N-glycosylation Type and Structure Analysis of HIV-1 Trimer 4571 and HIV-1 Broadly Neutralizing Antibodies (bNAbs) by Tandem Exoglycosidase Digestion

Authors

• Y Yang
• F Arnold
• J Cooper
• P Lei

Abstract

Glycosylation is one of the most common and complex post translational modification of eukaryotic protein. Oligosaccharides in glycoproteins influence many aspects of protein function such as half-life, potency, immunogenicity, efficacy etc. Therefore, characterization and monitoring of these carbohydrates is necessary for therapeutic and preventative glycoproteins. Oligosaccharide structural characterization is much more challenging than that of the other post-translational modifications due to its highly heterogeneous branched and isomeric nature. Mass spectrometry-based (MS) techniques are powerful tools and have been used successfully for the characterization of glycans, but even with the extensive application of tandem MS techniques, it is still extremely difficult from MS data alone to establish anomerity and branching configuration forms of complex oligosaccharides. On the other hand, application of series of exoglycosidases with high specificities can provide unambiguous oligosaccharide sequence. Each exoglycosidase selectively releases a specific type of monosaccharide from the nonreducing terminus based on its anomeric configuration and linkage to the remainder of the glycan chain. Tandem exoglycosidase digestion has been therefore considered as an orthogonal technique of mass spectrometry to characterize oligosaccharide structure. In this poster, N-linked oligosaccharides of a HIV-1-envelope (Env) protein vaccine candidate HIV Trimer 4571 and five HIV-1 broadly neutralizing antibodies (10E8VLS, CAP256V1LS, N6LS, VRC01 and VRC07-523) have been characterized by complementary of endoglycosydase deglycosylation followed by exoglycosidase sequencing for structural information of the monosaccharides sequence and the linkage type. From the data of nine panels of exoglycosidase treatment with the known specificity of the exoglycosidases, the oligosaccharide chain type and sequence were deduced.

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