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Nat Commun. 2017 Mar 28;8:14954. doi: 10.1038/ncomms14954.

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein.

Author information

1
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
2
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
3
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
4
Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.

Abstract

HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.

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