Induction of Antibodies Directed Against Branched Core O-Mannosyl Glycopeptides—Selectivity Complimentary to the ConA Lectin

Article abstract of DOI:10.1002/chem.201605627

Mammalian protein O-mannosylation, initiated by attachment of α-mannopyranose to Ser or Thr residues, comprise a group of post-translational modifications (PTMs) involved in muscle and brain development. Recent advances in glycoproteomics methodology and the “SimpleCell” strategy have enabled rapid identification of glycoproteins and specific glycosylation sites. Despite the enormous progress made, the biological impact of the mammalian O-mannosyl glycoproteome remains largely unknown to date. Tools are still needed to investigate the structure, role, and abundance of O-mannosyl glycans. Although O-mannosyl branching has been shown to be of relevance in integrin-dependent cell migration, and also plays a role in demyelinating diseases, such as multiple sclerosis, a broader understanding of the biological roles of branched O-mannosyl glycans is lacking in part due to the paucity of detection tools. In this work, a glycopeptide vaccine construct was synthesized and used to generate antibodies against branched O-mannosyl glycans. Glycopeptide microarray screening revealed high selectivity of the induced antibodies for branched glycan core structures presented on different peptide backbones, with no cross-reactivity observed with related linear glycans. For comparison, microarray screening of the mannose-binding lectin concanavalin A (ConA), which is commonly used in glycoproteomics workflows to enrich tryptic O-mannosyl peptides, showed that the ConA lectin did not recognize branched O-mannosyl glycans. The binding preference of ConA for short linear O-mannosyl glycans was rationalized in terms of molecular structure using crystallographic data augmented by molecular modeling. The contrast between the ConA binding specificity and that of the new antibodies indicates a novel role for the antibodies in studies of protein O-mannosylation.

Full text of DOI:10.1002/chem.201605627

A Journal of
Accepted Article
Title: Induction of Antibodies Directed Against Branched Core O-
Mannosyl Glycopeptides - Selectivity Complimentary to the ConA
Lectin
Authors: Jin Yu, Oliver C Grant, Christian Pett, Sabine Strahl, Robert J
Woods, and Ulrika Westerlind
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10.1002/chem.201605627
Chemistry - A European Journal
Induction of Antibodies Directed Against Branched Core O-Mannosyl Glycopeptides –
Selectivity Complimentary to the ConA Lectin
Jin Yu[a], Oliver C. Grant[b], Christian Pett[a], Sabine Stahl[c], Robert J. Woods[b] and Ulrika
Westerlind[a]*
[a] Gesellschaft zur Förderung der Analytischen Wissenschaften e.V. ISAS – Leibniz Institute for
Analytical Sciences
Otto-Hahn-Str. 6b, D-44227 Dortmund, Germany
Fax: (+49) 231-1392 4850
E-mail: ulrika.westerlind@isas.de
[b] Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA
30602, USA
[c] Centre for Organismal Studies (COS), Cell Chemistry, Heidelberg University, Im Neuenheimer
Feld 360, D-69120 Heidelberg, Germany
1
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10.1002/chem.201605627
Chemistry - A European Journal
Abstract
Mammalian protein O-mannosylation, initiated by attachment of α-mannopyranose to Ser or Thr
residues, comprise a group of post-translational modifications (PTMs) involved in muscle and brain
development. Recent advances in glycoproteomics methodology and the “SimpleCell” strategy, have
enabled rapid identification of proteins and specific sites modified with these PTMs. Despite the
enormous progress made, to date the biological impact of the mammalian O-mannosyl glycoproteome
remains largely unknown. Tools are still needed to investigate the structure, role and abundance of O-
mannosyl glycans. While O-mannosyl branching has been shown to be of relevance in integrin-
dependent cell-migration, and also plays a role in demyelinating diseases, such as multiple sclerosis, a
broader understanding of the biological roles of branched O-mannosyl glycans is lacking in part due to
the paucity of detection tools. In this work, a glycopeptide vaccine construct was synthesized and used
to generate antibodies against branched O-mannosyl glycans. Glycopeptide microarray screening
revealed high selectivity of the induced antibodies for branched glycan core structures presented on
different peptide backbones, with no cross-reactivity observed with related linear glycans. For
comparison, microarray screening of the mannose-binding lectin concanavalin A (ConA), which is
commonly used in glycoproteomics workflows to enrich tryptic O-mannosyl peptides, showed that the
ConA lectin did not recognize branched O-mannosyl glycans. The binding preference of ConA for
short linear O-mannosyl glycans was rationalized in terms of molecular structure using
crystallographic data augmented by molecular modeling. The contrast between the ConA binding
specificity and that of the new antibodies indicates a novel role for the antibodies in studies of protein
O-mannosylation.
2
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Introduction
Protein O-mannosylation comprises a group of modifications involved in important functions in brain
and muscle glycobiology.[1] In mammals, the protein O-mannosyltransferase 1 and 2 (POMT1,
POMT2) are responsible for the transfer of an initial mannose (Man) monosaccharide to Ser or Thr
residues on protein targets.[2] Subsequent addition of N-acetylglucosamine (GlcNAc) at the 2-position
of the core Man residue by the enzyme protein O-mannose β1,2-N-acetylglucosaminyl transferase 1
(POMGnT1) results in the formation of linear m1 core structures (GlcNAcβ1,2Manα).[3] Addition of a
second GlcNAc at the mannose 6-position by β1,6-N-acetylglucosaminyl transferase (GnT-Vb/GnT-
IX) forms the branched m2 core glycan (GlcNAcβ1,2[GlcNAcβ1,6]Manα).[4] The core m1 and m2
structures typically undergo further extension forming more complex O-glycan structures.[5] For
instance, the O-mannosyl core m1 glycans in the protein α-Dystroglycan (α-DG) have been found to
be modified with sialic acid (Sia, neuraminic acid, Neu5Ac) attached to a lactosamine
(3Galβ1,4GlcNAcβ) extension at the 4-position of the GlcNAc residues, resulting in a sialylated
tetrasaccharide epitope (Neu5Acα2,3Galβ1,4GlcNAcβ1,2Manα).[6] Other O-mannose core m1 and m2
elongated glycan epitopes found on α-DG contain fucosyl-N-acetyllactosamine, or a sulfated
trisaccharide (GlcA[3S]β1,3Galβ1,4GlcNAcβ) known as the human natural killer-1 (HNK-1)
epitope.[7] On core m1 and m2 O-mannosyl glycans, the HNK-1 epitope modulates the function of the
receptor protein-tyrosine phosphatase, known as RPTPβ.[4a] Increased HNK-1 epitope presentation by
core m2 branching results in RPTPβ phosphatase inhibition, causing increased levels of integrin-
dependent cell-migration.[4a,8] Further, RPTPβ is important for recovery from demyelinating diseases,
such as multiple sclerosis and spinal cord injury, and inhibition by HNK-1 core m2 branching is
believed to block the remyelination process.[9] A recent study has also showed that core m2 branching
is involved in increased prostate cancer tumor growth and metastasis.[10] A third O-mannosyl core
structure, the core m3 glycan (GlcNAcβ1,4Man) was recently discovered.[11] In α-DG, a GalNAc
residue may elongate the core m3 glycan (GalNAcβ1,3GlcNAcβ1,4Man). This core can be further
extended by phosphorylation of the terminal GalNAc, addition of two ribitol-5-phosphate groups, and
linking to repeating disaccharide units (-3Xylα1,3GlcAβ1-).[11,12] The GalNAc elongated core m3
glycans and ribitol-5-phosphate linkages are necessary for formation of the proteoglycan structures on
3
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Chemistry - A European Journal
α-DG, and are essential for stabilization of the dystrophin-glycoprotein complex, which acts as a
transmembrane linker between the extracellular matrix and intracellular cytoskeleton.[6a,11,12b,13]
Impaired glycosylation of α-DG has been shown to result in severe congenital muscular
dystrophies.[14]
α-DG was the first O-mannosylated protein found in mammals, however, a study of O-glycans in total
rabbit brain indicated that many more unidentified proteins carry O-mannosyl glycans, and led to the
conclusion that about 30% of the released O-glycans were mannosylated.[15] The high abundance of
protein O-mannosyl glycans in brain was also shown in a study of the mice brain proteome.[16]
Subsequently, a handful of additional mammalian glycoproteins were identified containing O-
mannosyl modifications, including the transmembrane protein CD24, a monoclonal IgG2 light chain
from CHO cells, phosphacan, tenascin-R, neurofascin 186, neurocan, brevican, versican, RPTPβ and
RPTPζ.[4a,17] Recently, the analytical “SimpleCell” strategy combined with glycopeptide enrichment
using the lectin concanavalin A (ConA) and LC-MS identification, showed that many proteins from
cultured breast cancer cells are modified with protein O-mannosylation.[18] In particular, a large family
of cadherin cell membrane receptors were found to be major carriers of O-mannosyl glycans.
Concurrently, LC-MS in combination with α-mannosidase treatment identified mono-O-mannosyl
glycans on T-cadherin from rabbit muscle and on recombinant epithelial E-cadherin from human
embryonic kidney cells.[19] O-mannosylation of E-cadherin has been shown to be crucial for the
function of cadherin mediated cell-cell adhesion.[19b] A recent study revealed that in gastric
carcinomas, decreased O-mannosylation unexpectedly affects the N-glycosylation pattern of E-
cadherin, thereby causing its dysregulation.[20] A monoclonal antibody (RKU1-3-5) specific for O-
linked α-mannose was later developed and applied in immunostaining of murine brain
glycoproteins,[21] leading to the observation that mono-O-mannosyl glycans are prevalent in inhibitory
GABAergic neurons and perineuronal nets. In the same study, using ConA glycopeptide enrichment,
α-mannosidase treatment and LC-MS based identification of (glyco)peptides, numerous murine brain
glycoproteins were found containing mono-O-mannosyl glycans, and among them members of the
cadherin and plexin superfamilies and the perineural net protein neurocan.
4
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In spite of the clear importance of O-mannosyl glycosylation, the tools available are still insufficient
for the specific detection, enrichment and identification of proteins containing these modifications. A
previous study, generated antibodies that were directed against
a
linear core m1
Galβ1,4GlcNAcβ1,2Manα-Thr glycopeptide.[22] The induced antibodies showed very high specificity
against the antigen structure, but retained some specificity for the peptide, making them unsuitable for
general detection of O-mannosyl glycans presented on diverse peptide backbones.
In the present work we aimed to generate antibodies directed against branched core m2
(GlcNAcβ1,2[GlcNAcβ1,6]Manα-Thr) O-mannosyl structures for applications in glycoprotein and
glycopeptide detection. A synthetic vaccine construct was designed carrying two core m2 glycans on a
tetrapeptide, with glycine residues flanking the carbohydrate epitopes. The glycopeptide was
conjugated via a C-terminal squarate linker to a mutant diphtheria toxin carrier protein (CRM197). An
efficient synthetic strategy was developed to prepare the branched core m2 glycosylated Fmoc-amino
acid building block that was applied in Fmoc-solid-phase peptide synthesis (Fmoc-SPPS) to generate
various glycopeptides. To evaluate the potential to use the induced core m2 antibodies in
glycoprotein/glycopeptide detection, detailed characterization of the specificity was undertaken
employing microarray screening of a library of 39 O-mannosyl glycopeptides. The glycopeptides
included short, extended, linear, and branched core glycans presented on a variety of peptides. The
glycopeptide microarray was also employed to evaluate the binding specificity of ConA to the
assorted O-mannosyl glycan structures. Computational carbohydrate grafting[23] was performed on the
O-mannosyl glycan – ConA crystal structure complexes (taken from PDBID 1TEI) in order to gain a
structural understanding of the observed specificities.
Results and Discussion
Synthesis: A limited number of chemical and chemoenzymatic preparations of linear core m1 and
core m3 O-mannosyl glycosylated amino acids and glycopeptides have been reported
previously.[12a,21,22,24] The first synthesis of core m2 structures is described here. A retrosynthetic
5
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analysis was carried out for the design of a straightforward route to the peracetylated
GlcNAcβ1,2[GlcNAcβ1,6]Manα-Thr Fmoc-SPPS amino acid building block 14, which was later
incorporated into the glycopeptide synthesis for the conjugation to CRM197 or for glycopeptide
immobilization on microarray slides (Figure 1). We focused on a convergent coupling approach using
a p-methoxybenzyl and N-trichloroethoxycarbonyl (NHTroc) protected thioglycoside donor 3 that
could extend the free 2- and 6-hydroxyl groups of a dimethoxybutane acetal protected Manα-Thr
acceptor 10 (disconnection A, Figure 1). For preparation of the orthogonal protected Manα-Thr amino
acid 10 the trichloroactamidate method was selected (disconnection B, Figure 1).[25]
((Figure 1))
The chemical synthesis commenced with preparation of the thioglycoside disaccharide donor 3. Here,
p-methoxybenzyl protection was selected instead of the more common benzyl protection to
circumvent a later hydrogenation step, which would be problematic in the presence of Fmoc groups on
the amino acid. Since the Troc group in later steps can be removed under mild conditions and is a
participating β-directing group[26] (desired for the coupling with the mannose acceptor 10) the known
GlcN3 thioglycoside 1[27] was converted to the desired GlcN-Troc-protected donor 3. Starting with p-
methoxybenzyl protection followed by azide zinc reduction and Troc-protection to give 3. For
preparation of the Manα-Thr amino acid 10, the 2,6-di-O-acetyl-3,4-O-dimethoxybutane-α-D-
mannopyranosyl trichloroacetamidate donor 7 first needed to be prepared. Starting from the known
Allyl 3,4-O-(2’,3’-dimethoxybutane-2’,3’-diyl)-α-D-mannopyranoside 4,[28] 2,6-acetyl protection,
anomeric deallylation with Wilkinson’s catalyst and reaction with trichloroacetonitrile in DBU was
performed. The obtained trichloroacetamidate donor 7 was then coupled to the Fmoc-Thr-O-tBu
amino acid 8[29] by trimethylsilyltriflate (TMSOTf) activation in diethyl ether to give 9. Selective
removal of the acetyl protecting groups on the mannose 2- and 6-positions using NaOMe in methanol
at pH 9.5 yielded the amino acid acceptor 10. By NIS/TfOH[30] activation at -50 °C, glycosylation took
place providing the GlcNAcβ1,2[GlcNAcβ1,6]Manα-Thr trisaccharide amino acid 11 in good yield.
The trisaccharide amino acid 11 was converted into a building block compatible with Fmoc-SPPS by
6
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Chemistry - A European Journal
three protecting group manipulation steps. Initially, the p-methoxybenzyl groups were removed by
CAN followed by acetylation. Subsequently the N-Troc groups were removed by reductive
elimination followed by acetylation to give 13. Simultaneous removal of the glycan dimethoxybutane
acetal and the threonine tert-butyl ester (tBu) was followed to give the desired Fmoc-SPPS
glycosylated amino acid 14. The mannose 3,4 hydroxyl groups of 14 were left unprotected during
peptide synthesis (Scheme 1).
((Scheme 1))
The obtained GlcNAcβ1,2[GlcNAcβ1,6]Manα-Thr Fmoc-SPPS amino acid 14 and the previously
described GlcNAcβ1-2Manα-Thr Fmoc-SPPS amino acid 15[22] were employed in the synthesis of the
glycopeptides 16-28. Starting from preloaded Fmoc-Gly-, Fmoc-Ala-, Fmoc-Asp(tBu)- or Fmoc-
Arg(Pbf) trityl resin (12.5 µmol), coupling of the standard Fmoc-amino acids (8 equivalents) was
carried out using HBTU/HOBt and the glycosylated Fmoc-Thr building blocks 14 and 15 (1.5
equivalents) were coupled using HATU/HOAt.[31] The glycosylated amino acids were pre-activated in
a smaller volume of solvent and manually added to the resin. Furthermore, the reaction time of the
glycosylated amino acids was extended (to 8h instead of 40 min). After full assembly of the
glycopeptides on the resin, and for later microarray surface immobilization, peptides 20-28 were
terminated by coupling of a triethylene glycol spacer.[32] Detachment from the resin was performed by
applying a mixture of trifluoracetic acid (TFA)/triisopropylsilane (TIPS)/H2O (90:5:5). Peptides 16-19
were instead capped at the N-terminal with Ac2O, HOBt and DIPEA followed by cleavage from the
resin, which allowed a spacer[33] to be connected to the C-terminal for later conjugation with an
immune carrier or for immobilization on a microarray surface. Insertion of a linker at the C-terminal
instead of the N-terminal aimed to avoid immunogenic response to a C-terminal amide or free
carboxylic acid in close proximity to the glycosylation sites. After a desalting step on C-18 cartridges,
the O-acetyl groups on all generated glycopeptides (16-28) were removed by treatment with catalytic
amounts of NaOMe in methanol at pH 9.5. Deprotected glycopeptides 16-28 were finally purified by
preparative HPLC. For vaccine construction and ELISA antibody titer determination, glycopeptide
protein-conjugates were prepared. The CRM197 immune carrier mutant originating from diphtheria
7
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Chemistry - A European Journal
toxin have successfully been used for immune stimulation of different glycoconjugates and was
therefore selected for conjugation to peptide 17 and later immunization. The glycopeptide 17, which
contained a spacer with a free amine at the C-terminal, was conjugated to diphtheria toxin CRM197 and
BSA by an initial coupling with diethylsquarate in EtOH/H2O (1:1) at pH 8 to give the squaric acid
monoamide 29. The coupling to BSA and CRM197 was performed in aqueous Na2HPO4 buffer at pH
9.5 to give the glycopeptide BSA 30 and CRM197 31 conjugates (Scheme 2).
((Scheme 2))
Immunization: For antibody induction against the GlcNAcβ1,2[GlcNAcβ1,6]Manα glycopeptide,
two rabbits (26559 and 26560) were immunized with the diphtheria toxin mutant CRM197 vaccine 31
together with a lipopolysaccharide cocktail (LPS, Span 80, Tween 80, mineral oil and aqua bidest.) as
adjuvant. Four booster immunizations were carried out over 8 weeks. Analysis of sera from the pre-
immune, first bleed and final bleed by ELISA coated with the GlcNAcβ1,2[GlcNAcβ1,6]Manα
glycopeptide BSA conjugate 30 showed that strong immune responses were directed against the
antigen structure (Figure S1).
Microarray analysis and molecular modeling: The binding specificities of the antibody sera were
evaluated by microarray analysis, against glycopeptides 16-28 and the previously synthesized O-
mannosyl glycopeptides 32-57[21,22,24e] (Table 1). The induced antibodies showed high selectivity
towards both the glycopeptide antigen structure 17 (microarray nr: 38) and all other peptides
presenting the branched core m2 GlcNAcβ1,2[GlcNAcβ1,6]Manα trisaccharide 19-28 (microarray nr:
15-23, 39), regardless of whether the glycans were presented as mono- or divalent structures on the
peptide backbone. Notably, neither the non-glycosylated peptide 18 (microarray nr: 33) nor the linear
core m1 GlcNAcβ1,2Manα disaccharide antigen peptide 16 (microarray nr: 37) were recognized by
the induced antibody sera. Nor did the antibodies cross-react with the other linear core m1
disaccharides (microarray nr: 5-14), the core m1 trisaccharide (microarray nr: 1-4), or α-mannose
monosaccharide (microarray nr: 24-32) modified peptides, such as sequences from α-DG, Plexin and
8
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Prothocadherin. Altogether we could conclude that new antibodies were generated, which recognized
branched core m2 structures with very high selectivity that should serve as useful tools for O-
mannosyl glycan detection (Figure 2 and Table 1).
((Figure 2))
((Table1))
The concanavalin A (ConA) lectin that commonly is used for enrichment of tryptic O-mannosyl
peptides was also evaluated on the glycopeptide microarray. Interestingly, the ConA recognition
pattern of the O-mannosyl glycopeptide structures contrasted that of the antibody sera. ConA showed
high reactivity to all α-mannose monosaccharide peptides (microarray nr: 24-32) and to all linear core
m1 GlcNAcβ1,2Manα disaccharide peptides (microarray nr: 5-14) except the diglycosylated antigen
peptide structure 16 (microarray nr: 37). Unlike the antibody sera, with the exception of glycans
conjugated to YAT*AVA (microarray nr: 4 and 17), no ConA binding recognition was observed for
branched core m2 (microarray nr: 15, 16, 18-23, 38, 39) and extended linear core m1 (microarray nr:
1-3) trisaccharide peptides (Figure 2 and Table 1). Notably, glycan presentation on the YAT*AVA
sequence consistently resulted in enhanced binding recognition, both in the case of ConA and the core
m2 antibody sera (Figure 2 and Table 1).
The observed selectivity of ConA towards the O-mannosyl glycopeptide array was rationalized by
molecular modeling based on an existing co-crystal structure of ConA (PDBID 1TEI)[34] bound to a
pentasaccharide ligand ((GlcNAcβ1,2Manα1,3[GlcNAcβ1,2Manα1,6]Man) that contains three Man
residues (core, 3-arm, and 6-arm) with GlcNAc residues β1,2-linked to the 3- and 6-arms. The Man
residues in the glycans in the microarray, could potentially align in any of the three Man orientations
in the co-crystal structure, thus each Man binding mode was examined for each arrayed glycan to
determine its ability to rationalize the Con A data (Figure 3).
9
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Using computational carbohydrate grafting,[23b] [36] putative bound structures for each of the arrayed O-
mannosyl glycans were generated by adding the relevant glycan branches to the pertinent components
(core, 3-arm, and 6-arm Man) of the crystallographic ligand, while attempting to resolve any steric
overlaps by adjusting the glycosidic linkages. The Gly-Spec webtool[35] (www.glycam.org/gr) was
used to perform this analysis. The predictions from grafting to the 6-arm were consistent with the
selectivity of ConA; namely that substitution at the 2-position of Man by GlcNAc would be tolerated,
while extension of the GlcNAcβ1,2Man disaccharide by Gal or modification at the 6-position of the
core Man by GlcNAc would not be (Figure 3). Thus, the linear glycans (array 5-14) that show affinity
for ConA were predicted to be able to bind in either the 6-arm or 3-arm alignments, while the
monosaccharide glycans (array 24-32) could potentially bind in any of the three Man positions. In
contrast, the linear core m1 trisaccharides (array 1-4) and branched glycans (array 15, 16, 18-23, 38,
39) that do not display affinity for ConA could only be tolerated in the 3-arm site (Figure 3). Grafting
to the Core Man residue indicated that the binding site could not tolerate substitution at the 2-position
of that residue, thus only glycopeptides 24-32 could bind via this site (Figure 3). This latter
observation is notable since studies based on N-linked glycans binding to ConA lead to the conclusion
that interactions within the 3-arm site are weaker than those within the 6-arm.[36] The binding observed
for the glycopeptide array elements 4 and 17 could be a combination of weak binding via the 3-arm
site and the consistently observed enhancing effect of the YAT*AVA peptide sequence.
((Figure 3))
Conclusion: In a number of cases, O-mannosyl core m2 branching has been shown to be important in
brain glycobiology, cell-migration, and tumor-cell growth and metastasis. In this study, core m2
glycans, glycopeptides and core m2 glycoconjugates were synthesized for the first time, and were used
to generate glycan specific antibodies and to evaluate the immune response. Immunizations with the
core m2 glycoconjugate vaccine 31 provided evidence that a strong immune response was directed to
the glycopeptide antigen structure 17. Evaluation of the binding specificity of the antisera using
glycopeptide microarrays showed highly specific recognition of the branched core m2 O-mannosyl
10
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10.1002/chem.201605627
Chemistry - A European Journal
glycans on different peptide backbones, without cross-reactivity with linear core m1 or
monosaccharide Manα modified peptides. We aim to further characterize and apply the new antibodies
as tools in future immunostaining experiments to explore functions of O-mannosyl branching in brain
biology and tumor progression. For comparison, the man-specific ConA lectin was also evaluated for
binding to the glycopeptide array. The data indicated that ConA recognized short core m1 and
monosaccharide Manα modified peptides, but showed little if any binding to branched core m2 or
extended linear core m1 trisaccharides. A computational examination of the ConA binding modes was
able to provide a structural rationalization for the array data. In conclusion, the specificity of the new
antibodies for branched O-mannosyl ligands stands in contrast to the binding specificity of ConA,
indicating a novel role for the antibodies in studies of protein O-mannosylation.
Methods:
Experimental details are given in supporting information.
Acknowledgements
This work was supported by Deutsche Forschungs Gemeinschaft grant number WE 4751/2-1 (to
U.W.) and Sonderforschungsbereich 1036, project 11 (to S.S.). S.S. is a member of the excellence
cluster CellNetworks. R.J.W. thanks the National Institutes of Health (U01 CA207824 and P41
GM103390) for support. Financial support by the Ministerium für Innovation, Wissenschaft und
Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und
Forschung des Landes Berlin, and the Bundesministerium für Bildung und Forschung is gratefully
acknowledged (ISAS, U.W.).
References
[1]
a) M. Lommel, S. Stahl, Glycobiology 2009, 19, 816-828; b) T. Endo, Glycoconj. J. 2004, 21,
3-7; c) C. M. Dobson, S. J. Hempel, S. H. Stalnaker, R. Stuart, L. Wells, Cellular and
Molecular Life Sciences 2013, 70, 2849-2857.
[2]
[3]
H. Manya, A. Chiba, A. Yoshida, X. Wang, Y. Chiba, Y. Jigami, R. U. Margolis, T. Endo,
Proc. Natl. Acad. Sci. 2004, 101, 500-505.
a) S. Takahashi, T. Sasaki, H. Manya, Y. Chiba, A. Yoshida, M. Mizuno, H.-K. Ishida, F. Ito,
T. Inazu, N. Kotani, S. Takasaki, M. Takeuchi, T. Endo, Glycobiology 2001, 11, 37-45; b) W.
Zhang, D. Betel, H. Schachter, Biochem. J. 2002, 361, 153-162.
11
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10.1002/chem.201605627
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[4]
a) K. L. Abbott, R. T. Matthews, M. Pierce, J. Biol. Chem. 2008, 283, 33026-33035; b) K.-I.
Inamori, T. Endo, J. Gu, I. Matsuo, Y. Ito, S. Fujii, H. Iwasaki, H. Narimatsu, E. Miyoshi, K.
Honke, N. Taniguchi, J. Biol. Chem. 2004, 279, 2337-2340; c) J. K. Lee, R. T. Matthews, J.-
M. Lim, K. Swanier, L. Wells, J. M. Pierce, J. Biol. Chem. 2012, 287, 28526-28536.
J. L. Praissman, L. Wells, Biochemistry 2014, 53, 3066-3078.
[5]
[6]
a) D. E. Michele, K. P. Campbell, J. Biol. Chem. 2003, 278, 15457-15460; b) M. D. Henry, K.
P. Campbell, Curr. Opin. Cell Biol. 1999, 11, 602-607.
[7]
[8]
a) C. T. Yuen, W. Chai, R. W. Loveless, A. M. Lawson, R. U. Margolis, T. Feizi, J. Biol.
Chem. 1997, 272, 8924-8931; b) N. R. Smalheiser, S. M. Haslam, M. Sutton-Smith, H. R.
Morris, A. Dell, J. Biol. Chem. 1998, 273, 23698-23703.
a) K. L. Abbott, K. Troupe, I. Lee, M. Pierce, Exp. Cell Res. 2006, 312, 2837-2850; b) I. Lee,
H.-B. Guo, M. Kamar, K. Abbott, K. Troupe, J.-K. Lee, G. Alvarez-Manilla, M. Pierce, J.
Neurochem. 2006, 97, 947-956.
[9]
K. Kanekiyo, K.-i. Inamori, S. Kitazume, K. Sato, J. Maeda, M. Higuchi, Y. Kizuka, H.
Korekane, I. Matsuo, K. Konke, N. Taniguchi, J. Neurosci. 2013, 33, 10037-10047.
T. Lange, S. Ullrich, I. Mueller, M. F. Nentwich, K. Stuebke, S. Feldhaus, C. Knies, O. J. C.
Hellwinkel, R. L. Vessella, C. Abramjuk, M. Anders, J. Schroeder-Schwarz, T. Schlomm, H.
Huland, G. Sauter, U. Schumacher, Clin. Cancer Res. 2012, 18, 1364-1373.
T. Yoshida-Moriguchi, L. Yu, S. H. Stalnaker, S. Davis, S. Kunz, M. Madson, M. B. A.
Oldstone, H. Schachter, L. Wells, K. P. Campbell, Science 2010, 327, 88-92.
a) K.-F. Mo, T. Fang, S. H. Stalnaker, P. S. Kirby, M. Liu, L. Wells, M. Pierce, D. H. Live,
G.-J. Boons, J. Am. Chem. Soc. 2011, 133, 14418-14430; b) M. Kanagawa, K. Kobayashi, M.
Tajiri, H. Manya, A. Kuga, Y. Yamaguchi, K. Akasaka-Manya, J. I. Furukawa, M. Mizuno, H.
Kawakami, Y. Shinohara, Y. Wada, T. Endo, T. Toda, Cell. Rep. 2016, 14, 2209-2223.
O. Ibraghimov-Beskrovnaya, J. M. Ervasti, C. J. Leveille, C. A. Slaughter, S. W. Sernett, K.
P. Campbell, Nature 1992, 355, 696-702.
[10]
[11]
[12]
[13]
[14]
[15]
D. Live, L. Wells, G.-J. Boons, ChemBioChem 2013, 14, 2392-2402.
W. Chai, C.-T. Yuen, H. Kogelberg, R. A. Carruthers, R. U. Margolis, T. Feizi, A. M.
Lawson, Eur. J. Biochem. 1999, 263, 879-888.
[16]
[17]
I. Breloy, S. Pacharra, C. Aust, F.-G. Hanisch, Biol. Chem. 2012, 393, 709-717.
a) C. Bleckmann, H. Geyer, A. Lieberoth, F. Splittstoesser, Y. Liu, T. Feizi, M. Schachner, R.
Kleene, V. Reinhold, R. Geyer, Biol. Chem. 2009, 390, 627-645; b) T. Martinez, D. Pace, L.
Brady, M. Gerhart, A. Balland, J. Chromatography A 2007, 1156, 183-187; c) K. Yahiro, A.
Wada, E. Yamasaki, M. Nakayama, Y. Nishi, J. Hisatsune, N. Morinaga, J. Sap, M. Noda, J.
Moss, T. Hirayama, J. Biol. Chem. 2004, 279, 51013-51021; d) D. R. Wing, T. Rademacher,
B. Schmitz, M. Schachner, R. A. Dweck, Biochem. Soc. Trans. 1992, 20, 386-390; e) S.
Pacharra, F.-G. Hanisch, I. Breloy, J. Proteome Res. 2012, 11, 3955-3964; f) S. Pacharra, F.-
G. Hanisch, M. Muehlenhoff, A. Faissner, U. Rauch, I. Breloy, J. Proteome Res. 2013, 12,
1764-1771; g) C. A. Dwyer, E. Baker, H. Hu, R. T. Matthews, Neuroscience 2012, 220, 47-
61; h) J. C. Trinidad, R. Schoepfer, A. L. Burlingame, K. F. Medzihradszky, Mol. Cell.
Proteomics 2013, 12, 3474-3488.
[18]
[19]
M. B. Vester-Christensen, A. Halim, H. J. Joshi, C. Steentoft, E. P. Bennett, S. B. Levery, S.
Y. Vakhrushev, H. Clausen, Proc. Natl. Acad. Sci. 2013, 110, 21018-21023.
a) P. R. Winterhalter, M. Lommel, T. Ruppert, S. Strahl, FEBS Letters 2013, 587, 3715-3721;
b) M. Lommel, P. R. Winterhalter, T. Willer, M. Dahlhoff, M. R. Schneider, M. F. Bartels, I.
Renner-Mueller, T. Ruppert, E. Wolf, S. Strahl, Proc. Natl. Acad. Sci. 2013, 110, 21024-
21029.
[20]
[21]
S. Carvalho, T. Oliveira, M. F. Bartels, E. Miyoshi, M. Pierce, N. Taniguchi, F. Carneiro, R.
Seruca, C. A. Reis, S. Strahl, S. S. Pinho, Oncotarget; Vol 7, No 40 2016.
M. F. Bartels, P. R. Winterhalter, J. Yu, Y. Liu, M. Lommel, F. Möhrlen, H. Hu, T. Feizi, U.
Westerlind, T. Ruppert, S. Strahl, PLoS One 2016, doi:10.1371/journal.pone.0166119.
J. Yu, U. Westerlind, ChemBioChem 2014, 15, 939-945.
a) M. B. Tessier, O. C. Grant, J. Heimburg-Molinaro, D. Smith, S. Jadey, A. M. Gulick, J.
Glushka, S. L. Deutscher, K. Rittenhouse-Olson, R. J. Woods, PLoS ONE 2013, 8, e54874; b)
O. C. Grant, H. M. K. Smith, D. Firsova, E. Fadda, R. J. Woods, Glycobiology 2014, 24, 17-
25.
[22]
[23]
12
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10.1002/chem.201605627
Chemistry - A European Journal
[24]
a) R. Sardzik, A. P. Green, N. Laurent, P. Both, C. Fontana, J. Voglmeir, M. J. Weissenborn,
R. Haddoub, P. Grassi, S. M. Haslam, G. Widmalm, S. L. Flitsch, J. Am. Chem. Soc. 2012,
134, 4521-4524; b) Y. Zhang, C. Meng, L. Jin, X. Chen, F. Wang, H. Cao, Chem. Commun.
2015, 51, 11654-11657; c) J. Seifert, T. Ogawa, Y. Ito, Tet. Lett. 1999, 40, 6803-6807; d) I.
Matsuo, M. Isomura, K. Ajisaka, Tet. Lett. 1999, 40, 5047-5050; e) J. Yu, M. Schorlemer, A.
Gomez Toledo, C. Pett, C. Sihlbom, G. Larson, U. Westerlind, J. Nilsson, Chem. Eur. J. 2016,
22, 1114-1124; f) K. Kawahira, H. Tanaka, A. Ueki, Y. Nakahara, H. Hojo, Y. Nakahara,
Tetrahedron 2009, 65, 8143-8153.
[25]
[26]
R. R. Schmidt, Angew. Chem. 1986, 98, 213-236.
a) U. Ellervik, G. Magnusson, Carbohyd. Res. 1996, 280, 251-260; b) Z. Zhang, I. R. Ollman,
X. S. Ye, R. Wischnat, T. Basov, C. H. Wong, J. Am. Chem. Soc. 1999, 121, 734-753.
R. R. Schmidt, M. Behrendt, A. Toepfer, Synlett 1990, 694-696.
R. D. Marwood, A. M. Riley, D. J. Jenkins, B. V. L. Potter, J. Chem. Soc., Perkin Trans. 1
2000, 1935-1947.
[27]
[28]
[29]
[30]
[31]
a) H. Kunz, in Preparative Carbohydrate Chemistry (ED.: S. Hanessian), Marcel Dekker,
New York, 1997, 265; b) A. Paquet, Can. J. Chem. 1982, 60, 976.
a) P. Konradsson, U. E. Udodong, B. Fraser-Reid, Tet. Lett. 1990, 31, 4313-4316; b) G. H.
Veeneman, J. H. van Boom, Tet. Lett. 1990, 31, 275-278.
a) L. Carpino, A. El-Faham, C. A. Minor, F. Albericio, J. Chem. Soc., Chem. Commun. 1994,
201-203; b) L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397-4398; c) V. Dourtoglou, B.
Gross, V. Lambropoulou, C. Zioudrou, Synthesis 1984, 572-574.
[32]
[33]
a) U. Westerlind, H. Schroder, A. Hobel, N. Gaidzik, A. Kaiser, M. Niemeyer Christof, E.
Schmitt, H. Waldmann, H. Kunz, Angew. Chem. Int. Ed. 2009, 48, 8263-8267; b) S. Keil, C.
Claus, W. Dippold, H. Kunz, Angew. Chem., Int. Ed. 2001, 40, 366-369.
M. Trester-Zedlitz, K. Kamada, S. K. Burley, D. Fenyö, B. T. Chait, T. W. Muir, J. Am.
Chem. Soc. 2003, 125, 2416-2425.
[34]
[35]
D. N. Moothoo, J. H. Naismith, Glycobiology 1998, 8, 173-181.
O. C. Grant, X. Xue, D. Ra, A. Khatamian, B. L. Foley, R. J. Woods, Glycobiology 2016, 26,
1027-1028.
[36]
[37]
O. C. Grant, M. B. Tessier, L. Meche, L. K. Mahal, B. L. Foley, R. J. Woods, Glycobiology
2016, 26, 772-783.
D. F. Thieker, J. A. Hadden, K. Schulten, R. J. Woods, Glycobiology 2016, 26, 786-787.
Schemes, Figures and Tables:
Figure 1. Disconnection approach for synthesis of core m2 branched O-mannosyl glycoconjugates,
glycopeptides and amino acid building blocks.
13
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10.1002/chem.201605627
Chemistry - A European Journal
Donor:
OH
O
OPMB
O
OPMB
O
a)
b)
HO
PMBO
PMBO
PMBO
PMBO
SPh
HO
SPh
SPh
N3
1 (64%)
N3
2 (63%)
NHTroc
3 (94%)
Acceptor:
OMe
RO
OR
O
OMe
OMe
AcO
OMe
O
OH
OAc
OAc
O
HO
AcO
c)
e)
f)
O
O
O
O
O
O
O
O
O
OMe
O
OMe
4
O
OMe
OR
OMe
7 (78%)
O
CCl3
OtBu
5 (93%) R=All
6 (92%) R=H
FmocHN
9 (84%) R=Ac
d)
NH
+
O
g
+
10 (78%) R=H
Core m2
amino acid:
PMBO
AcO
O
O
TrocHN
O
PMBO
AcO
O
AcO
AcO
PMBO
AcO
O
O
RHN
AcO
AcO
AcO
PMBO
PMBO
PMBO
O
O
AcO
O
O
AcO
AcO
AcO
AcHN
O
OMe
OMe
TrocHN
RHN
O
HO
HO
h)
i)
k)
O
O
O
3 + 10
O
O
AcHN
O
O
OMe
OMe
+
O
O
O
OtBu
OH
OtBu
FmocHN
11 (90%)
FmocHN
12 (91%) R=Troc
FmocHN
14 (62%)
O
O
O
j)
+
13 (85%) R=Ac
Scheme 1. Synthesis of the trisaccharide core m2 building block 14: a) NaH, 4-methoxybenzyl chloride, DMF,
r.t. 2 h; b) Zn, 1,4-dioxane/AcOH 10:1, r.t. 24 h; NaHCO3, 1,4-dioxane/H2O 2:1, TrocCl, r.t. 24 h; c) Pyr/Ac2O,
r.t. 24 h; d) Wilkinson’s catalyst, Tol/EtOAc/H2O 20:10:1, reflux 24 h; I2, THF/H2O 4:1, r.t. 2 h; e)
trichloroacetonitrile, DBU, DCM, 0 °C 3 h; f) Fmoc-Thr acceptor 5, TMSOTf, molecular sieves, Et2O, r.t. 30
min; g) NaOMe in MeOH, pH 9.5, r.t. 24 h; NaHCO3, 1,4-dioxane/H2O 1:1, Fmoc-OSu, r.t. 2 h; h) NIS, TfOH,
DCM, -50 °C 4 h; i) CAN, MeCN/H2O 9:1, r.t. 30 min; Pyr/Ac2O, r.t. 24 h; j) Zn, AcOH, r.t. 4 days; Pyr/Ac2O,
r.t. 24 h; k) TFA/DCM 3:1, r.t. 24 h.
14
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10.1002/chem.201605627
Chemistry - A European Journal
a)
SPPS
Fmoc
N
a) Fmoc-cleavage:
Piperidine (20%) in DMF,
b) Amino acid-coupling:
=
H
*
O
Fmoc-AA-OH, HBTU, HOBt, DIPEA/DMF,
or Glycosylated Fmoc-AA-OH, HATU, HOAt,
DIPEA/DMF
HO
HO
HO
O
HO
O
O
HO
O
HO
AcHN
O
HO
HO
HO
O
HO
AcHN
Resin-cleavage:
HO
HO
TFA/TIPS/H2O (15:0.9:0.9), 2.5 h
O
AcHN
O
HO
HO
Global deacetylation:
NaOMe, MeOH, pH 9.5, 20 h
= TentaGel resin trityl- Fmoc-
Gly\Ala\Arg(Pbf)\Asp(tBu)
GlcNAcβ1-2[GlcNAcβ1-6]Manα
GlcNAcβ1-2Manα
16: GT*T*G-Spacer-NH2, * = GlcNAcβ1-2Manα (28%)
17: GT*T*G-Spacer-NH2, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (3.6%)
18: GT*T*G-Spacer-NH2, * = H (24%)
antigen peptides
19: GT*T*A-Spacer-NH2, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (18%)
20: NH2-Spacer-YAT*AVA, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (67%)
21: NH2-Spacer-RGAIIQT*PTLA, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (52%)
22: NH2-Spacer-PVPGKPT*VTIR, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (21%)
23: NH2-Spacer-SQSLEET*ISPR, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (88%)
24: NH2-Spacer-SGPLDGGT*LLTIR, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (35%)
25: NH2-Spacer-NAPSGT*TVIHLNA, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (44%)
26: NH2-Spacer-NAPSGT*T*VIHLNA, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (52%)
27: NH2-Spacer-EPGGSYIT*T*VSATD, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (50%)
28: NH2-Spacer-QGPQAGGT*T*LTIHA, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα (46%)
other antigen structures
α-Dystroglycan
UPF0606 protein KIAA1549
Plexin-D1
Protocadherin Fat 4
Plexin-B2
b)
17: GT*T*G-Spacer-NH2, * = GlcNAcβ1-2[GlcNAcβ1-6]Manα
O
O
EtOH, H2O 1:1, 2h
EtO
OEt
50 mM Na2HPO4, pH 9.3, 72h
NH2
NH2 or CRM
BSA
O
O
30: BSA conjugate, n = 3
31: CRM197conjugate, n = 3
GT*T*G-Spacer-HN
N
H
n
*
Scheme 2. Synthesis of glycopeptides 16-28, BSA-conjugate 30 and CRM-conjugate 31.
15
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10.1002/chem.201605627
Chemistry - A European Journal
Figure 2. Microarray analysis and evaluation of binding to spotted peptides 16-28 and 32-57 with a) the core m2
polyclonal rabbit sera 26559 and 26560 and b) Biotin-ConA. The x-axis shows the microarray peptide spotting
ID numbers (details in Table 1) and y-axis show the relative fluorescence, readout by a) an Cy5 secondary anti-
rabbit antibody or b) Cy5-streptavidin.
Table 1. List of O-mannosyl peptides spotted on the microarray slides.
Microarray Compound
peptide Nr
Origin
Sequence
Glycan
Nr
32
33
34
35
36
37
38
39
40
41
42
43
44
45
22
21
20
23
24
25
26
28
27
46
47
48
49
50
51
52
53
54
18
55
55
57
16
17
19
1
2
3
4
5
6
7
8
α-Dystroglycan
α-Dystroglycan
antigen sequence
antigen sequence
α-Dystroglycan
SpPVPGKPT*VTIR
SpRGAIIQT*PTLG
SpGT*G
Galβ1-4GlcNAcβ1-2Manα
SpYAT*AVA
SpPVPGKPT*VTIR
SpRGAIIQT*PTLG
SpGT*G
α-Dystroglycan
antigen sequence
antigen sequence
UPF0606 protein KIAA1549
Plexin-D1
Protocadherin Fat 4
Protocadherin Fat 4
Plexin-B2
Protocadherin Fat 4
α-Dystroglycan
α-Dystroglycan
antigen sequence
UPF0606 protein KIAA1549
Plexin-D1
Protocadherin Fat 4
Protocadherin Fat 4
Plexin-B2
Protocadherin Fat 4
antigen sequence
α-Dystroglycan
α-Dystroglycan
UPF0606 protein KIAA1549
Plexin-D1
Protocadherin Fat 4
Protocadherin Fat 4
Plexin-B2
Protocadherin Fat 4
antigen sequence
antigen sequence
antigen sequence
antigen sequence
antigen sequence
antigen sequence
antigen sequence
SpYAT*AVA
GlcNAcβ1-2Manα
9
SpSQSLEET*ISPR
SpSGPLDGGT*LLTIR
SpNAPSGT*TVIHLNA
SpNAPSGT*T*VIHLNA
SpQGPQAGGT*T*LTIHG GlcNAcβ1-2Manα x2
SpEPGGSYIT*T*VSATD
SpPVPGKPT*VTIR
SpRGAIIQT*PTLG
SpYAT*AVA
SpSQSLEET*ISPR
SpSGPLDGGT*LLTIR
SpNAPSGT*TVIHLNA
SpNAPSGT*T*VIHLNA
SpQGPQAGGT*T*LTIHG
SpEPGGSYIT*T*VSATD
YAT*AV
SpPVPGKPT*VTIR
SpRGAIIQT*PTLG
SpSQSLEET*ISPR
SpSGPLDGGT*LLTIR
SpNAPSGT*TVIHLNA
SpNAPSGT*T*VIHLNA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
GlcNAcβ1-2[GlcNAcβ1-6]
Manα
GlcNAcβ1-2[GlcNAcβ1-6]
Manα x2
Manα
SpQGPQAGGT*T*LTIHG Manα x2
SpEPGGSYIT*T*VSATD
GTTGSp
SpGTG
SpYATAVA
non-glycan
YATAV
GT*T*GSp
GT*T*GSp
GT*T*ASp
GlcNAcβ1-2Manα
GlcNAcβ1-2[GlcNAcβ1-6]
Manα
16
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10.1002/chem.201605627
Chemistry - A European Journal
Figure 3. Structure-based rationalization of the observed binding specificity of ConA towards the arrayed
glycans; a) only the weaker 3-arm binding site tolerates linear core m1 trisaccharides and branched glycans,
which do not display affinity for ConA on the array; b) the Core Man does not tolerate substitution at the 2-
position of the bound Man; c) the 6-arm binding site does not tolerate substitution at the 4-position of the bound
GlcNAc, nor at the 6-position of the Man, which is consistent with the binding specificity of ConA amongst the
arrayed glycans; d) the co-complex of GlcNAcβ1-2Manα1-3[GlcNAcβ1-2Manα1-6]Manα with ConA from
PDBID 1TEI[34] shows the relative position of the 3-arm, 6-arm and monosaccharide binding sites. Glycans
represented in 3D-SNFG icon mode[37], the protein surface is in light grey, red arrows indicate the position of the
neighboring binding site(s).
Text table of contents:
Glycan Detection: Antibodies were generated for detection of core M2 O-mannosyl glycans, a class of protein modifications of importance
in tumor progression and brain biology. The binding specificities of the induced antibodies were evaluated on glycopeptide microarrays and
the binding recognition was compared with the lectin ConA. The observed binding data was further rationalized by molecular modelling.
The obtained antibodies were highly selective for core M2 modified glycopeptides and contrasted the binding specificity of ConA.
Key words: Carbohydrates • Glycosylation • Glycopeptides • Vaccines • Antibodies • Lectins
17
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