High-mannose (HM) type N-glycans, ranging from Man5GlcNAc2 to Man9GlcNAc2, are a major glycan component of HIV-1 envelope glycoprotein gp120. They play an active role in dendritic cell-mediated viral transmission by serving as the ligand for the dendritic cell-associated lectin (DC-SIGN). These high-mannose N-glycans are also targets for several broadly neutralizing antibodies including 2G12, PG9, PG16, and PGT128. Thus facile preparation of a library of structurally well-defined high-mannose N-glycans will facilitate functional evaluation and vaccine design. While pure chemical synthesis provides a way to make high-mannose N-glycans, it usually takes multiple tedious steps. We report here a top-down approach that consists of preparation of the full-size HM N-glycan, Man9GlcNAc2Asn, and its successive enzymatic degradation and chromatographic separation of the resulting distinct HM glycans. The starting material, Man9GlcNAc2Asn, was successfully obtained from soybean flour through isolation of crude soybean agglutinin (SBA) and thorough pronase digestion. It was then labeled at the free asparagine group with fluorenylmethyloxycarbonyl (Fmoc) group. Controlled digestion ofthe Fmoc-labeled Man9GlcNAc2Asn with an a-1, 2-mannosidase (cloned from bacteroides thetaiotaomicron) yielded a mixture of Man9-Man5GlcNAc2Asn-Fmoc. The distinct HM-glycoforms were successfully separated by normal phase high-performance liquid chromatography (NP-HPLC) and characterized by MALDI-TOF MS and HPAEC-PAD (high performance anion-exchange chromatography with pulsed amperometric detection) methods. This approach provides a facile means to obtain distinct high-mannose type oligosaccharides and isomers with sufficient quantities for various applications, including the synthesis of HIV glycopeptide antigens for epitope characterization and the preparation of HM-glycan microarrays for functional glycomics studies.

Chemoenzymatic synthesis of a high-mannose type N-glycan library for functional studies

247th National Meeting (2014)

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