WO2009141599A1

WO2009141599A1 - Enhanced glycosylation using modified endohexosaminidase

Info

Publication number: WO2009141599A1
Application number: PCT/GB2009/001248
Authority: WO
Inventors: Antony Fairbanks; Christoph Heidecke; James William Barnabas Moir
Original assignee: The University Of York
Priority date: 2008-05-20
Filing date: 2009-05-20
Publication date: 2009-11-26

Abstract

Replacement of the key glutamic acid 173 residue of Endo A by either glutamine or histidine produces mutant enzymes for which hydrolytic activity has either been eliminated, or significantly curtailed, yet which still effect transglycosylation of acceptor substrates by the use of oxazolines as glycosyl donors. The E173H histidine mutant was demonstrated to be more effective than wild type Endo A for the production of a single homogenous glycoform of ribonuclease B.

Description

ENHANCED GLYCOSYLATION USING MODIFIED ENDOHEXOSAMINIDASE

The invention relates to modified endohexosaminidases and their use in the tnodifi cation of polypeptide and peptide substrates.

Glycosylation of proteins is the most diverse form of post-translational modification, and can play a key role in protein folding,'¹¹ and can also crucially affect important protein properties.'^2"6' However since the biosynthesis of glycans is not under direct genetic control, glycoproteins are produced intracellular^ as heterogeneous mixtures of glycoforms, in which different oligosaccharide structures are linked to the same peptide chain. Access to pure single glycoforms of glycoproteins has now become a major scientific objective'⁷¹ since it is not only a prerequisite for more precise biological investigations into the different effects glycans have on protein properties, but also an important commercial goal in the field of glycoprotein therapeutics which are currently marketed as heterogeneous mixtures of glycoforms.

Access to single glycoforms of glycoproteins can be achieved by total synthesis of both glycan and polypeptide components, and some outstanding achievements in this area have recently been published.'⁸' ⁹¹ However such synthetic approaches are particularly arduous, and do not realistically represent a practical approach that could be applied to widespread and large-scale glycoprotein production. Alternative approaches based on bioengineering of cell lines in order to optimise production of glycoproteins bearing particular oligosaccharide structures have also been reported ⁽¹⁰' ¹¹⁾ and are being exploited commercially, though such approaches have no guarantee of complete glycan homogeneity.

An alternative method for achieving homogenous protein glycosylation involves the use of enzymatic catalysis,'¹²¹ and one particular class of enzymes which display considerable synthetic potential in this respect are the endohexosaminidases.'¹³¹ Endohexosaminidases are a class of enzyme which specifically cleave the chitobiose core [GlcNAcO(1-4)GlcNAc] of Λ/-linked glycans between the two /V-acetyl glucosamine residues, and since they cleave this linkage they can also be used to selectively synthesise it. Two members of this class which have been demonstrated to display useful synthetic glycosylation activity are Endo M from Mυcor /-//emaffs'^14'171 and Endo A from Arthrobacter protophormiae.¹™ ¹⁹¹ However since these enzyme-catalysed reactions are reversible, competitive product hydrolysis can greatly reduce synthetic efficiency, particularly when transglycosylations are undertaken using un- activated donors. Seminal work in the field by Shoda and co-workers demonstrated that carbohydrate oxazolines are useful activated glycosyl donors for these enzymes, presumably since they mimic the putative oxazolinium ions which are proposed intermediates in the enzymatic catalysed hydrolysis reaction.'²⁰' Subsequently extensive work from the group of Wang has detailed the efficient synthesis of a series of glycopeptides by transglycosylation with Endo ^ ^[21-^24] _an(j _{ngs a}|_{so recen}tiy reported the synthesis of single glycoforms of ribonuclease B using this approach. ^[25]

In order to circumvent the problem of competitive product hydrolysis previous work has focussed on the attempted development of irreversible glycosylation reactions using structurally modified oxazolines as glycosyl donors; the synthetic products of these reactions are generally not hydrolysed by the endohexosaminidase used to promote their synthesis'^26" ^28]; the enzymes therefore act as glycoligases. However another potential way to circumvent this problem is the use of specifically mutated enzymes, or glycosynthases, as developed by Withers'²⁹' and Planas'³⁰' which are not capable of product hydrolysis. To this end the production of specific mutants of Endo A was undertaken by site directed mutagenesis, and investigations were carried out into glycosylation reactions using these mutant enzymes.

The term 'glycosynthase' was first applied by Withers to retaining glycosidases in which the nucleophilic of the two catalytic acid residues in the enzyme active site had been replaced by site directed mutagenesis by a non-participating residue, for example by alanine. The use of an activated glycosyl donor, such as a glycosyl fluoride, then allowed this mutant enzyme to promote a synthetic reaction, but the mutant enzyme was not capable of hydrolysing the product glycosidic linkage as the key nucleophilic residue was absent.

Endo A is a member of the family 85 of the glycohydrolases. These enzymes, though they are retaining glycosidases, are thought to catalyse hydrolysis via a neighbouring group participation mechanism in which the carbonyl oxygen of the 2-acetamide group of the second GIcNAc residue is the actual nucleophile, rather than an enzyme bound aspartate or glutamate. These enzymes therefore do not possess a nucleophilic residue at the active site, and as such it is not possible to envisage the production of a glycosynthase¹³¹¹ along the lines of the accepted Withers and Planas precedents.

This disclosure relates to a process for the glycosylation of peptides and polypeptides that utilizes modified endohexosaminidases and also the modified endohexosaminidases. Statements of Invention

According to an aspect of the invention there is provided the use of an endohexosaminidase polypeptide comprising an amino acid sequence wherein the amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue to provide a modified endohexosaminidase polypeptide wherein said modified endohexosaminidase polypeptide has reduced or absent glycosidase activity and retained or enhanced glycosyltransferase activity in the glycosylation of a polypeptide or peptide substrate.

In a preferred embodiment of the invention said amino acid sequence is represented in Figure 7.

In a preferred embodiment of the invention said amino acid sequence is modified at amino acid residue 173 as represented in Figure 7, or an amino acid residue equivalent to amino acid residue 173 in a related endohexosaminidase polypeptide.

In a preferred embodiment of the invention said modification is an amino acid substitution.

Preferably said amino acid substitution is glutamic acid 173 for histidine.

Alternatively said amino acid substitution is glutamic acid 173 glutamine.

In a preferred embodiment of the invention said polypeptide is further modified by substitution of amino acid residue tyrosine 205; preferably said modification is substitution of tyrosine 205 with phenylalanine.

According to a further aspect of the invention there is provided a process for the manufacture of a glycosylated polypeptide or peptide comprising: i) forming a preparation comprising a modified endohexosaminidase polypeptide wherein said polypeptide has reduced or absent glycosidase activity and retained or enhanced glycosyltransferase activity, a GIcNAc comprising polypeptide or peptide and a donating oligosaccharide oxazoline; and ii) providing reaction conditions wherein said oligosaccharide is transferred to said polypeptide or peptide by said modified endohexosaminidase polypeptide. In a preferred method of the invention said modified endohexosaminidase polypeptide is represented by the amino acid sequence in Figure 1b wherein amino acid residue 173 is modified.

In a preferred method of the invention said modification is a substitution of amino acid residue glutamic acid 173 for histidine or glutamine.

In a preferred method of the invention said oligosaccharide oxazoline is a di, tri, tetra, penta, hexyl, hepta, octyl, nona, deca saccharide oxazoline.

In a preferred method of the invention said peptide is a peptide hormone selected from the group consisting of anti-diuretic hormone; oxytocin; gonadotropin releasing hormone, corticotrophin releasing hormone; calcitonin, glucagon, amylin, A-type natriuretic hormone, B-type natriuretic hormone, ghrelin, neuropeptide Y, neuropeptide YY_3-36. growth hormone releasing hormone, somatostatin; or homologues or analogues thereof.

In a preferred method of the invention said polypeptide is a chemokine.

The term "chemokine" refers to a group of structurally related low-molecular weight factors secreted by cells having mitogenic, chemotactic or inflammatory activities. They are primarily cation ic proteins of 70 to 100 amino acid residues that share four conserved cysteine residues. These proteins can be sorted into two groups based on the spacing of the two amino-terminal cysteines. In the first group, the two cysteines are separated by a single residue (Ox-C), while in the second group they are adjacent (C-C). Examples of member of the ¹C-X-C chemokines include but are not limited to platelet factor 4 (PF4), platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growth stimulatory activity protein (MGSA), macrophage inflammatory protein 2 (MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig alveolar macrophage chemotactic factors I and Il (AMCF-I and -II), pre-B cell growth stimulating factor (PBSF).and IP10. Examples of members of the ¹C-C group include but are not limited to monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP- 4), macrophage inflammatory protein 1 α (MIP-1-α), macrophage inflammatory protein 1β (MIP-1-β), macrophage inflammatory protein 1-γ (MIP-1-γ), macrophage inflammatory protein 3 α (MIP-3-α, macrophage inflammatory protein 3 β (MIP-3-β), chemokine (ELC), macrophage inflammatory protein-4 (MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78 β, RANTES, SIS-epsilon (p500), thymus and activation-regulated chemokine (TARC), eotaxin, I-309, human protein HCC-1/NCC-2, human protein HCC-3. In a further preferred method of the invention said polypeptde is a pro-angiogenic polypeptide.

A number of growth factors have been identified which promote/activate endothelial cells to undergo angiogenesis. These include vascular endothelial growth factor (VEGF A); VEGF B₁ VEGF C₁ and VEGF D; transforming growth factor (TGFb); acidic and basic fibroblast growth factor (aFGF and bFGF); and platelet derived growth factor (PDGF). VEGF is an endothelial cell-specific growth factor which has a very specific site of action, namely the promotion of endothelial cell proliferation, migration and differentiation. VEGF is a complex comprising two identical 23 kD polypeptides. VEGF can exist as four distinct polypeptides of different molecular weight, each being derived from an alternatively spliced mRNA. bFGF is a growth factor that functions to stimulate the proliferation of fibroblasts and endothelial cells. bFGF is a single polypeptide chain with a molecular weight of 16.5Kd. Several molecular forms of bFGF have been discovered which differ in the length at their amino terminal region. However the biological function of the various molecular forms appears to be the same. bFGF is produced by the pituitary gland.

In a preferred method of the invention said pro-angiogenic polypeptide is selected from the group consisting of: VEGF A, VEGF B, VEGF C, VEGF D, TGFb₁ aFGF and bFGF; and PDGF.

In a further preferred method of the invention said polypeptide is a growth factor.

Insulin-like growth factor 1 (IGF1) and its cognate receptor IGF1 R are, in combination with human GH, essential for normal growth and development. Additionally IGF1R has also been implicated in malignant transformation (Baserga et al 1997). The IGF1 , IGF2 and insulin receptors are closely related and IGF1R can also be activated by IGF2. IGF1 R consists of an alpha chain of approximately 740 residues disulphide linked to a transmembrane beta chain (9OkDa) which includes the cytoplasmic tyrosine kinase domain. Two alpha chains are disulphide linked so that the receptor forms an alpha2:beta2 tetramer on the membrane (Hubbard and Till, 2000). The alpha chain consists of several domains: two L domains, L1 (residues 1-150) and L2 (residues 300-460) are largely responsible for binding the hormone; the L domains are separated by a Cys-rich domain (151-299), and followed by fibronectin Type III domains (460-700) (Baserga R, Hongo A, Rubini M, Prisco M SValentis B (1997) "The IGF-1 receptor in in cell growth, transformation and apoptosis" Biochim Biophys Acta 1332: F105-F126); Hubbard SB & Till, JH (2000) "Protein tyrosine kinase structure and function." Annu. Rev. Biochem. 59:373-398). In a preferred method of the invention said ligand is a cytokine.

Cytokines, are involved in a number of diverse cellular functions. These include modulation of the immune system, regulation of energy metabolism and control of growth and development. Cytokines mediate their effects via receptors expressed at the cell surface on target cells. Cytokine receptors can be divided into three separate sub groups. Type 1 (growth hormone (GH) family) receptors are characterised by four conserved cysteine residues in the amino terminal part of their extracellular domain and the presence of a conserved Trp-Ser-Xaa-Trp-Ser motif in the C-terminal part. The repeated Cys motif is also present in Type 2 (interferon family) and Type III (tumour necrosis factor family).

In a preferred method of the invention said cytokine is selected from the group consisting of: growth hormones; leptin; erythropoietin; prolactin; interleukins (IL) IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11 , the p35 subunit of IL-12, IL-13, IL-15; granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF); cardiotrophin (CT-1); leukocyte inhibitory factor (LIF); interferon type I, Il or III .

In a preferred method of the invention said interferon is a type I interferon.

Preferably said type I interferon is selected from the group consisting of: interferon α, interferon β, interferon ε, interferon K and ω interferon.

In a preferred method of the invention said interferon α is selected from the group consisting of: IFNA 1, IFNA 2, IFNA 4, IFNA 5, IFNA 6, IFNA 7, IFNA 8, IFNA 10, IFNA 13, IFNA 14, IFNA 16, IFNA 17 and IFNA 21.

In a preferred method of the invention said polypeptide is a monoclonal antiobody or active binding fragment thereof.

According to a further aspect of the invention there is provided a modified endohexosaminidase polypeptide comprising an amino acid sequence as represented in Figure 7 wherein amino acid residue glutamic acid 173 is substituted for the amino acid histidine. In a preferred embodiment of the invention said polypeptide is further modified by substitution of amino acid residue tyrosine 205; preferably said substitution of amino acid residue tyrosine 205 with phenylalanine.

According to a further aspect of the invention there is provided a nucleic acid molecule that encodes a polypeptide according to the invention.

According to a further aspect of the invention there is provided an expression vector that includes a nucleic acid molecule according to the invention.

According to a further aspect of the invention there is provided a cell transformed or transfected with a nucleic acid molecule or vector according to the invention.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does 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.

An embodiment of the invention will now be described by example only and with reference to the materials, method, table and figures:

Table 1. Glycosylatioπ of 4 with donors 1-3 catalysed by WT Endo A and mutants E173H and E173Q; Figure 1a Enzymatic remodelling of a mixture of RNase B glycoforms to a single Man₃GlcNAc₂ glycoform using WT endo A and the E173H mutant. Figure 1b Catalytic mechanisms of family 85 endohexosaminidases; A) Hydrolytic mechanism of WT Endo A; B) Putative synthetic mechanism of mutant E173Q with an oxazoline donor; C) Putative synthetic / hydrolytic mechanism of mutant E173H with an oxazoline donor;

Figure 2 Oxazoline donors 1-3 used for glycosylation of GlcNAcAsn amino acid acceptor 4 using WT Endo A and mutants E173H and E173Q;

Figure 3 Time correlations of product yield for glycosylations of acceptor 4 with donors 1 (A- C), 2 (D-F), and 3 (G-I) using WT Endo A, mutant E173H, and mutant E173Q respectively;

Figure 4 ESI-MS spectra of dRNase B before and after reaction with oxazoline. (A) dRNase B. Charges for each peak indicate protein molecular mass is 13885 Da (expected size 13885 Da). (B) Product following reaction of dRNase B with oxazoline 3 in the presence of Endo A E173H. Charged species due to glycosylated RNase B are marked with asterisks and indicate protein with molecular mass 14575 Da (expected size 14575 Da). Charged species without asterisks are due to dRNase B;

Figure 5 Time course studies of the extent of glycosylation of dRNAse B with oxazoline 3 catalysed by WT Endo-A (σ) and the E173H mutant (D) at different substrate concentrations. Reactions were carried out in 50 mM potassium phosphate pH 6.5 at 37 ⁰C with oxazoline 3 in a 20-fold excess over dRNase B; Endo A and dRNAse B at concentrations of (A) 18 μM & 0.46 mM, (B) 50 μM & 1.3 mM and (C) 35 μM & 1.8 mM, respectively;

Figure 6 is Man₃-GlcNAc-oxazoline; and

Figure 7a is the amino acid sequence of the endohexosaminidase signal peptide; Figure 7b is the amino acid sequence of mature endohexosaminidases.

Materials and Methods

General Experimental

DEAE sephadex, Phenyl-Sepharose CL-4B, the CM52 column, and Sephadex G100 were purchased from Amersham BioSciences. Molecular weight markers were purchased from Invitrogen. All other chemicals were purchased from Sigma-Aldrich. Polyacrylamide gel electrophoresis was carried out by the standard methods in the presence of SDS¹. Proteins were visualized with Coomassie brilliant blue R-250. ESI-MS spectra were measured on an ABI Qstar tandem mass spectrometer. Preparation of samples for MS analysis was carried out by using a C18 ZipTip (Millipore).

HPLC analysis and purification

Oxazoline donors 1, 2, and 3 and acceptor 4 were synthesised as described previously. Analytical and semi-preparative HPLC was carried out on an Agilent-Hewlett Packard HPLC system (1050 Series) connected to an Agilent Variable Wavelength Detector (1100 Series) using DataApex Clarity software (version 2.4.1.43). All HPLC separations were performed at 23°C. Compounds for enzymatic reactions were purified using a Phenomenex Gemini™ 5D C18 column (250 * 10 mm). The column was eluted isocratically (MeCN/H₂O, 23:77) at a flow rate of 2 mL/min with detection at 210 nm for the donors and 257 πm for acceptor 4. Analytical runs were done using a Phenomenex Gemini™ 5D C18 column (250 * 4.6 mm). The column was eluted isocratically (MeCN/H₂O, 23:77) at a flow rate of 1 mL/min over 20 min with detection at 257 nm.

General procedure for enzymatic glycosylation of acceptor 4

For each experiment, 100 μg (207 nmol) of acceptor 4 and 621 nmol (3 eq.) of the oxazoline donor in phosphate buffer (0.1M, pH 6.5) were treated with the respective enzyme (total reaction volume: 50 μL) at 23°C. Aliquots (2 μL) were taken at appropriate intervals and analysed by HPLC. The yield was determined by integration of the product and acceptor peaks.

Expression and Purification of Recombinant Endo-A in E. col?*

E. coli BL21(DE3) was transformed with the pET23d-Endo-A plasmid, and the transformants were cultured in Luria-Bertani medium containing 100 mg/litre ampicillin at 37 ⁰C until the OD600 reached 0.5-0.8. The cells were then induced with IPTG to a final concentration of 1mM, and Endo A was expressed at 25 ⁰C overnight (16 h). The cells were collected and washed in 1OmM phosphate buffer (pH 7.0). Cell pellets were subjected to a freeze-thaw cycle, followed by sonication (process time 3 min, pulse time 3 sec, waiting time 10 sec, power level 7 on Misonix Sonicator 3000, NY). Insoluble materials were then removed by centrifugation. Purification of the recombinant Endo-A was carried out as previously described³'⁴ with a slight modification as follows (this allowed higher product purity to be achieved; Figure 1). The crude extract obtained was applied to a DEAE sephadex column (1.6 x 20 cm), which had previously been equilibrated with 10 mM phosphate buffer (pH 7.5). The column was washed with the same buffer, and the enzyme was then eluted in 4-ml fractions with a gradient eluent; formed from 10 mM phosphate buffer and 0.5 M NaCI in 10 mM phosphate buffer. The active fractions were combined, and ammonium sulfate was added to the enzyme solution to 1 M. The enzyme solution was applied to a Phenyl Sepharose CL-4B column (1.6 x 22 cm) equilibrated with 1 M ammonium sulfate in 10 mM phosphate buffer (pH 7.0). The enzyme was eluted with a gradient formed between 1 M ammonium sulfate and 40% ethyleneglycol in 10 mM phosphate buffer. The active fractions were combined and applied to DEAE sephadex column (1.6 x 20 cm) for a second round of anion exchange chromatography. The active fractions from this second round of anion exchange chromatography were combined, desalted, and concentrated with Vivaspin Ultra filtration (10,000, Sartorius Group).

Site-directed Mutagenesis

The mutants were generated by PCR using Pfu-polymerase (Promega, USA) and pET23d- Endo-A as a template according to the procedure for a QuikChange site-directed mutagenesis (Stratagene). The primers used were as follows:

Mut 5'-primer 3'-primer ant

5'GTCTGCCCCTTCTGTGTG ti I

OLJ 5'GGTTTATTAACCAACACACAG TTGGTTAATAAACC on

AAGGGGCAGAC

E17 5¹GGTTTATTAACCAACAGACAG 5'GTCTGCCCCTTCTGTCTG

3Q AAGGGGCAGAC TTGGTTAATAAACC

Mutations were confirmed by DNA sequencing. Plasmids containing the mutated Endo-A genes were introduced into E. coli BL21 (DE3). Expression and purification of the mutants was then carried out as described for the wild-type enzyme.

Example 1

Previous studies on Endo A revealed that tryptophan 216 was key for the transglycosylation activity ^[Z2\ and very recently it has been proposed'³³¹ that glutamate 173 is the catalytic residue that acts as a general acid to protonate the glycoside oxygen during the hydrolytic step, and as a general base to deprotonate the incoming hydrolytic water. Replacement of glutamate 173 with glycine, aspartate, and glutamiπe resulted in either extremely significant, or complete loss of hydrolytic activity,'³²' whilst replacement with alanine produced a hydrolytically inactive mutant, the activity of which could be rescued by the addition of azide or formate.¹³³¹ However we reasoned that mutants of Endo A that lack the general acid Glu173 may still be able to process activated glycosyl donors, and in particular an oxazoline could still serve as a transition state mimic and allow a synthetic reaction to occur. In the first step of the hydrolytic mechanism Glu173 acts as the general acid responsible for protonation of the outgoing D-glycosidic oxygen resulting in the formation of an intermediate oxazolinium ion (Figure 1b A). In the second step Glu173 acts as a general base, de-protonating the incoming water molecule. Without a proton donor residue at position 173 all hydrolytic activity should therefore be curtailed since glycosidic bonds cannot be broken without prior protonation of the anomeric oxygen atom. However an oxazoline may still be able to enter into the mechanistic pathway and could still be glycosylated by an incoming alcohol (or hydrolysed by incoming water) in the second step. Such a mutant enzyme would therefore be a glycosynthase; all hydrolytic activity is curtailed, but an activated oxazoline donor would still be processed.

Glycosylate of a model glycosyl amino acid bearing a GIcNAc residue was undertaken with a variety of N-glycan fragment oxazolines using wild type endohexosaminidase A (WT Endo A) and two mutant enzymes (E173H and E173Q) as biocatalysts; mutant E173Q acted as a glycosynthase and promoted irreversible glycosylation in the cases investigated, whilst mutant E173H displayed greatly reduced hydrolytic activity whilst retaining the ability to catalyse glycosylation. Mutant E173H was more efficient that WT Endo A for re-modelling of Ribonuclease B, and catalysed the formation of a homogenous Man₃GlcNAc₂ glycoform in an irreversible fashion.

It was decided to produce two mutants of Endo A in which glutamate 173 had been replaced by alternative amino acids; these were chosen as glutamine, a substitution which in fact had already previously been made and which had resulted in total loss of hydrolytic activity,'³²' and histidine. These choices were made on the basis of the following rationale. Glutamine is a non-acidic residue, and therefore should be incapable of promoting the hydrolytic reaction to any extent; the caveat being that it would also not be able to act as a general base and facilitate the synthetic reaction by aiding de-protonation of the incoming nucleophile. However it could still act as a hydrogen bond acceptor and facilitate nucleophilic attack on the oxazoline in this respect (Figure 1b B). Alternatively histidine could either act as a general acid, or as a general base, depending on its protonation state. However the pK_a of histidine is -6.0 as compared to ~4.1 for the side chain acid of glutamic acid; the hope was therefore that a histidine residue at position 173 would perhaps still act as a general base and facilitate the synthetic reaction, but that its lower pKa could mean a reduction in its ability to act as a general acid, with an accompanying reduction in the hydrolytic capability of the enzyme (Figure 1b C). Example 2

Two mutants of Endo A, E173Q and E173H, in which glutamate 173 was exchanged for glutamine and histidine respectively, were produced by site directed mutagenesis, and the proteins were overexpressed in E. coli and then purified. The three oxazolines 1-3 were accessed as glycosyl donors as previously described,'²⁶' ^2βl and these were then used together with glycosyl amino acid 4^[26' (Figure 2) in a series of enzyme catalysed reactions with VvT Endo A, and the two mutants E173H and E173Q.

Example 3

Reaction progress was in all cases carefully monitored by HPLC. Figure 3 details time course studies of the yield of glycosylated product formed for each of the three donors catalysed by each of the three enzymes. In terms of kinetics all reactions catalysed by WT Endo A were faster than those catalysed by the two mutant enzymes. For WT Endo A the maximum of product formation was reached after less than 30 minutes with all donors investigated, and there was little difference between the rates of reactions using the different donors (Figure 3A, Figure 3D, and Figure 3G). Reactions catalysed by the E173H mutant were much more substrate dependent. With the (1-3)-linked trisaccharide donor 1, maximum product formation was achieved only after 18 h (Figure 3, chart B), whilst with the (1-6)- linked trisaccharide donor 2 it was achieved after 4 h (Figure 3, chart E). Reaction with the tetrasaccharide donor 3 was the fastest and the maximum yield was achieved after ca. 2.5 h (Figure 3, chart H). Glycosylates catalysed by the E173Q mutant were even slower, and reaction times of about 20 h were required in order to reach the maximum yield with all of the donors investigated (Figure 3, charts C, F, and I).

The dependence of the rate of reaction on the donor structure, particularly observed using the E173H mutant, probably reflects the importance of the different parts of the oxazoline donor for binding to the enzyme active site. For example the presence of an α-mannose residue at position 6 of the central mannose (donor 2) leads to a much faster enzymatic reaction than with an α-mannose residue solely at position 3 (donor 1), whilst the tetrasaccharide donor 3, possessing α-mannose residues at both positions, reacts much faster than the two others. This relative ranking of the importance of the presence of mannose residues at branch points also correlated with the rates of product hydrolysis. Wild type Endo A hydrolysed all of the products 5, 6, and 7 at a significant rate, reproducing the acceptor 4 and releasing the donor as the free reducing sugar. However as can be seen (Figure 3A₁ 3D, and 3G)₁ the rate of hydrolysis increased in the order 5 < 6 < 7; an order which corresponded with the relative rates of glycosylation of the donors 1 < 2 < 3 observed using the mutant E173H. This rank order was also reflected in the maximum yields obtained for product formation (5 < 6 < 7) for glycosylates catalysed by both of the mutant enzymes (Table 1).

Figure 3 also clearly shows that the two mutations did indeed suppress the ability of the enzyme to hydrolyse the products. Replacement of glutamic acid 173 by histidine resulted in a mutant which still retained some hydrolytic activity (Figure 3B₁ 3E₁ and 3H), but for these substrates this was considerably reduced compared to that of WT Endo A (Figure 3A, 3D, and 3G). Indeed an assay using ribonuclease B (RNase B) as the substrate revealed the hydrolytic activity of the E173H mutant to be approximately 20% that of WT Endo A. Replacement of glutamic acid 173 by glutamine completely abolished product hydrolysis (Figure 3C, 3F, and 3I), and no hydrolytic activity was observed towards RNase B, confirming the previous report of Fujita et al.'³²¹ However, this E173Q mutant had also been found not to have any transglycosylation activity when chitobiose-Asn linked oligosaccharides were used as donors.¹³²¹ Findings here therefore once again underline the usefulness and higher activity of glycosyl oxazolines as donors in enzymatic glycosylates catalysed by eπdohexosaminidases. The E173Q mutant, previously considered to be inactive for transglycosylation, was in fact capable of processing these oxazolines efficiently though the overall efficiency of the process was substrate dependent (Table 1). With the (1- 3)-linked trisaccharide donor 1, the maximum yield of product 5 obtainable was a very modest 17 %, but using the (1-6)-linked trisaccharide donor 2 the synthetic efficiency improved, and product 6 could be obtained in 66 % yield. Moreover with the tetrasaccharide donor 3 synthetic efficiency improved once more, and 82 % yield of product 7 could be obtained in an irreversible reaction. Even better yields were achieved using the E173H mutant, though again synthetic efficiency was substrate dependant. Whilst the yield of 5 obtained using the (1-3)-linked trisaccharide donor 1 was only 55 %, use of the (1-6)-linked trisaccharide donor 2 produced 6 in 71 % yield, in an essentially irreversible process (Figure 3E). Most impressively use of the E173H mutant with tetrasaccharide donor 3 gave the pentasaccharide product 7 in quantitative yield, and with a dramatically reduced rate of subsequent hydrolysis as compared to WT Endo A. The time course plot of the reaction (Figure 3I) shows that the yield of product remained practically constant over a period of 6 h, illustrating that as long as there was oxazoline donor left in solution, product formation proceeded and was much faster than hydrolysis; only after all of the donor was exhausted did the yield decrease. This finding also indicates that the ratio of donor to acceptor used, which in these investigations was donor: acceptor 3:1 , could probably be reduced significantly without lowering the product yield, thus improving the overall efficiency of the process.

It should be borne in mind that the maximum yields that can be achieved with any of these enzymatic reactions are dependent on the relative rates of three processes: one synthetic and two hydrolytic. These are the rate of glycosylation, which forms the desired product, the rate of enzyme catalysed product hydrolysis, and the rate of direct hydrolysis of the oxazoline donor, which is presumably also catalysed by the enzyme. Suppression of product hydrolysis alone is therefore not sufficient to guarantee a good product yield, as can be seen from some of the present examples. For example during glycosylation of donors 1 and 2 catalysed by the E173H mutant (Figure 3B, 3E) it is apparent that after a certain period of time product formation ceases, presumably indicating that no oxazoline then remains in solution, it rather having been hydrolysed instead of transferred onto the acceptor. The improved synthetic efficiency observed using larger oxazoline donors might indicate that the relative rate of glycosylation vs. direct oxazoline hydrolysis becomes more favourable as more extended oxazoline donors are used. Under normal circumstances product hydrolysis is also faster with more extended oligosaccharides, and so the overall efficiency of the synthetic processes then becomes limited by product hydrolysis. However because product hydrolysis is totally suppressed with mutant enzymes such as E173Q, and to a lesser extent with E173H, then the reaction profile and overall synthetic efficiency improves, and is especially good for the largest oxazoline donor that has so far been investigated.

Example 4

Attention then turned to the potential application of these enzymes for protein glycosylation. Ribonuclease B (RNase B) had recently been remodelled by Wang and co-workers using WT Endo A,^[2Sl and in this respect would serve as good system for comparison of the ability of mutant and wild type enzymes to effect glycoprotein remodelling. The studies detailed above indicated that of the oxazoline donors available the tetrasaccharide oxazoline 3 was the most useful. Furthermore they also indicated that kinetically the E173H mutant was the more efficient of the two mutants so far produced.

Commercially available bovine RNase B was firstly enzymatically trimmed back by treatment with Endo H to produce dRNase B, a single protein glycoform bearing a GIcNAc residue at the sole N-linked glycosylation site. dRNase B was then used as a substrate for glycosylation with tetrasaccharide oxazoline 3 using both WT Endo A and the E173H mutant (Figure 1a). In both cases the enzyme was able to effect production of a single glycoform product, Man₃GlcNAc₂ RNase B, which was characterised by mass spectrometry (Figure 4: calculated mass of the Man₃GlcNAc₂ glycoprotein 14575; found 14575). The progress of the reaction was monitored in both cases, and at three different concentrations of the d RNase B substrate (Figure 5).

Both enzymes efficiently catalysed the formation of the product Man₃GlcNAc₂ glycoform of RNase B; the maximum yields obtainable were 72% and 84% using WT Endo A and the E173H mutant respectively. The time course study revealed that WT Endo A effected transglycosylation of dRNase B more rapidly than the E173H mutant. However WT Endo A catalysed hydrolysis of the product glycoprotein, whereas the E173H mutant did not. Therefore after a certain time period, which was dependent on the concentrations of substrates used, the E173H mutant became the more efficient catalyst, ultimately allowing formation of the glycoprotein product in a higher yield.

Example 5

Human GM-CSF produced in P.pastoris (Sargramostim - Genscript Corporation) is heterogeneously glycosylated at amino acids asparagine 27 and asparagine 37. Also, the first four amino acids of the yeast-produced polypeptide are missing compared to the native amino acid sequence.

0.5mg GM-CSF was dissolved at a final concentration of 4 mg/mL in 5OmM citrate buffer pH 5.5 containing 100μg/mL bovine serum albumin (BSA) and incubated with 500U Endo H_f (1,000,000 U/mL - New England Biolabs) for 4h at 37⁰C to cleave the chitobiose core of the high-mannose glycosylation, leaving single N-acetylglucosamine (GIcNAc) residues attached to asparagines 27 and 37. EndoH_f and cleaved oligosaccharides were removed from the reaction by size-exclusion chromatography using a Superdex S75 column (GE Healthcare) equilibrated in phosphate-buffered saline (PBS) pH7.4, containing 100μg/mL BSA. The partially deglycosylated target was collected as a single peak, recovering 230μg GM-CSF in 6mL PBS/BSA.

This was concentrated to 50μL by centrifugal ultrafiltration using an Amicon Ultra4 (3,000 Oa cut-off - Millipore) then lyophilised and reconstituted in a 9.75μL reaction containing (final concentration) 1.8mM GM-CSF, 36 mM Man₃-GlcNAc-oxazoline and 35 μM Endo A mutant E173Q in 5OmM Tris-HCI pH 8.0, and incubated at 37⁰C. 1 μL was removed for LC-MS analysis at to, and a further 0.5μL at 16h. At 18h a further 300μg Man₃-GlcNAc-oxazoline and 7.5μg EndoA mutant E173Q were added to the reaction. The incubation was maintained at 37°C and further aliquots were taken for analysis after 24 and 40 hours.

Without glycosylation, the truncated polypeptide has a theoretical molecular weight of 14039 Da. Addition of one or two GIcNAc residues would yield species of theoretical mass 14242 Da and 14445 Da, respectively. These values agree well with the two species observed at to: 14243 Da and 14446 Da. At t=16h, t=24h and t=40h two species were observed with masses 14931 Da and 15825 Da, respectively. These values correspond well with the theoretical masses of Man₃-GlcNAc₂-GM-CSF (14932 Da) and (Man₃-GlcNAc₂)₂-GM-CSF (15825 Da).

References

[I] A. Helenius, M. Aebi, Science 2001, 291, 2364-2369.

[2] D. F. Wyss, J. S. Choi, J. Li, M. H. Knoppers, K. J. Willis, Science 1995, 269, 1273.

[3] J. D. Aplin, J. C. Wriston, CRC Crit. Rev. Biochem. 1981, 10, 259.

[4] T. Misaizu, S. Matsuki, T. W. Strickland, M. Takeuchi, A. Kobata, S. Takasaki,

Blood 1995, 86, 4097-4104.

[5] S. Elliot, A. Lorenzini, S. Asher, K. Aoki, D. Brankow, L. Buck, L. Busse, D. Chang,

J. Fuller, J. Gant, N. Hernday, M. Hokum, S. Hu, A. Knudten, N. Levin, R. Komorrowski, F.

Martin, R. Navarro, T. Osslund, G. Rogers, N. Orgers, G. Trail, J. Egrie, Nat. Biotechnol.

2003, 21, 414-421.

[6] A. Varki, Glycobiology 1993, 3, 97-130.

[7] C. S. Bennett, C-H. Wong, Chem. Soc. Rev. 2007, 36, 1227-1238.

[8] C. Unverzagt, C. Piontek, S. Mezzato, D. Varon, N. Lombana, M. Schaffrath, C.

Poehner, M. Puttner, P. Ring, C. Heinlein, A. Martin, F. X. Schmid, J. Peptide Sci. 2006, 12,

139-139.

[9] C. Piontek, D. Varon, C. Heinlein, S. Mezzato, C. Poehner, C. Unverzagt,

Biopolymers 2007, 88, 596-596.

[10] S. R. Hamilton, R. C. Davidson, N. Sethuraman, J. H. Nett, Y. W. Jiang, S. Rios, P.

Bobrowicz, T. A. Stadheim, H. J. Li, B. K. Choi, D. Hopkins, H. Wischnewski, J. Roser, T.

Mitchell, R. R. Strawbridge, J. Hoopes, S. Wildt, T. U. Gerngross, Science 2006, 313, 1441-

1443.

[I I] H. J. Li₁ N. Sethuraman, T. A. Stadheim, D. X. Zha, B. Prinz, N. Ballew, P. Bobrowicz, B. K. Choi, W. J. Cook, M. Cukan, N. R. Houston-Cummings, R. Davidson, B. Gong, S. R. Hamilton, J. P. Hoopes, Y. W. Jiang, N. Kim, R. Mansfield, J. H. Nett, S. Rios, R. Strawbridge, S. Wildt, T. U. Gerngross, Nat. Biotechnol. 2006, 24, 210-215.

[12] K. Witte, P. Sears, C-H. Wong, J. Am. Chem. Soc. 1997, 119, 2114-2118. [13] K. Haneda, M. Takeuchi, M. Tagashira, T. Inazu, K. Toma, Y. Isogai, M. Hori, K.

Kobayashi, M. Takeuchi, K. Takegawa, K. Yamamoto, Carbohydr. Res. 2006, 341, 181-190.

[14] K. Yamamoto, S. Kadowaki, J. Watanabe, H. Kumagai, Biochem. Biophys. Res.

Commun. 1994, 203, 244-252.

[15] K. Haneda, T. Inazu, K. Yamamoto, H. Kumagai, Y. Nakahara, A. Kobata,

Carbohydr. Res. 1996, 292, 61-70.

[16] K. Yamamoto, K. Fujimori, K. Haneda, M. Mizuno, T. Inazu, H. Kumagai,

Carbohydr. Res. 1998, 305, 415.

[17] M. Mizuno, K. Haneda, R. Iguchi, I. Muramoto, T. Kawakami, S. Aimoto, K.

Yamamoto, T. Inazu, J. Am. Chem. Soc. 1999, 121, 284.

[18] K. Takegawa, M. Tabuchi, S. Yamaguchi, A. Kondo, I. Kato, S. Iwahara, J. Biol.

Chem. 1995, 270, 3094-3099.

[19] J.-Q. Fan, L. H. Huynh, B. B. Reinhold, V. N. Reinhold, K. Takegawa, S. Iwahara,

A. Kondo, I. Kato, Y. C. Lee, Glycoconjugate J. 1996, 13, 643-652.

[20] M. Fujita, S.-i. Shoda, K. Haneda, T. Inazu, K. Takegawa, K. Yamamoto, Biochim.

Biophys. Acta 2001, 1528, 9-14.

[21] B. Li, Y. Zeng, S. Hauser, H. J. Song, L. X. Wang, J. Am. Chem. Soc. 2005, 127,

9692-9693.

[22] H. Li, B. Li, H. Song, L. Breydo, I. V. Baskakov, L.-X. Wang, J. Org. Chem. 2005,

70, 9990-9996.

[23] Y. Zeng, J. S. Wang, B. Li, S. Hauser, H. G. Li, L. X. Wang, Chem. Eur. J. 2006,

12, 3355-3364.

[24] L. X. Wang, H. J. Song, S. W. Liu, H. Lu, S. B. Jiang, J. H. Ni, H. G. Li,

ChemBioChem 2005, 6, 1068-1074.

[25] B. Li₁ H. J. Song, S. Hauser, L. X. Wang, Org. Lett. 2006, 8, 3081-3084.

[26] T. W. D. F. Rising, T. D. W. Claridge, N. Davies, D. P. Gamblin, J. W. B. Moir, A. J.

Fairbanks, Carbohydr. Res. 2006, 341, 1574-1596.

[27] T. W. D. F. Rising, T. D. W. Claridge, J. W. B. Moir, A. J. Fairbanks,

ChemBioChem 2006, 7, 1177-1180.

[28] T. W. D. F. Rising, C. D. Heidecke, J. W. B. Moir, Z. Ling, A. J. Fairbanks, Chem.

Eur. J. 2008, accepted for publication.

[29] L. F. Mackenzie, Q. R. Wang, R. A. J. Warren, S. G. Withers, J. Am. Chem. Soc.

1998, 120, 5583-5584.

[30] C. Malet, A. Planas, FESS Lett. 1998, 440, 208-212.

[31] During preparation of this manuscript a paper was published reporting the glycosynthase activity of an N175A mutant of Endo M using oxaxoline donors. See: M. Umekawa, W. Huang, B. Li₁ K. Fujita, H. Ashida, L.-X. Wang, K. Yamamoto, J. Biol. Chem. 2008, 283, 4469-4479.

[32] K. Fujita, K. Takegawa, Biochem. Biophys. Res. Commun. 2001, 283, 680-686. [33] K. Fujita, R. Sato, K. Toma, K. Kitahara, T. Suganuma, K. Yamamoto, K. Takegawa, J. Biochem. 2007, 142, 301-306.

Claims

1. The use of an endohexosaminidase polypeptide comprising an amino acid sequence wherein the amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue to provide a modified endohexosaminidase polypeptide wherein said modified endohexosaminidase polypeptide has reduced or absent glycosidase activity and retained or enhanced glycosyltransferase activity in the glycosylate of a polypeptide or peptide substrate.

2. Use according to claim 1 said amino acid sequence is represented in Figure 7.

3. Use according to claim 2 wherein said amino acid sequence is modified at amino acid residue 173 as represented in Figure 7, or an amino acid residue equivalent to amino acid residue 173 in a related endohexosaminidase polypeptide.

4. Use according to any of claims 1-3 wherein said modification is an amino acid substitution.

5. Use according to claim 4 wherein said amino acid substitution is glutamic acid 173 for histidine.

6. Use according to claim 4 wherein said amino acid substitution is glutamic acid 173 glutamine.

7. Use according to any of claims 3-6 wherein said polypeptide is further modified by substitution of amino acid residue tyrosine 205.

8. Use according to claim 7 wherein said modification is substitution of tyrosine 205 with phenylalanine.

9. A method for the manufacture of a glycosylated polypeptide or peptide comprising: i) forming a preparation comprising a modified endohexosaminidase polypeptide wherein said polypeptide has reduced or absent glycosidase activity and retained or enhanced glycosyltransferase activity, a GIcNAc comprising polypeptide or peptide and a donating oligosaccharide oxazoline; and ii) providing reaction conditions wherein said donating oligosaccharide oxazoline is transferred to said polypeptide or peptide by said modified endohexosaminidase polypeptide.

10. A method according to claim 9 wherein said modified endohexosaminidase polypeptide is represented by the amino acid sequence in Figure 7 wherein amino acid residue 173 is modified.

11. A method according to claim 10 wherein said modification is a substitution of amino acid residue glutamic acid 173 for histidine orglutamine.

12. A method according to any of claims 9-11 wherein said oligosaccharide oxazoline is a di, tri, tetra, penta, hexyl, hepta, octyl, nona or deca saccharide oxazoline.

13. A method according to any of claims 9-12 wherein said peptide is a peptide hormone selected from the group consisting of anti-diuretic hormone; oxytocin; gonadotropin releasing hormone, corticotrophin releasing hormone; calcitonin, glucagon, amylin, A-type natriuretic hormone, B-type natriuretic hormone, ghrelin, neuropeptide Y, neuropeptide YY₃. ₃₆, growth hormone releasing hormone, somatostatin; or homologues or analogues thereof.

14. A method according to any of claims 9-12 wherein said polypeptide is a chemokine.

15. A method according to any of claims 9-12 wherei said polypeptide is a pro- angiogenic polypeptide.

16. A method according to claim 15 wherein said pro-angiogenic polypeptide is selected from the group consisting of: VEGF A, VEGF B, VEGF C₁ VEGF D, TGFb, aFGF and bFGF; and PDGF.

17. A method according to any of claims 9-12 wherein said polypeptide is a growth factor.

18. A method according to any of claims 9-12 wherein said polypeptide is a cytokine.

19. A method according to claim 18 wherein said cytokine is selected from the group consisting of: growth hormone; leptin; erythropoietin; prolactin; interleukins (IL) IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11 , the p35 subunit of IL-12, IL-13, IL-15; granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM- CSF); ciliary neurotrophic factor (CNTF); cardiotrophin (CT-1); leukocyte inhibitory factor (LIF); interferon type I, Il or III .

20. A method according to claim 19 wherein said interferon is a type I interferon.

21. A method according to claim 20 wherein said type I interferon is selected from the group consisting of: interferon α, interferon β, interferon ε, interferon K and ω interferon.

22. A method according to claim 21 wherein said interferon α is selected from the group consisting of: IFNA 1 , IFNA 2, IFNA 4, IFNA 5, IFNA 6, IFNA 7, IFNA 8, IFNA 10, IFNA 13, IFNA 14, IFNA 16, IFNA 17 and IFNA 21.

23. A method according to any of claims 9-12 wherein said polypeptide is a monoclonal antibody or active binding fragment thereof.

24. A modified endohexosaminidase polypeptide comprising an amino acid sequence as represented in Figure 7 wherein amino acid residue glutamic acid 173 is substituted for the amino acid histidine.

25. A polypeptide according to claim 24 wherein said polypeptide is further modified by substitution of amino acid residue tyrosine 205.

26. A polypeptide according to claim 25 wherein said substitution of amino acid residue tyrosine 205 with phenylalanine.

27. A nucleic acid molecule that encodes a polypeptide according to claim 24 or 25.

28. An expression vector that includes a nucleic acid molecule according to claim 27.

29. A cell transformed or transfected with a nucleic acid molecule or vector according to claim 27 or 28.