The present invention characterises PNGase L and describes its use in removing N-glycans from glycosylated proteins. Corresponding methods are also provided.

Background

N-glycosylation is a common form of post translational protein modification. It results in the formation of a glycoprotein, in which an N-glycan is attached to an asparagine residue of the protein. Glycans have a wide variety of functions, including protecting the protein from proteases and binding interactions. Given that glycans can modulate protein function, removal of N-glycans from proteins may be desirable in certain circumstances.

All N-glycans have a core structure (Figure 1a), but some details of their decoration vary according to the organism that synthesised them. For instance, glycosylated proteins from mammalian, insect and plant cells vary in their N-glycan profile. Mammalian-derived glycosylated proteins comprise a-1 ,6 core fucosylated N-glycans; insect proteins comprise a mixture of a-1 ,6 and a-1 ,3 core fucosylated N-glycans; and plant proteins comprise a-1 ,3 core fucosylated N-glycans only. Mammalian proteins also contain glycans with sialic acid caps which can vary, and hydrolyse, under acid conditions.

The Peptide-N4-(N-acetyl-p-glucosaminyl)asparagine amidase (PNGase) enzyme family hydrolyses the p-aspartylglucosaminyl linkage between an N-glycan and the asparagine residue to remove the glycan from the protein. These enzymes are produced naturally by many organisms for different functions, for example, to acquire N-glycans for nutrients or to recycle or remodel glycoproteins.

A number of PNGase enzymes have been previously characterised (Figure 1c), the majority of which only remove a subset of N-glycans. For example, PNGase F from Elizabethkingia menigoseptica is capable of removing a-1 ,6 core fucosylated N-glycans only (e.g. from glycosylated proteins expressed in mammalian systems). It is the most common PNGase that is used by the scientific community. By contrast, PNGase F type II (also from Elizabethkingia menigoseptica) and PNGase A are specific to removal of a -1 ,3 core fucosylated N-glycans (such as those found on glycosylated proteins expressed by plant cells). PNGase A was originally characterised from almonds, but homologues of this enzyme are now also sold commercially. Glycoproteins must be pre-treated with trypsin before PNGase A can effectively be used. Of the characterised PNGases, only PNGase H+ from Terriglobus roseus is capable of removing both a-1 ,6 core fucosylated N-glycans and a-1 ,3 core fucosylated N-glycans. However, the low pH conditions required by PNGase H+ may adversely affect the glycoprotein and/or N-glycan products of the reaction, which restricts its use. Acid hydrolysis of sialic acid caps, as well as protein denaturation are particular problems associated with PNGases that have a low optimum pH.

There is a need for an improved means for removing N-glycans from glycoproteins.

Brief summary of the disclosure

The PNGase family encompasses several known and well characterised enzymes. Family members have relatively low sequence homology and there is no identifiable correlation between sequence and N-glycan specificity. The diverse nature of the family means that identifying new family members and predicting their activity is not straight forward. Over two thousand predicted PNGases exist within sequence databases that have not been tested thus far.

The inventors have identified, tested and characterised the enzymatic specificity and activity profile of a predicted PNGase, PNGase L (SEQ ID NO:2). SEQ ID NO:2 has previously been identified as a putative PNGase sequence, originating from a wood bacteria, Flavobacterium akiainviviens. However, no testing or characterisation of the encoded protein has previously been performed.

The invention is based on the surprising finding that PNGase L is capable of efficiently removing N-glycans from glycoproteins expressed by plant, mammalian or insect cells. It therefore has broad specificity, and can surprisingly remove both a-1 ,3 and a-1 ,6 core fucosylated N-glycans from glycosylated proteins. Advantageously, PNGase L has also been found to effectively remove both a-1 ,3 and a-1 ,6 core fucosylated N-glycans at neutral pH. The inventors have also found that, surprisingly, PNGase L does not discriminate against types of N-glycan, so can remove both complex and high-mannose N-glycans efficiently. Based on these activities PNGaseL should also target hybrid N-glycans as well. To the best of the inventors’ knowledge, PNGase L is the only PNGase to date with such broad specificity that also functions at neutral pH. It therefore provides an improved means for removing N- glycans from glycoproteins without adversely affecting the N-glycan and/or glycoprotein products. PNGase L is provided herein as a novel means for efficiently removing N-glycans from glycoproteins expressed by plant, mammalian or insect cells. SEQ ID NO:2 represents the full length PNGase L protein encoded by the open reading frame (ORF), including the signal peptide. The corresponding mature protein (without the signal peptide) is shown as SEQ ID NO:1.

In one aspect, the use of a PNGase enzyme is provided for removing a-1 ,6 core fucosylated N-glycans from a glycosylated protein, wherein the PNGase:

(i) comprises an amino acid sequence as shown in SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto; and

(ii) is capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8.

Suitably, the PNGase enzyme may comprise an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence which has at least 70% sequence identity thereto.

Suitably, the N-glycans that are removed from the glycoprotein may be oligomannose N- glycans, complex N-glycans, hybrid N-glycans, or a combination thereof.

Suitably, the PNGase enzyme may be used for removing a-1 , 3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from the glycosylated protein.

Suitably, the glycoprotein may have been produced by a vertebrate cell. Suitably, the vertebrate cell may be a mammalian cell.

Suitably, the glycoprotein may have been produced by an invertebrate cell. Suitably, the invertebrate cell may be an insect cell.

In another aspect, the use of a PNGase enzyme is provided for removing a-1 , 3 core fucosylated N-glycans from a glycosylated protein, wherein the PNGase:

(i) comprises an amino acid sequence as shown in SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto; and

(ii) is capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8.

Suitably, the PNGase enzyme may comprise an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence which has at least 70% sequence identity thereto. Suitably, the N-glycans that are removed from the glycoprotein may be oligomannose N- glycans, complex N-glycans, hybrid N-glycans or a combination thereof.

Suitably, the glycoprotein may have been produced by a plant, slime mold, insect or parasite.

Suitably, the glycoprotein may be a secreted glycoprotein, ora membrane bound glycoprotein.

Suitably, the PNGase may be used over a pH range of about pH6 to about pH8.

Suitably, the PNGase may be used in the production of proteins, in the production of glycans, in the characterisation of glycosylation proteins, in glycome analysis, in the preparation of a milk product, or in the preparation of a gelling agent.

Suitably, the production of proteins may comprise the production of antibodies, antibody fragments, or derivatives thereof.

In another aspect, a method is provided for removing a-1 ,6 core fucosylated N-glycans from a glycosylated protein, the method comprising: a) contacting a nucleic acid that encodes a PNGase enzyme comprising the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto, with a cell under conditions in which the nucleic acid is incorporated into the cell and the PNGase is expressed; or b) culturing a genetically modified cell comprising a nucleic acid that encodes a PNGase enzyme comprising the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto, under conditions in which the PNGase is expressed; or c) providing a PNGase that comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which has at least 70% sequence identity thereto; and contacting the PNGase with the glycosylated protein under conditions in which the PNGase is capable of removing a-1 ,6 core fucosylated N-glycans from the glycosylated protein.

Suitably, the glycoprotein may have been produced by a vertebrate cell. Suitably, the vertebrate cell may be a mammalian cell.

Suitably, the glycoprotein may have been produced by an invertebrate cell and the PNGase removes a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from the glycosylated protein. Suitably, the invertebrate cell may be an insect cell. In another aspect, a method is provided for removing a-1 ,3 core fucosylated N-glycans from a glycosylated protein, the method comprising: a) contacting a nucleic acid that encodes a PNGase enzyme comprising the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto, with a cell under conditions in which the nucleic acid is incorporated into the cell and the PNGase is expressed; or b) culturing a genetically modified cell comprising a nucleic acid that encodes a PNGase enzyme comprising the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto, under conditions in which the PNGase is expressed; or c) providing a PNGase that comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which has at least 70% sequence identity thereto; and contacting the PNGase with the glycosylated protein under conditions in which the PNGase is capable of removing a-1 ,3 core fucosylated N-glycans from the glycosylated protein.

Suitably, the glycoprotein may have been produced by a plant, slime mold, insect or parasite.

Suitably, the PNGase enzyme may comprise an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence which has at least 70% sequence identity thereto.

Suitably, the N-glycans that are removed from the glycoprotein may be oligomannose N- glycans, complex N-glycans, hybrid N-glycans, or a combination thereof.

Suitably, the step of contacting the PNGase with the glycosylated protein may occur at a pH range of about pH6 to about pH8.

Suitably, the method may further comprise isolating the deglycosylated protein or cleaved glycan products.

Suitably, the method may further comprise characterizing the deglycosylated protein and/or the glycan cleavage products.

Suitably, the glycoprotein may be a secreted glycoprotein or a membrane bound glycoprotein, optionally wherein the glycoprotein is an antibody, antibody fragment, or derivative thereof. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

Brief description of the Figures

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1a and b show types of core N-glycan structures and currently characterised PNGase specificities. The core of an N-glycan structure is composed of a chitobiose core with a pi tlinkage between the GIcNAcs, this is capped with a pi ,4-linked mannose and bonded to this are two a-linked mannose “arms” (a1 , 3 and cd ,6). This pentasaccharide is dubbed Man3, which is variably decorated depending on the source. B) Man3 can be built on in different ways. Firstly, additional a-linked mannose sugars can be added to produce high-mannose N- glycans, where the pattern of linkages is conserved throughout organisms. Alternatively, complex N-glycans can be produced which broadly consist of GIcNAcs pi ,2-linked to the mannose, then p-linked galactose on to the GIcNAcs. The variation in complex N-glycan structures produced by different organisms is considerable and there are particular features associated with different eukaryotes. For instance, the linkage between the Gal and GIcNAc is variable between plants and mammals, with either a pi ,3 or pi ,4, respectively. Consequently, the fucose that can also decorate that GIcNAc is an a1 ,4 or 3 for plants and mammals, respectively. In mammals, the galactose is commonly decorated with sialic acids, which can be different types and be attached through variable linkages. The chain of sugars extending from the mannose arms are referred to as “antenna” and analysis of complex N- glycans in mammals from different glycoproteins reveals there can be up to four antennae, with a third pi ,4-linked and fourth pi ,6-linked antenna attached to the a1 ,3 and a1 ,6 mannose arms, respectively. Further decorations or alterations to this description are possible, including sulfation, GalNAc rather than GIcNAc, and polyLacNAc extensions. In mammals, an a1 ,6- fucose is common on the first GIcNAc, whereas in plants only a1 ,3-fucose are found and in insects both are common decorations. Plants also commonly have a xylose pi ,2-linked to the central mannose. N-glycans can also come in hybrid structures, where one arm is high- mannose and the other in complex.

Figure 1c provides a summary of the specificities of different PNGase enzymes.

Figure 2 shows a summary of the results. Three PNGase enzymes were tested against a panel of nine substrates under three different conditions. Some samples for PNGase L have some missing products, a: low release of glycans with branch sialic acids, b: low bisecting glycans released, c: low neutral glycans released, d: less release in general. Each reaction was repeated in triplicate.

Figure 3 shows activity for the PNGase enzymes against IgG.

Figure 4 shows activity for the PNGase enzymes against horseradish peroxidase.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

Various aspects of the invention are described in further detail below.

Detailed Description

The invention is based on the characterisation of a newly identified PNGase with unusually broad N-glycan specificity. The new PNGase can advantageously be used to remove N- glycans at neutral pH. It can effectively remove N-glycans from native glycoproteins as well as from denatured forms. This broad N-glycan specificity is surprising for a putative PNGase from a Flavobacterium spp. Accordingly, use of a PNGase enzyme for removing N-glycans from a glycosylated protein is provided herein. In one embodiment, the PNGase may be used for removing a-1 ,6 core fucosylated N-glycans from a glycosylated protein. In an additional or alterative embodiment, the PNGase may be used for removing a-1 ,3 core fucosylated N-glycans from a glycosylated protein.

As used herein, “PNGase” refers to an enzyme from the peptide-N4-(N-acetyl-p- glucosaminyl)asparagine amidase family (generally classified as EC 3.5.1.52 in accordance with the Enzyme Nomenclature Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology). PNGases are enzymes that remove N-glycans from glycosylated proteins. More specficially, PNGases catalyze a chemical reaction that cleaves a N4-(acetyl-beta-D-glucosaminyl)asparagine residue in which the glucosamine residue may be further glycosylated, to yield a (substituted) N-acetyl-beta-D- glucosaminylamine and a peptide containing an aspartate residue. The most commonly used commercial PNGases are PNGase A (almond) and PNGase F (E. coli expressed). Further details on the function and characterisation of PNGases can be found in Du et al., 2015; and Sun et al., 2015, for example.

The PNGase provided herein originates from a wood bacteria, Flavobacterium akiainviviens. Further details of the bacterium may be found in Kuo et al., 2013. The PNGase provided herein is also referred to herein as “PNGase L”. PNGase L comprises an amino acid sequence as shown in SEQ ID NO:1 , which represents the mature enzyme, without a signal peptide. The full length amino acid sequence for PNGase L (including signal peptide) is shown in SEQ ID NO:2.

In one example, the PNGase described herein is a wild-type PNGase enzyme. The terms "natural" and "wild type" as used herein mean a naturally-occurring enzyme. That is to say an enzyme expressed from the endogenous genetic code and isolated from its endogenous host organism and/or a heterologously produced enzyme which has not been mutated (i.e. does not contain amino acid deletions, additions or substitutions) when compared with the mature protein sequence (after co- and post-translational cleavage events) endogenously produced. Natural and wild-type proteins may be encoded by codon optimised polynucleotides for heterologous expression, and may also comprise a non-endogenous signal peptide selected for expression in that host. The PNGase described herein may be obtainable (may be obtained) from a bacterium, preferably from Flavobacterium spp. Preferably, the PNGase may be obtainable (preferably obtained) from Flavobacterium akiainviviens.

As would clear to a person of skill in the art, variants of the sequences shown in SEQ ID NO:1 or SEQ ID NO:2 may also be functional. Such functional variants are also provided herein.

The term "variant" means a protein with one or more amino acid alterations (i.e. amino acid deletions, additions or substitutions) when compared with the natural or wild-type sequence within the mature protein sequence (SEQ ID NO:1 or SEQ ID NO:2). Functional variants are preferably at least as biologically active as the sequences recited herein (SEQ ID NO:1 or SEQ ID NO:2, as appropriate). The term "biologically active" refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.

A PNGase enzyme that has utility within the context provided herein therefore may comprise an amino acid sequence as shown in SEQ ID NO:1 , or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:1. The term “variant” also encompasses homologues.

Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:1 , or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO:1 may therefore be a conservative amino acid sequence variant of SEQ ID NO:1 , wherein the variant has PNGase L activity (e.g. is capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about PH8).

Non-functional variants are amino acid sequence variants of SEQ ID NO: 1 that do not have PNGase L activity (i.e. cannot remove a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:1 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and non-functional allelic variants) are well known to a person of ordinary skill in the art.

PNGase enzymes that may be used as described herein may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:1 or portions or fragments thereof. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:1), or portions or fragments thereof. As stated previously, such sequence variation (compared to the amino acid sequence shown in SEQ ID NO:1) is permitted provided that the variant has PNGase L activity (e.g. is capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about PH8).

The PNGase provided herein may therefore:(i) comprise an amino acid sequence as shown in SEQ ID NO:1 , or an amino acid sequence which has at least 60% sequence identity thereto; and (ii) be capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8.

For example, the PNGase provided herein may therefore:(i) comprise an amino acid sequence as shown in SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto; and (ii) be capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8. This encompasses PNGases with the required function and at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ I D NO: 1 .

A PNGase enzyme that has utility within the context provided herein therefore may comprise an amino acid sequence as shown in SEQ ID NO:2, or functional variants (or functional fragments) thereof. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:2. The term “variant” also encompasses homologues.

Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO:2 may therefore be a conservative amino acid sequence variant of SEQ ID NO:2, wherein the variant has PNGase L activity (e.g. is capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about PH8).

Non-functional variants are amino acid sequence variants of SEQ ID NO: 2 that do not have PNGase L activity (i.e. cannot remove a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and non-functional allelic variants) are well known to a person of ordinary skill in the art.

PNGase enzymes that may be used as described herein may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2 or portions or fragments thereof. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:2), or portions or fragments thereof. As stated previously, such sequence variation (compared to the amino acid sequence shown in SEQ ID NO:2) is permitted provided that the variant has PNGase L activity (e.g. is capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about PH8).

The PNGase provided herein may therefore:(i) comprise an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence which has at least 60% sequence identity thereto; and (ii) be capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8.

For example, the PNGase provided herein may therefore:(i) comprise an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence which has at least 70% sequence identity thereto; and (ii) be capable of removing a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8. This encompasses PNGases with the required function and at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2. The PNGases described herein have PNGase L activity. In the context of enzymes, the term “activity” is used to refer to the physiological function of the enzyme i.e. its ability to catalyse a specific reaction. As used herein, “PNGase L activity” refers to the ability to remove a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of about pH6 to about pH8. In other words, enzymes with “PNGase L activity” have broad spectrum specificity for N-glycans, and are able to remove both a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from glycoproteins. For the avoidance of doubt, this does not mean that the enzyme must be capable of removing every a-1 ,3 core fucosylated N-glycan and a-1 ,6 core fucosylated N-glycan from every substrate, under all conditions - it merely means that the enzyme’s activity is not solely restricted to removal of a-1 ,3 core fucosylated N-glycans or a-1 ,6 core fucosylated N-glycans only.

PNGases described herein that have PNGase L activity are able to remove both a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from glycoproteins over a pH range of about pH6 to about pH8. In other words, when a pH of about pH6 to about pH8 is used, these enzymes are still able to remove both a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from glycoproteins. This is advantageous for the resultant protein and/or glycan products, as denaturation of the products is minimised (compared to other PNGases that are only able to remove both a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from glycoproteins at much lower (acidic) pH ranges, for example). For the avoidance of doubt, being able to “remove both a-1 ,3 core fucosylated N-glycans and a- 1 ,6 core fucosylated N-glycans from glycoproteins over a pH range of about pH6 to about pH8” does not mean that the enzyme must be capable of removing every a-1 ,3 core fucosylated N-glycan and a-1 ,6 core fucosylated N-glycan from every substrate over the stated pH range - it merely means that the enzyme’s activity is not solely restricted to removal of a-1 ,3 core fucosylated N-glycans or a-1 ,6 core fucosylated N-glycans only and that it is able to do so at the stated pH range. As would be clear to a person of skill in the art, stating that an enzyme functions “over a pH range of about pH6 to about pH8” does not mean that the enzyme only functions over this range - it may also be capable of exerting its activity at lower (or higher) pH values.

Several suitable assays may be used to determine whether a PNGase or a putative PNGase has PNGase L activity. For the avoidance of doubt, the following test may be used (and is preferred herein):

For the “standard” conditions, use small 250 pl Eppendorfs and up to 100 pg of glycoprotein, dry sample if it exceeds 9 pl, and then make sample back up to 9 pl with ultrapure water. Add 1 pl of 10x denaturation solution (5 % SDS, 400 mM dithiothreitol), vortex, briefly centrifuge, and incubate at 100 °C for 10 mins. Cool to room temperature and briefly centrifuge again to get all the liquid to the bottom of the Eppendorf. Add 2 pl of 10x PNGaseF reaction buffer (500 mM sodium phosphate, pH 7.5)*, 2 pl of NP-40 (10 % solution), increase volume to 20 pl with 6 pl of ultrapure water, add 1 pl of enzyme. Close Eppendorf, mix gently, briefly centrifuge, and incubate overnight. The incubation time required can vary according to sample, but for the data presented here the reactions were left overnight at 37 °C. “Denaturing” conditions, were the same protocol as described, but no denaturing solution or NP-40, which were replaced with ultrapure water. “Native” conditions removed the denaturing solution, NP-40, and the heating step from the described protocol. *For the PNGase A reactions the supplied reaction buffer was used to allow optimum activity, 10x GlycoBuffer 3 (500mM sodium acetate, pH 6).

As used herein, “glycoprotein” refers to a protein which contains oligosaccharide chains (glycans) covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a co-translational or posttranslational modification. This process is known as glycosylation. Secreted extracellular proteins and membrane bound proteins are often glycosylated. The glycoproteins described herein may therefore be secreted glycoproteins or membrane bound glycoproteins. The phrases “glycoprotein”, “target glycoprotein”, “glycoprotein substrate”, “glycoprotein of interest” and “glycoprotein sample” may be used interchangeably herein, unless the context specifically indicates otherwise.

The PNGases described herein are used to remove N-glycans from glycoproteins. As used herein “removing” N-glycans, refers to spatially separating the N-glycan from the glycoprotein. In this context, the term “removing” may be used interchangeably with “cleaving” or “releasing”, unless the context specifically indicates otherwise. The PNGase removes the specified N- glycans to produce a (at least partially) deglycosylated protein and N-glycan products.

The PNGases described herein are used to remove a-1 ,3 core fucosylated N-glycans and/or a-1 ,6 core fucosylated N-glycans from glycoproteins. The structure of a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans are well known in the art. See for example Du et al. 2015 J. Agric. Food Chem, 63, 48, 10550-10555.

The a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans present on a glycoprotein may include oligomannose N-glycans, complex N-glycans, hybrid N-glycans, or a combination thereof. Oligomannose N-glycans are also referred to as “high mannose” N- glycans herein. High mannose N-glycans comprise two N-acetylglucosamines with many mannose residues, often almost as many as are seen in the precursor oligosaccharides before it is attached to the protein. Complex N-glycans include bi-antennary, tri-antennary, tetra- antennary, penta-antennary, hexa-antennary, hepta-antennary etc N-glycans. They may have almost any number of the other types of saccharides, including more than the original two N- acetylglucosamines. Advantageously, the PNGases described herein can remove oligomannose N-glycans and complex N-glycans from glycoproteins, without any obvious preference or bias in specificity. They are also able to remove neutral and sialylated glycans efficiently. Furthermore, based on these activities the PNGases described herein should also remove hybrid N-glycans from glycoproteins.

Depending on the glycoprotein, the PNGases described herein may remove a-1 ,6 core fucosylated N-glycans only (e.g. when these are the only N-glycans available for removal from the glycoprotein); a-1 ,3 core fucosylated N-glycans only (e.g. when these are the only N- glycans available for removal from the glycoprotein), or a mixture of a-1 ,3 core fucosylated N- glycans and a-1 ,6 core fucosylated N-glycans (e.g. when both of these N-glycans are available for removal from the glycoprotein). The PNGases may therefore be used with different glycoprotein substrates, to produce different (at least partially) deglycosylated protein and/or N-glycan products.

Typically, the PNGase described herein will be used to remove the N-glycans from a plurality of glycoproteins simultaneously. In other words, the PNGase will typically be contacted with a sample that comprises a plurality of glycoproteins. The glycoproteins in the sample may be the same or they may be different (e.g. the sample may comprise of mix of at least two different glycoproteins). The PNGase therefore typically acts on a glycoprotein sample.

The PNGase described herein is capable of removing at least 10% of the a-1 ,3 core fucosylated N-glycans in the glycoprotein sample. Preferably, the enzyme is capable of removing at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the a-1 ,3 core fucosylated N-glycans in the glycoprotein sample. The enzyme may remove at least 95% or about 100% of the a-1 ,3 core fucosylated N-glycans in the glycoprotein sample.

The PNGase described herein is also capable of removing at least 10% of the a-1 ,6 core fucosylated N-glycans in the glycoprotein sample. Preferably, the enzyme is capable of removing at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the a-1 ,3 core fucosylated N-glycans in the glycoprotein sample. The enzyme may remove at least 95% or about 100% of the a-1 ,6 core fucosylated N-glycans in the glycoprotein sample. The target glycoprotein may be a glycoprotein that has been produced by a cell. In this context, the terms, “produced by” and “expressed by” can be used interchangeably, unless the context specifically indicates otherwise.

For example, the target glycoprotein may be a glycoprotein that has been produced by a vertebrate cell. For example, the glycoprotein may have been produced by a mammalian cell, such as a mammalian cell line, e.g. a human cell line, a mouse cell line, etc. The glycoprotein may be a protein that is naturally produced by the vertebrate cell (i.e. naturally occurring in the cell), or it may be a protein that is not naturally produced by the vertebrate cell. In other words, it may be a native (endogenous) protein or a recombinant (exogenous) protein for the particular vertebrate cell that has produced it. When a protein is produced by a vertebrate cell such as a mammalian cell, a-1 ,6 core fucosylated N-glycans may be added post- translationally (or co-translationally) to generate a glycoprotein comprising a-1 ,6 core fucosylated N-glycans. The PNGases described herein may be used to remove a-1 ,6 core fucosylated N-glycans from these glycoproteins.

In a different example, the target glycoprotein may be a glycoprotein that has been produced by an invertebrate cell. For example, the glycoprotein may have been produced by an insect cell, such as an insect cell line etc. The glycoprotein may be a protein that is naturally produced by the invertebrate cell (i.e. naturally occurring in the cell), or it may be a protein that is not naturally produced by the invertebrate cell. In other words, it may be a native (endogenous) protein or a recombinant (exogenous) protein for the particular invertebrate cell that has produced it. When a protein is produced by an invertebrate cell such as an insect cell, a-1 ,6 core fucosylated N-glycans and a-1 ,3 core fucosylated N-glycans may be added post- translationally (or co-translationally) to generate a glycoprotein comprising a-1 ,6 core fucosylated N-glycans and a-1 ,3 core fucosylated N-glycans. The PNGases described herein may be used to remove a-1 ,6 core fucosylated N-glycans and a-1 ,3 core fucosylated N- glycans from these glycoproteins.

In a further example, the target glycoprotein may be a glycoprotein that has been produced by a cell wherein a-1 ,3 core fucosylated N-glycans are added during protein glycosylation. For example, the glycoprotein may have been produced by a plant cell (or a slime mold, or parasite), or by an insect cell (which adds both a-1 ,3 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans during protein glycosylation) etc. The glycoprotein may be a protein that is naturally produced by the cell (i.e. naturally occurring in the cell), or it may be a protein that is not naturally produced by the cell. In other words, it may be a native (endogenous) protein or a recombinant (exogenous) protein for the particular cell that has produced it. When a protein is produced by such a cell, a-1 ,3 core fucosylated N-glycans may be added post-translationally (or co-translationally) to generate a glycoprotein comprising a- 1 ,3 core fucosylated N-glycans. The PNGases described herein may be used to remove a-1 ,3 core fucosylated N-glycans from these glycoproteins.

The cell may be a cell line, a tissue, or an organism. The glycosylated protein may therefore be produced in vitro or in vivo. The use of the PNGase (in removing the N-glycans from glycoproteins) is typically in vitro.

Advantageously, N-glycans can be removed from glycoproteins by the PNGases described herein using conditions that include a neutral pH. As used herein, “neutral pH” refers to a range of pH values that is from about pH 6 to about pH 8. In other words, the conditions to which the glycoprotein is subjected during removal of the N-glycans can be controlled to maintain a pH in the range of from about pH 6 to about pH 8. This can minimise denature of the glycoprotein, and the resultant (at least partially) deglycosylated protein and N-glycan products. The phrase “from about pH 6 to about pH 8” includes any pH value within this range, as well as any subranges. For example, it includes from about pH 6.5 to about pH 7.5, or from about pH 6.8 to about pH 7.2. It also includes distinct pH values within the range e.g. about pH 6, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.7, about 7.9, about 8.0 etc.

The PNGases described herein have several different uses in the art. For example, the PNGases may be used in the production of proteins, in the production of glycans, in the characterisation of glycosylation proteins, in glycome analysis, in the preparation of a milk product, or in the preparation of a gelling agent etc. The use of PNGases in these applications is well known. The known methods can therefore be adapted to suit the PNGases described herein, using methods routinely used in the art.

Specific examples in which the PNGases described herein would be particularly useful include the following.

In one example, the PNGases may be used to remove N-glycans from proteins such as biopharmaceuticals (e.g. antibodies) that are expressed using mammalian, insect or plant cells. Removal of N-glycans from such proteins may be used to control product quality (e.g. to ensure the glycan profiles for such protein products are within approved limits) and/or to maintain consistency in protein function (e.g. remove host glycans that may impact protein function). It therefore provides a new means for improving protein production processes.

PNGases described herein may also be used in the removal and subsequent analysis of glycans (“glycome analysis”) from one or more proteins. At the moment researchers typically digest the peptide and then apply techniques such as mass spectrometry to determine glycan structures. This destroys the original protein and doesn’t allow the glycan function to be explored. The PNGases described herein provide a new means for glycome analysis and/or the production of N-glycans themselves, which may have uses in food science areas (e.g. as an agent to provide improved gel firmness in e.g. milk- based foods (see US10098367). Glycome analysis may also be relevant for example in the examination of human saliva and other body fluids for forensic identification (see Kailemia et al. 2017 Anal Bioanal Chem, 409 (2)395-410 and WO2016200099).

N-glycans from both plants and animals have been linked to allergies, such as hayfever, pet allergies and food allergies. The PNGases described herein may be used for research into such allergic responses, for example to study the mechanisms and tissues responsible for a glycan-based allergy. They also provide a potential treatment for such allergies.

Methods for removing N-glycans using the PNGases described herein are also provided herein. The methods are typically in vitro.

The method may be for removing a-1 ,6 core fucosylated N-glycans from a glycosylated protein. Additionally, or alternatively, the method may be for removing a-1 ,3 core fucosylated N-glycans from a glycosylated protein. As described in detail above, the PNGases described herein are capable of removing both a-1 ,6 core fucosylated N-glycans and a-1 ,3 core fucosylated N-glycans from glycosylated proteins, therefore proteins having one or both of these types of N-glycans can be treated (i.e. deglycosylated) in accordance with the methods described herein.

The methods described herein comprise contacting a PNGase as described herein with a glycosylated protein under conditions in which the PNGase is capable of removing a-1 ,6 core fucosylated N-glycans (and/or removing a-1 ,3 core fucosylated N-glycans) from the glycosylated protein. As stated in much more detail above, the PNGase comprises the amino acid sequence of SEQ I D NO: 1 , or an amino acid sequence which has at least 60% or at least 70% sequence identity thereto, and has PNGase activity (i.e. is capable of removing a-1 ,6 core fucosylated N-glycans and a-1 ,6 core fucosylated N-glycans from a glycosylated protein over a pH range of from about pH 6 to about pH 8).

Suitable conditions for contacting the PNGase with the glycosylated protein are readily identifiable to a person of skill in the art, using routine methods known to them. Non-limiting examples of suitable conditions include contacting the PNGase with the glycosylated protein in the presence of an aqueous buffer. Appropriate non limiting aqueous buffers include sodium phosphate, sodium citrate and sodium acetate. Other appropriate aqueous buffers may readily be identified by a person of skill in the art.

For example, the PNGase may be contacted with the glycosylated protein for at least 10 minutes (typically for at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, at least 180 minutes etc).

For example, the PNGase may be contacted with the glycosylated protein at a temperature range from about 25°C to about 39°C. For example, the PNGase may be contacted with the glycosylated protein at a temperature of about 35°C to about 38, or about 37 °C.

The PNGase may be brought into contact with the glycoprotein using any suitable means. For example, the PNGase enzyme may be added to the glycoprotein (or the glycoprotein added to the PNGase enzyme). In other words, the enzyme may be provided as an enzyme reagent for use in the method. Alternatively or additionally, the PNGase may be produced by a cell that is in contact with the glycoprotein (for example, the PNGase may be expressed by the same cell as the glycoprotein, or it may be expressed by another cell that is in contact with the glycoprotein, or it may be expressed by another cell that is in contact with the cell that is expressing the glycoprotein such that a mixture of cells (one or more that is expressing the glycoprotein and one or more that is expressing the PNGase) are used). In such examples, the cell that is expressing the PNGase may be a genetically modified cell that encodes the PNGase, or it may be a cell that is transfected, transduced or transformed with a nucleic acid that encodes the PNGase, wherein the transfection, transduction or transformation step is part of the method itself.

The methods described herein may therefore include the step of: a) contacting a nucleic acid that encodes a PNGase enzyme comprising the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto, with a cell under conditions in which the nucleic acid is incorporated into the cell and the PNGase is expressed; or b) culturing a genetically modified cell comprising a nucleic acid that encodes a PNGase enzyme comprising the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence which has at least 70% sequence identity thereto, under conditions in which the PNGase is expressed; or c) providing a PNGase that comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which has at least 70% sequence identity thereto; and then contacting the PNGase with the glycosylated protein under conditions in which the PNGase is capable of removing the specified N-glycans from the glycosylated protein.

Appropriate conditions for contacting a nucleic acid with a cell such that the nucleic acid is incorporated into the cell and the PNGase is expressed are well known in the art. Appropriate conditions for culturing a genetically modified cell comprising a nucleic acid encoding a PNGase such that the PNGase is expressed are also well known in the art. See for example EP1776455B1 , which provides general methods for the expression of enzymes, provision of suitable expression vectors, regulatory sequences, constructs, host cells, organisms, transformation methods etc for the production of appropriate volumes of enzyme.

As described in more detail elsewhere herein, methods that remove a-1 ,6 core fucosylated N- glycans from the glycosylated protein are particularly useful when the glycoprotein has been produced by a vertebrate cell, such as a mammalian cell. Furthermore, methods that remove a-1 ,6 core fucosylated N-glycans and a-1 ,3 core fucosylated N-glycans from the glycosylated protein are particularly useful when the glycoprotein has been produced by an invertebrate cell, such as an insect cell. Finally, methods that remove a -1 ,3 core fucosylated N-glycans from the glycosylated protein are particularly useful when the glycoprotein has been produced by a plant, slime mold, insect or a parasite. See for example Feasley et al., Glycoconj J (2015) 32:345-359; and Hykollari et al., J Proteome Res 2013, 12, 1173-1187.

As described in more detail elsewhere herein, the methods can advantageously perform the step of contacting the PNGase with the glycosylated protein at a pH range of about pH6 to about pH8. This is particularly useful when it is important to prevent or minimize denaturation of the protein and/or N-glycan products.

The PNGase for use in the methods described herein may be immobilized. By immobilizing te enzyme it is possible to reuse it more easily.

The methods described herein may include the additional step of recovering the deglycosylated protein or cleaved glycan products. This may include isolating the deglycosylated protein or cleaved glycan products from the rest of the reaction components/reagents. Standard methods for recovery and isolation of the deglycosylated protein or cleaved glycan products are known. See for example WO2015184325.

The methods described herein may also include the additional step of characterizing the deglycosylated protein and/or the glycan cleavage products. Methods for characterising the protein and/or glycan cleavage products are also well known (see for example Du et al. 2015 J. Agric. Food Chem, 63, 48, 10550-10555).

General definitions

The PNGase that is described herein may be in an isolated form. The term "isolated" means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.

The PNGase that is described herein may be in a purified form. The term "purified" means that the sequence is in a relatively pure state - e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.

The PNGase may be a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques. Recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.

The PNGase may be a synthetic sequence - i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.

The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for the PNGases described herein.

The terms protein, peptide and polypeptide are used interchangeably herein.

As used herein, a “naturally-occurring” polypeptide refers to an amino acid sequence that occurs in nature. A “non-essential” (or “non-critical”) amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence of SEQ ID NOs:1 or 2) without abolishing or, more preferably, without substantially altering a biological activity, whereas an “essential” (or “critical”) amino acid residue results in such a change. For example, amino acid residues that are conserved are predicted to be particularly non-amenable to alteration, except that amino acid residues within the hydrophobic core of domains can generally be replaced by other residues having approximately equivalent hydrophobicity without significantly altering activity.

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 have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential (or non-critical) amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSLIM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2,

3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSLIM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of

4, and a frameshift gap penalty of 5.

Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed with the N BLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http://www.ncbi.nlm.nih.gov>. The polypeptides described herein can have amino acid sequences sufficiently or substantially identical to the amino acid sequences of SEQ ID NO:1 or 2. The terms “sufficiently identical” or “substantially identical” are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.

The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

A nucleotide sequence described herein may be prepared using recombinant DNA techniques (i.e. recombinant DNA). Alternatively, the nucleotide sequence could be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).

A nucleotide sequence encoding an enzyme which has the specific properties as defined herein may be identified and/or isolated and/or purified from any cell or organism producing said enzyme. Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme or a part of the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for the enzyme (e.g. maltose for a glucosidase (maltase) producing enzyme), thereby allowing clones expressing the enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S.L. et al., (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.

The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Due to degeneracy in the genetic code, nucleotide sequences may be readily produced in which the triplet codon usage, for some or all of the amino acids encoded by the original nucleotide sequence, has been changed thereby producing a nucleotide sequence with low homology to the original nucleotide sequence but which encodes the same, or a variant, amino acid sequence as encoded by the original nucleotide sequence. For example, for most amino acids the degeneracy of the genetic code is at the third position in the triplet codon (wobble position) (for reference see Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN 0-7167-1920-7) therefore, a nucleotide sequence in which all triplet codons have been "wobbled" in the third position would be about 66% identical to the original nucleotide sequence however, the amended nucleotide sequence would encode for the same, or a variant, primary amino acid sequence as the original nucleotide sequence.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

Aspects of the invention are demonstrated by the following non-limiting examples.

EXAMPLES

The inventors have characterised PNGase L and have found it to have an unexpectedly broad specificity towards plant, insect, and mammalian-type N-glycans at a neutral pH. They carried out a series of assays using a variety of substrates and conditions alongside commercially available PNGase F and A to exemplify its broad activity and utility.

Materials and Methods

For the “standard” conditions: Use small 250 pl Eppendorfs and up to 100 pg of glycoprotein, dry sample if it exceeds 9 pl, and then make sample back up to 9 pl with ultrapure water. Add 1 pl of 10x denaturation solution (5 % SDS, 400 mM dithiothreitol), vortex, briefly centrifuge, and incubate at 100 °C for 10 mins. Cool to room temperature and briefly centrifuge again to get all the liquid to the bottom of the Eppendorf. Add 2 pl of 10x PNGaseF reaction buffer (500 mM sodium phosphate, pH 7.5)*, 2 pl of NP-40 (10 % solution), increase volume to 20 pl with 6 pl of ultrapure water, add 1 pl of enzyme. Close Eppendorf, mix gently, briefly centrifuge, and incubate overnight. The incubation time required can vary according to sample, but for the data presented here we left the reactions overnight at 37 °C, especially as we were using a range of condition we wanted to assess how well the enzymes could do without time as a factor.

“Denaturing” conditions, were the same protocol as described, but no denaturing solution or NP-40, which were replaced with ultrapure water. “Native” conditions removed the denaturing solution, NP-40, and the heating step from the described protocol.

*For the PNGase A reactions we used the supplied reaction buffer to allow optimum activity, 10x GlycoBuffer 3 (500mM sodium acetate, pH 6).

The clean-up steps and fluorescent labelling were completed according to Ludger SOP. The reactions were cleaned up using LC-PBM-96 plates: https://www.ludger.com/glycan-clean- up/. The glycans were labelled using LT-KPROC-96 https://www.ludger.com/products/glycan_labeling_kits.php and cleaned up using LC-PROC-

96 https://www.ludger.com/products/glycan_purification_kits.php

Procainamide-labelled glycans were analysed by LC-FLD-ESI-MS. Sample was injected into a Waters ACQUITY LIPLC Glycan BEH Amide column (2.1 x 150 mm, 1.7 pm particle size, 130 A pore size) at 40 °C on a Dionex Ultimate 3000 UHPLC instrument with a fluorescence detector (Aex = 310 nm, >tem = 370 nm) attached to a Bruker Amazon Speed ETD. Mobile phase A was a 50 mM ammonium formate solution (pH 4.4) and mobile phase B was neat acetonitrile. Analyte separation was accomplished by a gradient running from 85 to 57% mobile phase B over 105 min at a flow rate of 0.4 ml min-1. The Amazon Speed was operated in the positive sensitivity mode using the following settings: source temperature, 180 °C; gas flow, 4 I min-1 ; capillary voltage, 4,500 V; ICC target, 200,000; maximum accumulation time, 50.00 ms; rolling average, 2; number of precursor ions selected, 3; scan mode, enhanced resolution; mass range scanned, 400 to 1 ,700.

Results

Nine different substrates were used to compare PNGase L activity to the known activity of PNGase F and PNGase A. In addition to the variation in core decoration displayed by plants, insects, and mammals, the antenna of N-glycans can be split into five main groups. The first is high-mannose N-glycans (HMNG), which have between 5 and 9 a-linked mannose and these structures are the same throughout all organisms (Figure 1 b). The second is plant-type complex N-glycans and the third is mammalian complex N-glycans, which is the most variable type. The final two groups are hybrid versions, where one antenna is HMNG and one is complex (Figure 1 b). The specificity of PNGases is usually limited to the decoration of the Man3 core structure, however, the substrates used in this work aimed to test the specificity of the three PNGases against as many substrate variables as possible. Previous work on PNGases has shown that they have greater activity against denatured protein compared to native Sun et al. J Biol Chem 2015 290, 7452-62. To explore the best assay conditions for PNGase L, the inventors also carried out all assays under three conditions: denaturing with boiling and detergent (the commercial PNGase F method), denaturing with just boiling, and native protein.

A summary of the results is shown as a heat map in Figure 2. These results show that for PNGase A, activity was limited to plant-type N-glycans and some high-mannose N-glycans could be released under native conditions. No removal of mammalian N-glycans was observed. These observations are in line with what would be expected. Using the commercial PNGase F conditions, PNGase F was active against mammalian and high-mannose N-glycans, but completely inactive against glycoproteins from plant sources. These observations are in line with what would be expected. Under boiling-only conditions, the efficiency of PNGase F was reduced for half of the mammalian glycoproteins. Under native conditions, the efficiency of PNGase F was reduced significantly for all samples tested, except for the plasma substrate (with mammalian complex N-glycans) and RNaseB substrate (with high-mannose N-glycans).

In contrast to the PNGase A and F, PNGase L had activity against glycoproteins from both mammalian and plant sources. There wasn’t a significant difference in activity when comparing the boiling with detergent and just boiling conditions, but activity was significantly lower under native conditions. In comparison to PNGase F, PNGase L doesn’t release the full repertoire of N-glycan structures present. These include less release in general, low bisecting structures being released, and low release of glycans with branching sialic acids. This is likely to do with the conditions tested, which had previously been optimised for PNGase F.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Sequences SEQ ID NO:1 (PNGase L mature protein sequence, without signal peptide):

QESTSLQVFDNAVFYNMYGGLVENEPVPEGAIRLRNTTVAKPLTDEQIAAFGNTLTL NVTAA SLCDNYDRIGNVNLAFVPAGQTTYEFGAVTERIELGRFITPFMVPDGDVEVPYTWDISHV LNI LH H PVLAEQYDFWI EFEI AGYQGGPGQGGAAVEYPTICAN RQDVYRGSLELVSSGTYEEQL VVFDALSHKFELKNYTLDGTDVLGQTEKTFTFYLNQPVENAKFYFINSNHGSNSGGEEYV R RWHYVYLDNAQKLSYRPGGLSCVPFFDYNTQSNCIYYLCDGTNNTRPDTNSAWSWNNWC PGDKVPTRVVELGNLEAGEHSFKMRVPAAQFTGAQGYFPLSAYFTGEVDVLNTETFAAAA FTISPNPVNDVATITANGQEIKAVSVTNTLGQVVLTGATDRLDLSALQNGIYWRVDFANG TT

GVQKIVKN

SEQ ID NO:2 (PNGase L protein sequence, including signal peptide):

MKKTLLALSFAAFFGFGANAQESTSLQVFDNAVFYNMYGGLVENEPVPEGAIRLRNT TVAK PLTDEQIAAFGNTLTLNVTAASLCDNYDRIGNVNLAFVPAGQTTYEFGAVTERIELGRFI TPF MVPDGDVEVPYTWDISHVLNILHHPVLAEQYDFWIEFEIAGYQGGPGQGGAAVEYPTICA N RQDVYRGSLELVSSGTYEEQLVVFDALSHKFELKNYTLDGTDVLGQTEKTFTFYLNQPVE N AKFYFINSNHGSNSGGEEYVRRWHYVYLDNAQKLSYRPGGLSCVPFFDYNTQSNCIYYLC DGTNNTRPDTNSAWSWNNWCPGDKVPTRWELGNLEAGEHSFKMRVPAAQFTGAQGYF PLSAYFTGEVDVLNTETFAAAAFTISPNPVNDVATITANGQEIKAVSVTNTLGQVVLTGA TDR

LDLSALQNGIYWRVDFANGTTGVQKIVKN

References

Du et al. 2015 J. Agric. Food Chem, 63, 48, 10550-10555

US10098367

Kailemia et al. 2017 Anal Bioanal Chem, 409 (2)395-410

WO2016200099

Feasley et al., Glycoconj J (2015) 32:345-359

Hykollari et al., J Proteome Res 2013, 12, 1173-1187

Sun et al. J Biol Chem 2015 290, 7452-62

Kuo et al., Int J of Systematic and Evolutionary Microbiology, (2013), 63, 3280-3286 EP1776455B1

WO2015184325

General references

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Matthes et al., (1984) EM BO J. 3, p 801-805. US 4,683,202

Saiki R K et al., (Science (1988) 239, pp 487-491).

Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN 0-7167-1920-7)

Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994) Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991)