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

• Published in: Nature communications Vol. 8; no. 1; pp. 14954 - 13
• Main Authors: Cao, Liwei, Diedrich, Jolene K., Kulp, Daniel W., Pauthner, Matthias, He, Lin, Park, Sung-Kyu Robin, Sok, Devin, Su, Ching Yao, Delahunty, Claire M., Menis, Sergey, Andrabi, Raiees, Guenaga, Javier, Georgeson, Erik, Kubitz, Michael, Adachi, Yumiko, Burton, Dennis R., Schief, William R., Yates III, John R., Paulson, James C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.03.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/296; 631/326/596; 631/45/1268; 692/699/255/1901; 82; 82/58; 82/81; Antibodies; Chromatography; Glycoproteins; Humanities and Social Sciences; Mass spectrometry; multidisciplinary; Peptides; Proteomics; Research centers; Science; Science (multidisciplinary); Scientific imaging; Vaccines; Viruses; La Jolla California; United States > US; California
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms14954

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. The analysis of site-specific glycosylation of HIV Envelope glycoprotein (Env) is challenging as it contains 25–30 glycosylation sites with multiple glycan forms at each site. Here the authors present a generally applicable mass spectrometry-based method for site-specific analysis of protein glycosylation that they apply to the analysis of the HIV-1 Env.