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Human OGA binds substrates in a conserved peptide recognition groove

Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.


INTRODUCTION
The O-GlcNAc (O-linked N-acetylglucosamine) modification is a dynamic and reversible form of protein glycosylation occurring on specific serine and threonine residues of intracellular proteins [1,2]. Since the initial discovery of O-GlcNAc [3], technological advances have greatly facilitated its detection, and proteomics studies [4][5][6] have shown that a significant proportion of cellular proteins are O-GlcNAcylated. However, the functional importance of O-GlcNAc is only just emerging, with evidence to suggest that it may regulate protein activity in a manner analogous (and complementary) to phosphorylation [7]. O-GlcNAc levels are known to respond dynamically to nutrient availability [1] and stress [8], and to undergo changes during the cell cycle [9] and development [10]. O-GlcNAc has been shown to be associated with a range of human diseases [2]. Strikingly, only two enzymes orchestrate the O-GlcNAc modification. Both the OGT (O-GlcNAc transferase) and its antagonistic OGA (O-GlcNAcase or β-N-acetylglucosaminidase) are encoded by single genes in metazoa [11,12]. Little is known about the mode of substrate recognition of either enzyme, although studies have hinted at the existence of a defined O-GlcNAc peptide sequon [2,5]. In addition, it is thought that, for a single OGT to modify specific sites on a multitude of proteins, interactions with adapter proteins may be instrumental in achieving selectivity and regulation of activity [13].
OGA was first identified as a neutral-pH hexosaminidase, which was later associated with the gene MGEA5 (meningiomaexpressed antigen 5) [14]. The domain structure of hOGA (human OGA) is frequently oversimplified by designating an N-terminal hexosaminidase followed by a C-terminal domain with sequence homology with HATs (histone acetyltransferases) (referred to as the HAT domain in the present paper). A further putative domain may exist in the stretch of over 300 amino acids in between the hexosaminidase and HAT domains. hOGA (Ser 405 ) is itself a substrate for OGT [15]. Residues 404-548 have been shown to be required for interaction with OGT [16], and it is thought that the formation of such a complex negatively regulates O-GlcNAc removal.
Pharmacological inhibition of OGA has been an invaluable tool for investigating the cellular functions of O-GlcNAc; notably the non-selective inhibitor PUGNAc [O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino-N-phenylcarbamate] has been used extensively to raise cellular O-GlcNAc levels [17]. Advances in understanding of the OGA reaction mechanism [18], in conjunction with structures of bacterial OGAs [19,20], have informed the development of a number of highly potent and selective inhibitors for hOGA [21].
Despite these recent advances in our understanding of OGA structure, mechanism and inhibition, we do not understand how OGA interacts with the protein part of its substrates. Such interactions could constitute the basis of substrate specificity in hOGA. In the present paper, we reveal that hOGA possesses a conserved substrate-binding groove that is required for the interaction with O-GlcNAc proteins. Different substrates require distinct surface residues for binding, affording a first glimpse into the substrate selectivity of human O-GlcNAcase.

Cloning, expression and structure determination of OgOGA (Oceanicola granulosus OGA)
The O. granulosus gene for hyaluronoglucosaminidase (ZP_01156836.1) was cloned from genomic DNA into pGEX6-P1 for expression as a GST (glutathione transferase) fusion in Escherichia coli. The protein OgOGA was purified by affinity, anion-exchange and size-exclusion chromatography (see the Supplementary material at http://www.BiochemJ. org/bj/432/bj4320001add.htm). OgOGA was crystallized at 1.9 mg/ml from PEG [poly(ethylene glycol)]/MgCl 2 solutions (see the Supplementary Online Data). The structure of the apoenzyme was solved using MAD (multiwavelength anomalous diffraction) phasing with a lead derivative and the structure of a PUGNAc complex was obtained by molecular replacement. Diffraction data and model statistics are shown in Supplementary Table S1 at http://www.BiochemJ.org/bj/432/bj4320001add.htm.

Glycopeptide synthesis and detection
A 3,4,6-triacetyl-O-GlcNAc-Fmoc (fluoren-9-ylmethoxycarbonyl)-Ser-OH derivative was synthesized as described previously [22]. Microwave-assisted solid-phase peptide synthesis was performed with a CEM Liberty instrument on low-load Rink amide MBHA (methylbenzhydrylamine) resin 100-200 mesh (Novabiochem) using standard Fmoc chemistry protocols on a 0.05 mmol scale. The structural homogeneity of the glycopeptides was verified by high-resolution mass spectra taken on Bruker micro-TOF (time-of-flight) instrument in the direct injection mode. The same instrument was used for the monitoring of the enzymatic deglycosylation in the LC (liquid chromatography)-MS mode using an Agilent 1200 binary analytical HPLC system equipped with a Waters X-Bridge C 18 3 μm particles, 3.2 mm×50 mm column and diode array UVvisible detector. Typically, the reaction was diluted to 0.3 ml with water, then passed through a 10 kDa molecular-mass cutoff concentrator to remove protein. Then, 10 μl of the flowthrough was taken for the analysis using gradient elution (flow rate 0.3 ml/min) with a 5-95 % linear gradient of acetonitrile (0.1 % ammonia) in water (0.1 % ammonia).

Colorimetric and fluorimetric OGA assays
Initial rates of hydrolysis of pNP-GlcNAc (p-nitrophenyl-β-Nacetylglucosamine) were measured in buffer containing 25 mM Tris/HCl (pH 7.5) and 0.1 mg/ml BSA, with substrate concentrations ranging from 0 to 10 mM with a DMSO concentration of 2 % throughout the assay. Reactions were performed with 5 nM enzyme in a 50 μl assay volume. After 30 min of incubation at 37 • C, the reaction was stopped by addition of 100 μl of 3 M glycine/NaOH (pH 10.3), and product concentration was measured at A 405 . 4MU-GlcNAc (4-methylumbelliferyl-β-Nacetylglucosamine) assays were performed in the same buffer, with substrate concentrations ranging from 0 to 8 mM, and the enzyme concentration was reduced to 0.2 nM and the assay time to 20 min. Product formation was determined fluorimetrically (λ ex = 360 nm/λ em = 460 nm). Inhibition constants for OGA inhibitors were obtained with 4MU-GlcNAc with substrate concentrations equivalent to the K m . All experiments were performed in triplicate, and results were analysed and plotted using GraphPad Prism.
Kinetic parameters of 4MU-GlcNAc turnover by wild-type and mutant hOGA were determined as described above, with enzyme concentrations of 20 nM and a maximum substrate concentration of 2 mM (1 % DMSO in the assay). Competition assays with peptides were performed at 200 μM 4MU-GlcNAc. K m determination for GlcNAc-modified peptides was performed following an approach using established equations for multisubstrate enzyme kinetics to derive kinetic constants of a substrate whose turnover cannot be detected directly [24]. The fluorigenic reporter substrate and the GlcNAcylated peptide are considered as competitive inhibitors of each other. From the initial hydrolysis rates of 4MU-GlcNAc in the presence and absence of the peptide, the Michaelis constant for the peptide (K m ) can be determined from eqn (1): where v i /v 0 is the relative activity in the presence of inhibitor, K m and S are the Michaelis constant and substrate concentration of the reporter substrate respectively, and S is the concentration of the peptide.
In vitro removal of O-GlcNAc was performed in a 5 μl volume in 25 mM Tris/HCl and 25 mM GST (pH 7.5), and contained 10 μM O-GlcNAcylated substrate protein and 1 μM wild-type or mutant hOGA. Reaction mixtures were incubated for 4-6 h at 37 • C, and a 1 μl aliquot was blotted for O-GlcNAc with CTD110.6 (Abcam). DM-17 rabbit anti-hOGT antibody was purchased from Sigma. GST and TAB1 antisera were obtained from the Division of Signal Transduction and Therapy [28].

Recombinant hOGA is active on O-GlcNAc-modified proteins and peptides in vitro
Although hOGA has been shown to be active on a GlcNAcmodified peptide substrate [29], most studies have been restricted to measuring the enzyme activity in vitro using pseudosubstrates, or monitoring OGA activity in vivo by Western blot analysis of cellular O-GlcNAc levels after overexpression, knock down or inhibition of OGA. We aimed to demonstrate in vitro activity of hOGA on recombinant glycoproteins. Substrates were prepared by in vitro glycosylation of the previously characterized O-GlcNAc proteins TAB1 and the transcription factors FoxO1 and CREB (see Supplementary Figure S1). OGA assays were then Table 1 Table 1). hOGA activity on these peptides was confirmed by LC-MS. The affinity of hOGA for these peptides was then quantified by their ability to compete with the reporter substrate 4MU-GlcNAc ( Figure 1B and Table 1). The resulting K m values for the hOGA-Ser 405 -and TAB1-Ser 395 -derived peptides are in the highmicromolar and low-millimolar range respectively, somewhat decreasing in affinity with increasing peptide length. The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

Discovery of a bacterial OGA with improved similarity to the human enzyme
hOGA is known to possess an N-terminal catalytic domain (residues 60-366) and the C-terminal HAT domain (residues 707-916) ( Figure 2A). However, sequence analysis of putative metazoan OGAs shows that they also possess a low-complexity N-terminus (residues 1-59), and a 'middle' domain (residues 367-707), also containing a low-complexity region (residues 396-553). Only the catalytic domain is represented in the available (bacterial) OGA structures, yet any of the additional hOGA domains could contribute to substrate recognition/specificity. In the absence of a hOGA structure to guide our substratespecificity studies, we aimed to identify new bacterial OGAs with improved sequence identity and extending to beyond just the catalytic domain. A BLAST search identified a predicted protein from O. granulosus (OgOGA) that not only shows improved sequence identity (37 %) with the hOGA catalytic domain without any alignment gaps ( Figure 2B), but also, intriguingly, possesses a 200-residue C-terminal domain that shares approx. 20 % identity with the hOGA middle domain (Figures 2A and 2B). An OgOGA structure would reveal the position of this domain with respect to the active site and serve as a guide to identifying residues or domains involved in glycoprotein substrate recognition.
To investigate the possible OGA activity of OgOGA, the gene was cloned from genomic DNA and expressed in E. coli. The enzyme shows OGA activity with pNP-GlcNAc

OgOGA possesses a conserved peptide-binding groove and 'stalk'
We next explored the structural similarities of OgOGA with hOGA to allow exploitation of OgOGA as a structural guide for hOGA mutagenesis studies. OgOGA was crystallized and its structure solved, including a complex with PUGNAc (see Supplementary Table S1). The OgOGA structure reveals an N-terminal TIM barrel domain and a C-terminal α-helical domain (Figures 2A and 2C). The OgOGA catalytic domain is similar to that of CpNagJ (Clostridium perfringens NagJ) [RMSD (root mean square deviation) = 1.5 Å (1 Å = 0.1 nm) for 254 C α s; Figure 2C]. The α4-β5, β3-α3, β4-α4 and β5-α5 loops are of different lengths and/or adopt different conformations. Strikingly, sequence alignments show that these loops match hOGA, but not CpNagJ, in length and conservation ( Figure 2B). The C-terminal domain, hereafter referred to as the stalk domain, possesses good sequence similarity with hOGA ( Figure 2B) and contains a core four-helical bundle ( Figure 2C). Unexpectedly, this domain is structurally similar to the third domain of CpNagJ (RMSD = 1.9 Å for 110 C α s, yet <10 % sequence identity; Figure 2C), although the loops connecting the helices differ in length and conformation. In particular, the α10-α11 loop is nine residues longer in CpNagJ compared with OgOGA (and, by alignment, hOGA; Figure 2B), reaching into the CpNagJ active site, which is impossible in OgOGA (and thus unlikely in hOGA). Similarly, the OgOGA structure suggests structural homology between the stalk domain of Bacteroides thetaiotaomicron OGA [20] and hOGA (see Supplementary Figure S3 at http://www.BiochemJ.org/bj/432/bj4320001add.htm).
The OgOGA active site is formed by the TIM barrel β-α loops ( Figure 2C), with the catalytic loop residues Asp 115 /Asp 116 on β4-α4 conserved with hOGA, compatible with the observed OGA activity ( Figure 2C). This loop does not fully engage with the PUGNAc inhibitor, presumably related to the disordered phenylcarbamate group. Ligand-induced conformational changes in the β4-α4 loop have also been observed for CpNagJ [30]. Upon PUGNAc binding to OgOGA, the β7-η1 loop shifts by up to 4.7 Å exposing the GlcNAc-binding site, associated with small displacements of the neighbouring β8-α8 and β1-α1 loops. Residues hydrogen-bonding with the sugar hydroxy groups (Gly 8 , Tyr 160 , Asp 226 and Asn 254 ) or the N-acetyl moiety (Asn 221 ) are conserved in hOGA (Figures 2B and 2E). Similarly, the residues that show van der Waals/stacking interactions with the sugar and the N-acetyl group (Tyr 10 , Tyr 160 and Trp 219 ) are conserved in hOGA. Strikingly, the surface around the active site, together with the stalk domain, forms a groove lined by side chains that are highly conserved/identical between OgOGA and hOGA and also among metazoan OGAs ( Figure 2D).

Residues lining the conserved binding groove are required for hOGA-substrate interactions
We next tested the hypothesis that the conserved groove represents the binding site for O-GlcNAc glycoprotein substrates by expressing GST-fusions of 20 individual hOGA point mutants of residues lining the groove (Figures 2B and 2D). To control for effects on protein folding and/or catalysis rather than protein substrate binding, we measured 4MU-GlcNAc hydrolysis by these mutants (Figure 3A). Mutations affecting 4MU-GlcNAc hydrolysis are mostly located close to the sugar/catalytic machinery (Tyr 69 ) or occupy positions shown to undergo a conformational change upon inhibitor binding (Tyr 286 and Asp 287 in the β7-η1 loop) ( Figure 2). Strikingly, de-O-GlcNAcylation assays with the TAB1, FoxO1 and CREB glycoprotein substrates identified mutants of residues some distance from the active site (I176L, S190A, S190Q, T217A, E218S, F223S and K289A) with reduced activity against one or more of the protein substrates, yet unimpaired 4MU-GlcNAc hydrolysis ( Figure 3).

Conclusions
Since its discovery more than three decades ago, it has become clear that O-GlcNAc is an important post-translational modification found on hundreds of intracellular proteins and plays a role in signalling pathways. However, it is not clear what determines specificity in the targeting and cycling of O-GlcNAc on these proteins. We have investigated whether OGA specifically recognizes the peptide component of its glycoprotein substrates. Our findings show that, by similarity to the structurally related bacterial OgOGA, hOGA possesses a putative substrate-binding groove that is highly conserved within metazoan OGAs, in addition to a stalk domain in close proximity to the active site. Indeed, mutations targeting this groove identified residues that are not required for hydrolysis of the 4MU-GlcNAc pseudosubstrate, but affected hydrolysis of glycoprotein substrates. Although a limited set of glycoprotein substrates (TAB1, FoxO1 and CREB) and peptides (derived from TAB1 and hOGA itself) was tested, these mutational studies hinted at differential substrate recognition. The differences between the residues required for the turnover of these substrates may reflect differences in the molecular environment of the O-GlcNAc site(s)   The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

Figure 3 Activity of hOGA groove mutants
Twenty individual mutants of hOGA were generated based on the structural information obtained from OgOGA. (A) To evaluate the structural integrity of the hOGA mutants, the catalytic efficiency of 4MU-GlcNAc turnover was determined and compared with the wild-type (k cat /K m = 7000 M − 1 ·s − 1 ) and the catalytically impaired D175N mutant (negative control). hOGA mutants were also incubated with O-GlcNAc substrate proteins TAB1, FoxO1 and CREB, and deglycosylation was observed by Western blotting. (B) Visualization of the correlation between the mutants' ability to hydrolyse the pseudosubstrate 4MU-GlcNAc and glycoprotein substrates. Catalytically impaired mutants (k cat /K m for 4MU-GlcNAc 50 % of wild-type) locate in the areas shaded in red. Mutants unaffected in both 4MU-GlcNAc turnover and protein deglycosylation (cut-off 10 % reduction of wild-type activity) are shown in the blue areas. Mutations reducing OGA activity on protein substrates only are depicted in the yellow areas. Location of the mutations is shown on the surface around the active site of OgOGA (coloured by domain, see Figure 2A). The mutated amino acids are shaded according to correlation plots, thus amino acids affecting protein substrate recognition are highlighted in yellow. The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. on these proteins/peptides, and could tune O-GlcNAc cycling rates. Together, the results of the present study constitute the first evidence for specific interactions between hOGA and its substrate proteins beyond the sugar moiety, and may form the basis of an improved understanding of the enzyme's substrate specificity and the functional implications thereof.

AUTHOR CONTRIBUTION
The scientific hypothesis was devised by Marianne Schimpl, Alexander Schüttelkopf and Daan van Aalten. The experimental design was by Marianne Schimpl, Alexander Schüttelkopf, Vladimir Borodkin and Daan van Aalten. Experimentation was by Marianne Schimpl, Alexander Schüttelkopf and Vladimir Borodkin. Interpretation of results was by Marianne Schimpl, Alexander Schüttelkopf and Daan van Aalten who also wrote the paper.
The resulting construct was transformed into E. coli BL21(DE3) pLysS cells, which were then grown overnight at 37 • C in 80 ml of Luria-Bertani medium supplemented with 100 μg/ml carbenicillin. This culture was then used to inoculate 8 litres of autoinduction medium [2] with carbenicillin, in which the cells were grown for 48 h at 25 • C before harvesting by centrifugation at 3500 g for 30 min. Cells were washed, then resuspended in 50 ml of 25 mM Tris/HCl (pH 7.5) and 250 mM NaCl and lysed by passage through a continuous-flow cell disrupter at 207 MPa (Constant Systems). After centrifugation and filtration, the soluble fraction of the lysate was incubated for 2 h at 4 • C with 4 ml of glutathione-Sepharose beads (GE Healthcare), then washed with lysis buffer. After addition of 400 μg of GSTtagged PreScission protease, the beads were incubated overnight at 4 • C. Beads were removed, and the filtrate was dialysed into 25 mM Tris/HCl (pH 7.5) before loading on to a 5 ml HiTrap Q FF column (GE Healthcare) equilibrated in the same buffer. After washing protein was eluted using a 0-500 mM NaCl gradient. Fractions containing OgOGA were pooled and concentrated to <5 ml for gel filtration using a XK26/60 Superdex 75 gelfiltration column equilibrated in 25 mM Tris/HCl (pH 7.5) and 150 mM NaCl. The resulting fractions containing pure OgOGA as verified by SDS/PAGE were pooled and concentrated to 1.9 mg/ml.

OgOGA structure determination
OgOGA was crystallized at 20 • C using sitting-drop vapour diffusion with drops of 1 μl of protein solution mixed with 1 μl of mother liquor over a 60 μl reservoir of 18 % poly(ethylene glycol) 4000, 100-250 mM MgCl 2 and 100 mM Tris/HCl (pH 8.5). Crystals appeared overnight as hexagonal prisms. Crystals were cryoprotected using a mixture of 80 % mother liquor and 20 % ethylene glycol. For the PUGNAc complex structure, solid PUGNAc was added to a drop containing crystals for 30 min before cryoprotection. For phasing, 0.17 μl of a solution of 200 mM lead acetate in mother liquor was added to a drop containing crystals for 1 h before cryoprotection. All data were collected at the European Synchrotron Radiation Facility (Grenoble, France) at a temperature of 100 K, and processed using HKL software [3] (Supplementary Table S1). OgOGA crystals are of space group C2 with one protein molecule per asymmetric unit. Data for the PbAc 2 derivative were collected at beamline BM14 with λ = 0.946 Å (peak) and λ = 0.950 Å (inflection point) and processed/scaled to 1.9 Å and 2.0 Å respectively. Phases were calculated using SHELXC/D/E [4,5] through the HKL2MAP interface [6], WarpNtrace [7] built 264 of a possible 447 residues. After manual completion using Coot [8], REFMAC5 [9] and other CCP4 software [10], the resulting model was used for molecular replacement into the native 2.0 Å dataset. Model building and refinement proceeded as for the derivative structure and resulted in a final model with an R work of 0.186 and an R free of 0.222 with good geometry throughout (Supplementary Table S1). The PUGNAc complex was solved by molecular replacement with the derivative structure and refined as above to an R work of 0.242 and an R free of 0.279 with good geometry. Both reported structures exhibit two poorly ordered regions, one comprising several residues directly preceding helix α4 (modelled in the apo structure), the other covering part of the α13-α14 loop (lacking in both models). Additionally, the 12 C-terminal residues are disordered in the PUGNAc complex. Interestingly, the residues lacking from the α13-α14 loop would not be able to span the distance (31.4 Å, in principle covered by five amino acids, i.e. 6.28 Å per amino acid) between the built amino acids on either side of the gap; it is possible that the actual connection is between two monomers and the protein thus forms a (sub)domain-swapped dimer (distance = 13.2 Å, giving an average of 2.64 Å span per residue). This has not been modelled as such, as OgOGA is monomeric in solution (by gel-filtration; results not shown). Alternatively, the distance between the disordered ends of the loop could be explained by the effects of an unknown proteolytic event. Finally, the termini of helices α13 and α14 are 29 Å apart, which can easily be covered by the α13-α14 loop (21 residues, ∼ 1.3 Å per residue) after a degree of conformational change.
Ligand co-ordinates and topologies were generated with PRODRG [11]; during model building, it became apparent that the ligand electron density covered only the (pseudo)sugar ring and the rest of the molecule was either disordered or, more likely, had been hydrolysed. Consistent with the latter hypothesis, the ligand was modelled as a tetrahydropyran-2-one derivative. Figure 1(B) of the main text was created using Aline [12], Figures 2(C)-2(E) of the main text and the surface views in Figure 3(B) of the main text were rendered using PyMOL (DeLano Scientific http://www.pymol.org).

Figure S1 In vitro O-GlcNAcylation of proteins with OGT generates OGA substrates
GlcNAcylation reactions on GST (control), TAB1, GST-FoxO1 or GST-CREB. Full-length OGT was used for GST and CREB, a truncated ( 9) construct for FoxO1 and a GST fusion thereof (GST-9) for TAB1. Reactions contained 3.75 mM UDP-GlcNAc and recombinant OGT in 50 mM Tris/HCl (pH 7.5) plus 1 mM DTT (dithiothreitol). Enzyme/substrate protein ratios were 1:20, total protein concentration was 0.5 mg/ml. The reaction was allowed to proceed for 3 h at 37 • C. The top panel shows the different constructs of OGT, detected with DM-17 rabbit hOGT antiserum, the middle panel shows O-GlcNAcylation, as detected with the CTD110.6 antiserum, and the bottom panel shows total protein staining. This was necessary as the TAB1 used in this experiment did not carry a GST tag, but also serves to illustrate the presence of degradation products in the FoxO1 and CREB samples, some of which appear to bear the O-GlcNAc modification, as seen in the middle panel.

Glycopeptide synthesis
The 2,3,4,6-tetracetyl-O-GlcNAc-Fmoc-Ser derivative was synthesised according to the published procedure [13]. Solidphase peptide synthesis was performed on automated microwaveassisted peptide synthesizer CEM Liberty on low-load Rink amide MBHA (methylbenzhydrylamine) resin 100-200 mesh (Novabiochem) using standard Fmoc chemistry protocols on 0.05 mmol scale. Fmoc deprotection was executed with 20 % piperidine-DMF (dimethylformamide) at 70 • C for 3 min. Coupling of Fmoc amino acids (four equivalents) was mediated by PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) (four equivalents) in the presence of DIPEA (N,N-di-isopropylethylamine) (four equivalents) in DMF/Nmethyl-2-pyrrolidone 4:1 at 70 • C for 10 min except for serine and histidine that were coupled at 50 • C to prevent epimerization. Acetylation was achieved after removal of Fmoc protection from the N-terminus with 20 % acetic anhydride in pyridine at 70 • C for 5 min. After completion of the synthesis, the resin was collected in a 15 ml disposable PP (polypropylene) reservoir (Biotage) fitted with a PP fritted disk and PTFE [poly(tetrafluoroethylene)] stopcock. The resin was successively washed with DCM (dichloromethane) (3 × 5 ml) and methanol (3 × 5 ml). The acetate protections from the sugar residue were then removed by treatment with a solution of hydrazine hydrate in methanol 1:4 (2 ml) for 1 h at room temperature (23 • C). The reagent was filtered off and the treatment was repeated twice for 30 min with the same amount of fresh reagent. Finally, the resin was successively washed with methanol (3 × 5 ml) and DCM (3 × 5 ml). The resin was then treated with a cleavage cocktail (2 ml) made of TFA (92 %), water (2.5 %), phenol (2.5 %), DODT (3,6-dioxa-1,8-octanedithiol) (2.5 %) and TIS (tri-isopropylsilane) (1 %) for 2 h at room temperature. The cleavage cocktail was drained off and collected, and the resin was washed with neat TFA (trifluoroacetic acid) (2 × 1 ml). The combined filtrate was concentrated in vacuo to approx. 1 ml. The peptides were precipitated with ice-cold diethyl ether (35 ml) and recovered by centrifugation at 3000 g for 15 min. The pellet was washed twice with fresh diethyl ether (35 ml), then blow dried with argon, dissolved in water/acetic acid/acetonitrile (3:1:0.5, by vol.) (5 ml) and freeze-dried. The crude glycopeptides were purified by reverse-phase HPLC on a 21.1 × 100 Luna C 18 (2) column (Phenomenex) using a linear gradient of 5-95 % acetonitrile in water (0.1 % ammonia). UV detection was at 214 nm. Appropriate fractions were collected and freeze-dried to afford the target glycopeptides in 50-70 % yield based on the resin-loading capacity. The structural homogeneity of the glycopeptides was verified by high-resolution mass spectra taken using a Bruker micro-TOF (time-of-flight) instrument in the direct injection mode.