Synthesis of a Glycopeptide Vaccine Conjugate for Induction of Antibodies Recognizing O‐Mannosyl Glycopeptides

Abstract

In spite of the clear importance of protein O-mannosylation in brain glycobiology, tools are lacking for specific detection, enrichment, and identification of proteins containing these modifycations. We envisioned inducing antibodies that specifically recognize O-mannose glycans on proteins and peptides. With this in mind, we prepared a glycopeptide vaccine construct containing the N-acetyllactosamine-extended mannose motif Galβ1-4GlcNAcβ1-2ManαThr, found as a common core structure on almost all mammalian O-mannosyl glycoproteins identified. O-mannose glycosylated amino acid building blocks and the corresponding glycopeptides were prepared by chemical synthesis and then conjugated to an immune carrier protein. After administration of the synthetic vaccine into rabbits, strong immune responses were obtained. Further evaluation by ELISA neutralization experiments and glycopeptide microarrays showed that the induced antibodies were highly specific to the glycopeptide antigen.

Human chitotriosidase CHIT1 cross reacts with mammalian‐like substrates

“…0 LacNAc, LacNAcb1-6Galb1-3-GalNAc and LacNAcb1-2-Man-(sp)(Gly)Thr(Gly) monitored by UPLC-MS For derivatization, oligosaccharides were dissolved in PBS (pH8.5) at 50 lM (LacNacb1-6 0 LacNAc and extended core 2 [13,14] or 100 lM (LacNAc-Man, [15] Fig. 2).…”

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

“…[12a,21,22,24] The first synthesis of core m2 structures is described here. A retrosynthetic 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 CRM 197 or for glycopeptide immobilization on microarray slides (Figure 1).…”

“…Insertion of al inker at the C-terminal instead of the N-terminal avoids immunogenic response to aC -terminal amide or free carboxylic acid in close proximity to the glycosylation sites. After ad esalting step on C-18 cartridges, the O-acetyl groups on all generated glycopeptides (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) were removed by treatment with catalytic amountso fN aOMe in methanola t pH 9.5. Deprotected glycopeptides 16-28 were finally purified by preparative HPLC.…”

“…A previous study generated antibodies that were directed against a linear core m1 Galβ1,4GlcNAcβ1,2-Manα-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.…”

Facile Chemoenzymatic Synthesis of O‐Mannosyl Glycans

“…Most recently, Cao and coworkers prepared six core M1 structures ( M100 , M101 , M102 , M104 , M105 , and M102G ) by employing a chemoenzymatic approach. [7a] Glycopeptides harboring basic O-mannosyl structures (e.g., M101 , [8] M102 , [9] core M0, [7e] M000 , [10] or core M3 [11] ) have also been generated. Nevertheless, the majority of O-mannosyl glycans (especially core M2 branched structures) are still not accessible, which has hampered our in-depth understanding of O mannosylation.…”