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

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 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.

Keywords: antibodies; carbohydrates; glycopeptides; lectins; vaccines.

Figure 1

Disconnection approach for synthesis of core m2 branched O-mannosyl glycoconjugates, glycopeptides, and amino acid building blocks.

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 shows the relative fluorescence, readout by a) a Cy5 secondary anti-rabbit antibody or b) Cy5-streptavidin.

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 PDB ID 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).

Scheme 1

Synthesis of the trisaccharide core m2 building block 14: a) NaH, 4-methoxybenzyl chloride, DMF, RT 2 h; b) Zn, 1,4-dioxane/AcOH 10:1, RT 24 h; NaHCO₃, 1,4-dioxane/H₂O 2:1, TrocCl, RT 24 h; c) Pyridine/Ac₂O, RT.24 h; d) Wilkinson’s catalyst, Tol/EtOAc/H₂O 20:10:1, reflux 24 h; I₂, THF/H₂O 4:1, RT 2 h; e) trichloroacetonitrile, DBU, dichloromethane, 0°C 3 h; f) Fmoc-Thr acceptor 5, TMSOTf, molecular sieves, Et₂O, RT 30 min; g) NaOMe in MeOH, pH 9.5, RT 24 h; NaHCO₃, 1,4-dioxane/H₂O 1:1, Fmoc-OSu, RT 2 h; h) NIS, TfOH, dichloromethane, −50°C 4 h; i) CAN, MeCN/H₂O 9:1, RT 30 min;Pyridine/Ac₂O, RT 24 h; j) Zn, AcOH, RT 4 days; Pyridine/Ac₂O, RT 24 h; k) TFA/dichloromethane 3:1, RT 24 h.

Scheme 2

Synthesis of glycopeptides 16–28, BSA-conjugate 30 and CRM-conjugate 31.