has GGT activity of about 80,000 dpm, which corresponds to GGT activity measured in one milliliter of mouse serum (SaIo et al., 2006a). Fragment analysis by CD spectroscopy (Jasco J-715 spectropolarimeter equipped with a microprocessor for spectral accumulation and data manipulation) indicated that the mutations of the LH3 N- terminal fragment did not cause secondary structural changes.

Immunofluorescence and light microscopy

HT- 1080 cells transfected with the LH3 siRNA expression vector, pRNA U6.1/Neo or pSilencer 2.1-U6 neo, were observed by light microscopy (Nikon). For the cell morphology study, HT- 1080 cells, either overexpressing full-length human LH3 and mutants or treated with the LH3 fragments in medium, were grown on glass coverslips, fixed with 4% paraformaldehyde at room temperature for 15 min and stained as described elsewhere (Heikkinen et al., 2000) with a monoclonal antibody against either the polyhistidine tag (Sigma) or collagen IV (DAKO) at a dilution of 1 : 100. Alexa Fluor 488 or 594 (1 : 100, Molecular Probes) was used as the secondary antibody, and the staining was detected by fluorescence microscopy (Nikon). Cells transfected with the empty pcDNA3 vector or untreated cells were used as controls. Other cell lines treated with the LH3 fragment in medium were stained with antibodies against procollagen type I (Kellokumpu et al., 1997) or collagen type IV (DAKO). The recovery experiment was carried out by

seeding IxIO ⁵ HT- 1080 cells on coverslips in 35 mm plates and treating the cells for 24h with media or media containing the DXD fragment. All media were then changed either to 10% FCS DMEM or to 10% FCS DMEM containing the LH3 N-terminal fragment. The cells were fixed as described above after 24 h, 48 h, 72 h, respectively, and stained with a type IV collagen antibody. For cytoskeletal protein staining, 1 x 10 ⁵ of HT- 1080 cells were grown on coverslips and treated with the LH3 fragments for 48 h without changing the medium. The cells were fixed and stained as above with monoclonal antibody against vimentin, tubulin, and actin (Sigma), at a 1 :100 dilution. Actin cytoskeleton was stained with Alexa Fluor 568 phalloidin (1 :20, Molecular Probes) that binds specifically to F-actin. Confocal microscopy images were acquired with an Olympus Fluoview 1000 confocal microscope (Olympus, Japan).

Flow cytometric analysis

The proliferation assay was carried out with HT- 1080 cells incubated with the LH3 N- terminal fragment or the DXD fragment (about 3μg/ml), and with HT- 1080 cells stably overexpressing the LH-deficient mutant of LH3 (clone H 14-3) using the cells transfected with empty pcDNA3 vector (clone 9) as a control. The cells (about Ix 10 ⁵ ) were grown in 6x 35 mm dishes and maintained in 10% FCS DMEM. The medium was changed every 24 h. The cells were trypsinized and counted daily using the absolute counting function of a CyFlow Space flow cytometer (Partec, Germany) for up to 4 days.

In cell cycle analysis HT- 1080 cells were seeded at a density of l ^χ 10 ⁵ cells/35 mm plate in triplicate in 10% FCS DMEM containing either the LH3 N-terminal fragment or the DXD fragment (3μg/ml of the purified fragment). The treatment was maintained for 48 h with one medium change at 24 h. Untreated cells were used as the control. The cells were harvested and resuspended in 100 μl of 3.4 mM sodium citrate buffer, pH 7.6, containing 0.1% Nonidet P40 and 1.5 mM spermine tetrahydrochloride. The DNA was stained by propidium iodide and analyzed with a CyFlow Space flow cytometer (Partec, Germany). The fraction of every cell cycle phase was calculated.

Cell surface binding analysis

IxIO ⁶ HT- 1080 cells /10 cm plates were grown for 18 h to about 50% confluency. The cells were treated with the LH3 N-terminal fragment or the DXD fragment (3 μg/ml of the purified fragment) added in culture medium for 3 h. The cells were washed twice by PBS and homogenized in buffer containing 20 mM Tris-HCl, pH 8, 0.4 M NaCl, 1 mM DTT. The cell lysates were collected after centrifugation at 14,000 rpm for 30 min at +4 °C. Any his-tagged proteins in the lysates and the added fragments in media were concentrated by Nickel purification as described elsewhere (Heikkinen et al., 2000). The cell pellet fractions were resuspended in the same buffer and separated by 15% SDS-PAGE for Western blotting analysis.

Non-permeabilized cell staining was carried out by using the affinity purified polyclonal antibody PLOD3 (ProteinTech Group, Inc.) against the human LH3 at a working solution of lOμg/ml. IxIO ⁵ HT- 1080 cells were inoculated into 35 mm plates with coverslips and cultivated with the LH3 N-terminal fragments as described above for 7 h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS, pH 7.4, for 3 min at room temperature, blocked in 1% bovine serum albumin in PBS, pH 7.4 for 1 h at room temperature. Goat anti-rabbit Alexa Fluor 488 (Molecular Probes) was used as the secondary antibody at a 1 :100 dilution. Wheat germ agglutinin (WGA), Texas Red-X conjugate (lOμg/ml, Molecular Probes) was used as a cell surface marker. Hoechst 33258 (1 μg/ml, Sigma- Aldrich) was used for nuclei staining. The cell surface staining was checked by confocal microscopy (Olympus Fluoview 1000).

Other methods

LH, GT and GGT activity assays were done as described elsewhere (Kivirikko and Myllyla, 1982, Myllyla et al., 1975). Western blot analysis was carried out using monoclonal antibodies against the His tag (Sigma) or polyclonal antibodies, hLH3Nl, against the purified human LH3 N-terminal fragment (Davids Biotechnologie, Germany). The proteins were fractionated under reducing conditions by 10% or 15% SDS-PAGE, blotted onto an Immobilon-P membrane (Millipore), and incubated with the primary antibody. Anti-mouse (Zymed) or anti-rabbit (P .A.R.I. S) IgG peroxidase conjugate was

used as the secondary antibody. Bound antibodies were visualized using the ECL detection system (Amersham Pharmacia Biotech) and x-ray film (Eastman Kodak Co). The QuickChange site-directed mutagenesis kit (Stratagene) was used to make mutations in the cDNA sequences. The nucleotide changes of the mutations were confirmed by sequencing.

Results

LH3 functions as LH, GT and GGT in HT- 1080 cells

In order to verify that LH3 modifies lysyl residues in collagens in cellulo, we have generated wild-type, LH-deficient, and glycosyltransferase-deficient human LH3 cDNA constructs in a mammalian pcDNA3 expression vector, and successfully transfected them separately into HT- 1080 cells. These cells produce mainly type IV collagen (Alitalo et al, 1980, Wang et al., 2000)that is highly hydroxylated and glycosylated (Kivirikko et al., 1992, Ayad et al., 1998), and thus a good read-out molecule for the study. Western blot analysis showed clearly the overexpression of human LH3 and its mutated variants in the transfected HT- 1080 cells under constant selection pressure (Fig. IA) or with stably transfected clones (Fig. IB). Stably transfected clones were easily established with all constructs except the glycosyltransferase-deficient mutant (Fig. IB). The transfected cell lines were used to analyze cellular LH, GT and GGT activities as well as the amount of lysyl modifications in collagenous proteins.

LH activity was measured by the assay not separating LH3 from the other two LH iso forms, therefore the activity given represents the total LH activity of the cells. Our results indicate that LH activity was increased about 1.30 fold in cells stably transfected with the wild-type LH3 (clone HO-12), and about 1.34 fold in cells transfected with the glycosyltransferase-deficient LH3 under selection pressure. In cells stably transfected with a LH-deficient construct (clone H 14-3), the LH activity was reduced to about 54% compared to that of a pcDNA3 transfected clone (clone 9). Analysis of the amounts of the hydroxy lysyl residues in collagenous proteins from cell culture medium by amino acid analysis revealed that the overexpression of wild-type LH3 resulted in about a 1.5 fold increase in the hydroxylation of lysyl residues compared to that in the vector control. No increase was found in the hydroxylysine content of the medium of cells transfected with the LH-deficient clone (not shown). The results indicate that the increase of hydroxylysine

in collagenous proteins is due to the overexpression of the LH activity of LH3. Overexpression of LH3 also slightly increased the glycosylation of hydroxy Iy syl residues in collagenous sequences in cells (Table 4). Approximately a 10% increase in the content of GGH and GH was observed, when the cells were stably expressing wild-type LH3 (clone HO- 12), the increase was also found with LH-defϊcient LH3 (clone H 14-3) revealing that LH3 -dependent glycosyltransferases are able to increase the glycosylation of hydroxy Iy syl residues in cells. GGT activity increased about 6 fold and GT activity 2 fold in the HT- 1080 cells stably overexpressing wild-type human LH3 or the LH-defϊcient LH3 when compared with control cells transfected with the empty vector (clone 9) (Table 4).

Table 4 GT/GGT activities and galactosylation (GH) and glucosylation (GGH) of hydroxylysyl residues in HT- 1080 cells stably overexpressing LH3 and the LH-deficient mutant of LH3 ^a

Construct GT% GGT% GH%" GGH% ^C pcDNA3 100 100 70.6±2.8 42.0±9.0

(clone 9)

Non-mutated LH3 201±56 647±218 75.9±6.1 52.7±30.1

(clone HO- 12)

LH-deficient mutant 218±126 603±293 79.0±4.9 46.6±16.1

(clone H 14-3) aMean value from three experiments ±SD, assayed as described in experimental procedures. bGalactosylhydrosylysine as percentage of hydroxylated lysyl residues. cGlucosylgalactosylhydroxylysine as percentage of galactosylated hydroxylysine residues.

RNA interference analysis was carried out in order to confirm that LH3 is responsible for GT and GGT activities of cells. This is a very specific method to target the knock down to a certain gene. As shown elsewhere (Brummelkamp et al, 2002) one nucleotide change in the targeting sequence is enough to fail the suppression of the gene, therefore it is probable that homologous genes like the other LH isoforms remain unaffected in the treatment.

Knocking down of the LH3 gene by siRNA cocktails, a vector- free system consisting of a mixture of specifically targeted small interference RNAs, resulted in efficient reduction of

GGT and GT activities after delivery of siRNAs into the cells. A difference was observed between the knocking down efficacy of the two siRNA cocktails (LH3 siRNAs- 1 and LH3 siRNAs-2), suggesting that the more effective targeting sequence of LH3 is located in the medial region of the molecule (not shown). The effect of RNA interference was transient, GGT/GT activities returned to normal levels after 24 h to 48 h. The effect could be prolonged when the targeted sequences were used sequentially in double transfections. Only 20% of GGT and 17% of GT activities were obtained, compared to that of the negative control, when the cells were transfected with LH3 siRNAs- 1 and LH3 siRNAs-2 in a consecutive manner with a 24 h interval and followed by a further 48 h cultivation. The negative control siRNA, which had no significant homology to any known gene sequences from mouse, rat or human, was composed of a 19 bp scrambled sequence with 3'dT overhangs. In order to prolong the effect of RNAi we used also a vector-based siRNA and co-transfected three LH3 siRNA expression constructs in pRNAU6.1/Neo into HT- 1080 cells. This enabled us to target three different sequences at the 5' and medial regions of LH3 transcript simultaneously thus making the knock-down more specific. After 10 days (Fig. 2) GT and GGT activities were reduced to the level of about 15-18% of the untreated cells, the cells appeared round and died very quickly in two days. It should be noted that transfection of the vector alone (Fig. 2) as negative control also caused reduction of GGT and GT activities in the cells compared to untreated cells; however, the reduction was less than that in LH3 siRNAs transfected cells and both activities were recovered after culturing the cells for a longer time, whereas LH3 siRNA expression remained the activities at a very low level leading finally to the cell death. Taken together, our data clearly show that disruption of LH3 reduces both glycosyltransferase activities in HT- 1080 cells, indicating that LH3 is responsible for GGT and GT activities in these cells.

The addition of a glycosyltransferase-deficient LH3 fragment to the culture medium changes the cell morphology and slows cell growth

As demonstrated recently (SaIo et al, 2006a and 2006b), LH3 is secreted from cells and is located on the cell surface and in the extracellular space in some tissues. This extracellular localization differentiates LH3 from the other LH isoforms, although the functions of LH3 in the extracellular space is not known. In order to study the extracellular functions we exposed cell surfaces to medium containing an excess amount of the glycosyltransferase- deficient LH3 N-terminal fragment, and then determined the consequences of the treatment

to the cell. The same amount of a LH3 N-terminal fragment with GGT activity was used as a control. We treated HT- 1080 cells with a purified 30 kDa LH3 N-terminal fragment deficient in the glycosyltransferase activities (the DXD fragment) by adding the fragment (0-4 μg/ml) into the medium. Our preliminary experiments revealed that the effect was concentration dependent, a 3 - 4 μg/ml concentration of the DXD fragment arresting cell growth already after 24 h incubation (see later in Fig. 5A), 3 μg/ml reducing the cell number by 12% and 4 μg/ml by 34%, when compared to the LH3 N-terminal fragment. A fragment concentration of 3 μg/ml was used in further studies.

We first determined whether the LH3 N-terminal fragment and/or its DXD mutated form bound to the cell surface when added to the cell medium. HT- 1080 cells were incubated with the fragments (3 μg/ml) for 3 hours at 37 ⁰ C. The fragments remaining in the medium served as a molecular weight control. The cell lysate and the cell pellet were collected after sonication and centrifugation. The His-tagged proteins in lysate and medium were concentrated by Ni-NTA column. All fractions were resuspended in SDS sample loading buffer, and analyzed by Western blotting with the antibody hLH3Nl against the purified human LH3 N-terminal fragment (Fig. 3A). The results indicated a major 30 kDa band in the cell pellet fraction, indicating that the fragments are bound to the surface of HT- 1080 cells (Fig. 3 A, lane 1-3). The same sized bands in medium confirmed that similar amounts of both fragments were used in the experiment (Fig. 3A, lane 4-6). A faint band was also observed in the cell lysate after Nickel purification, indicating that a trace amount of the fragment was internalized into the cell (Fig. 3A, lane 7-9) and suggesting that endocytosis is associated with the phenomenon. Both fragments, mutated and wild-type, behaved similarly. Furthermore, immunofluorescence stainings of non-permeabilized HT- 1080 cells treated with the DXD fragment or with the LH3 N-terminal fragment revealed an overlay of the LH3 staining with cell surface marker confirming that the fragments are bound to the cell surface (Fig. 3B, panel F, I).

As seen in figures 4 and 5 A, the DXD fragment in HT- 1080 cell medium affected the cells very rapidly, inducing cell stretching (Fig. 4) as well as a significant inhibition of cell proliferation. The number of cells in DXD treated plates was significantly lower, when compared with untreated cells (Fig. 5A). As seen in figure 5 A, treatment of HT- 1080 cells with the wild-type N-terminal fragment of LH3 (3 μg/ml of the purified fragment) showed a tendency to stimulate cell proliferation. In order to confirm that the glycosyltransferase

activities, not LH activity, of LH3 are responsible for stimulating cell proliferation, we overexpressed the full length LH-deficient LH3 (clone H 14-3) in HT- 1080 cells, and compared the cell growth with the vector transfected cells (clone 9). The data given in Fig. 5B, indicate that overexpression of the glycosyltransferase activities in the cells cause an increase in cell proliferation. Our additional data (not shown) demonstrate that overexpressing LH-defϊcient LH3 in cells produces a marked increase of LH-deficient LH3 protein and GGT activity in the cell medium. Taken together, the data suggest that the glycosyltransferase activities of LH3 promote cell proliferation, and the presence of LH3 glycosyltransferase activities in the extracellular space is required for cell growth and viability.

To test whether the reduction of cell proliferation caused by the DXD fragment of LH3 was due to arrest of cell division at a specific stage in the cell cycle we analysed the DNA content by flow cytometry. There was, however, no obvious difference (not shown) in DNA content between the untreated cells and those incubated with the LH3 N-terminal fragment or the DXD fragment in medium.

Table 5 shows the effect on cell growth by a treatment as described herein.

Table 5. Effect on cell growth after the treatment of the LH3N fragment or the DXD fragment

3: HT1080 cells were grown (lxl0 ⁶ /10 cm dish) in 10% FCS DMEM with the LH3N fragment or the DXD fragment (DDDDD 187- 19 IADADD mutation) (about 3ug/ml) for 48 h. f : promotion of cell growth; J,: inhibition of cell growth HT- 1080, birosarcoma cells; SK-N-SK, neuroblastoma cells; MG-63, osteosarcoma cells; 293, embryonic kidney cells

Cellular cytoskeleton after the LH3 treatments

Actin filaments and microtubules are involved in the cell morphology change after the addition of the glycosyltransferase-deficient LH3 fragment in the culture medium

Actin and vimentin are the most widely distributed proteins to form cytoskeletal networks in cells. The networks are frequently associated with microtubules. The integrins are the cell surface receptors that are the main mediators in cell-extracellular matrix adhesions. The cytoplasmic domains of integrins interact with actin filaments. Therefore remodeling of the actin cytoskeleton represents a key element of the response to extracellular stimuli. Microtubules also play a crucial role in many cellular functions, including maintenance of cell shape, cell signaling and cell division. Different microtubule-associated proteins (MAPs) influence the assembly and stability of microtubules and the association of microtubules with other cell structures. MAPs are targets of many extracellular signals, participating in many signal transduction pathways and their binding to microtubules being regulated by phosphorylation.

In an attempt to establish whether cell morphology change is due to the disorganization of the cytoskeletal components in cells, we examined the cytoskeleton, a dynamic structure that maintains cell shape, enables cellular motion, and plays important roles in both intracellular transport and cellular division. The organization of actin, tubulin and vimentin, the major types of protein filaments mechanically supporting cellular membranes, was studied in HT- 1080 cells by using immunofluorescence staining after the treatment of the cells by addition of the DXD fragment or the LH3 N-terminal fragment to the cell medium. Our results showed that the vimentin cytoskeleton remained unchanged in the treated cells compared to the controls (Fig. 6C). However, actin networks underwent a clear reorganization following the DXD treatment (Fig. 6A). The fϊlopodia, the rod-like cell surface projections filled with bundles of parallel actin filaments, were severely disrupted in the DXD fragment treated cells with almost no cell-cell contact seen at the cell periphery whereas the protrusions were well-developed in the untreated controls and the LH3N fragment treated cells (Fig. 6A). As shown in Fig. 6B, the overall architecture of the microtubule network was not considerably altered. However, brighter perinuclear tubulin staining around the centrosome, with much less staining extending towards the filopodia, was observed in the DXD treated cells than in the untreated controls and in the LH3N

fragment treated cells, thus confirming the lack of cell-cell contacts observed in the F-actin staining. Thus, the data clearly revealed cytoskeletal changes in the HT- 1080 cells after the DXD fragment had been added to the cell culture medium, suggesting that LH3 associated glycosyltransferase activity is an important extracellular factor in maintaining cell shape, in controlling cell migration and in regulating cell-extracellular matrix adhesions.

The morphological change caused by the DXD fragment in the extracellular space is a reversible process and dependent on cell type

To examine whether the change in cell morphology caused by treatment with the DXD fragment of LH3 can be reversed, we incubated the DXD treated HT- 1080 cells for 24-72 h either with normal culture medium or with medium containing the LH3 N-terminal fragment (3 μg/ml of the purified fragment). Interestingly, after the DXD fragment was withdrawn from the medium, the morphology of the cells gradually returned to normal and the cells started to grow and divide. The morphology of most of the cells was normal 24 h after removal of the DXD fragment of LH3, and no abnormal, stretched cells were observed at 48 h (not shown). The addition of the LH3 N-terminal fragment with glycosyltransferase activities to the medium did not accelerate the process, compared to cell medium without any additions. We also carried out a competition experiment by adding LH3 N-terminal fragment to the medium simultaneously with the DXD fragment. When the fragments were added in equal amounts (3 μg/ml) into the medium, the LH3 N- terminal fragment did not prevent the morphology change caused by the DXD fragment. More studies are required to study whether the fragments are bound to different partners, have different binding affinities or whether the DXD fragment has a higher impact on cell behaviour compared to the LH3 N-terminal fragment.

In order to see, if the DXD fragment of LH3 has a similar effect on various cultured cells, we treated other cell lines with the DXD fragment in culture medium (3 μg/ml of the purified fragment). As seen in Figure 7A for 293 cells, the DXD treatment clearly prevented cell proliferation and changed the cell morphology within 48 h, whereas only a very mild effect, if any, was detected for adult lung fibroblasts (Fig. 7B), adult skin fibroblasts, and the osteosarcoma cells (not shown). The results indicate that certain cells are more sensitive to the treatment than others, revealing some cell-type specificity.

Disturbance of the glycosyltransferase activities of LH3 inside the cell results in cell death

In order to confirm that a lack of glycosyltransferase activity of LH3 is responsible for cell growth arrest, we also investigated the effects of disturbing GGT activities inside the cells. We were not able to establish stably transfected cell lines overexpressing glycosyltransferase-deficient LH3, but we have data from transfected cells under constant selection pressure. The cells transfected with glycosyltransferase-deficient LH3 grew as a pool of cells, and, as shown earlier (Fig. IA), they overexpressed glycosyltransferase- deficient LH3 under constant selection pressure. Immunofluorescence staining using an anti-polyhistidine antibody showed that the morphology of these cells was altered after about one month of transfection. They were much more stretched and required cell-cell attachment, which are features of stressed cells (Fig. 8, panel C). Cells transfected with the empty vector or other constructs (wild type LH3 or LH-deficient mutant) grew and divided normally (Fig. 8, panels A, B, D). As described above RNAi was also used to disturb the glycosyltransferase activity of LH3 intracellularly. After being co-transfected with the three LH3 siRNA expression constructs in pRNAU6.1/Neo the cells showed growth retardation, an abnormal rounded shape as described above (Fig. 2B), and finally cell death, whereas the cells transfected with the empty pRNAU6.1/Neo vector grew normally with no obvious morphological change. The same experiments were carried out in Cos7 cells, revealing similar findings (not shown).

Taken together, the results indicate the importance of the glycosyltransferase activities of LH3 for cell growth and viability. The overexpression of glycosyltransferase-deficient LH3 intracellularly and the application of the mutated LH3 N-terminal fragment (the DXD fragment) extracellularly were causing similar consequences like growth retardation, morphological change, and cell death. A delay of appearance of growth retardation was evident however when glycosyltransferase-deficient LH3 was inside the cells.

In order to see, if the LH3 glycosyltransferase activity in extracellular space could regulate the level of endogenous LH3, we have manipulated HT-1080 cells (fibrosarcoma) and CCD- 19LU cells (normal lung fibroblasts) by adding the purified active LH3 N-terminal fragment (LH3 N fragment) containing the glycosyltransferase activities of LH3 (GT and GGT activities) into the culture medium for 48 hours. The endogenous LH3 was then

stained with affinity purified polyclonal PLOD3 antibody (Proteintech Group, Inc) and checked by electron microscopy. In normal lung fibroblasts (Fig. 17 A. CCD- 19LU cell), the expression of endogenous LH3 was significantly reduced after manipulation of the LH3 N fragment (Fig. 17A. bottom panel), whereas in fibrosarcoma cells (Fig. 17 B. HT- 1080 cell) no reduction was observed (Fig. 17 B. bottom panel) suggesting a defect regulation mechanism in controlling of the expression level of the endogenous LH3 in tumour cells compared to the normal ones.

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