Global site-specific N-glycosylation analysis of HIV envelope glycoprotein
Liwei Cao, Jolene K. Diedrich, Daniel W. Kulp, Matthias Pauthner, Lin He, Sung-Kyu Robin Park, Devin Sok, Ching Yao Su, Claire M. Delahunty, Sergey Menis, Raiees Andrabi, Javier Guenaga, Erik Georgeson, Michael Kubitz, Yumiko Adachi, Dennis R. Burton, William R. Schief, John R. Yates III & James C. Paulson
Nature Communications 8, Article number: 14954 (2017)
Glycomics HIV infections Mass spectrometry Virology
Received: 12 October 2016 Accepted: 15 February 2017 Published online: 28 March 2017
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.
We thank Dr Ian A. Wilson and Dr Peter S. Lee for providing recombinant influenza virus haemagglutinin. This work was supported by NIH R01AI113867 (J.C.P., J.Y., W.R.S.); NIH UM1 AI100663 (D.R.B.); the International AIDS Vaccine Initiative (W.R.S., D.R.B.); and NIH P41 GM103533 (J.Y.).
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
Liwei Cao & James C. Paulson
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA
Jolene K. Diedrich, Lin He, Sung-Kyu Robin Park, Claire M. Delahunty, John R. Yates III & James C. Paulson
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA
Daniel W. Kulp, Matthias Pauthner, Devin Sok, Ching Yao Su, Raiees Andrabi, Javier Guenaga, Erik Georgeson, Michael Kubitz, Dennis R. Burton, William R. Schief & James C. Paulson
Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA
Daniel W. Kulp, Matthias Pauthner, Devin Sok, Sergey Menis, Yumiko Adachi, Dennis R. Burton & William R. Schief
L.C., J.R.Y. and J.C.P. designed the research. L.C. prepared samples for MS analysis. J.K.D., L.C. and C.M.D. performed the MS analysis. L.C., L.H. and S.R.P. analysed the data. D.W.K., M.M., D.S., C.Y.S., S.M., R.A., J.G., E.G., M.K. and Y.A. expressed and purified Env proteins. D.R.B., W.R.S., J.R.Y. and J.C.P. supervised the project. L.C. and J.C.P. wrote the manuscript.
The authors declare no competing financial interests.
Correspondence to James C. Paulson.