title: Global N-Glycan Site Occupancy of HIV-1 gp120 by Metabolic Engineering and High-Resolution Intact Mass Spectrometry.
abstract: A vital step in HIV vaccine development strategies has been the observation that some infected individuals generate broadly neutralizing antibodies that target the glycans on the surface of HIV-1 gp120. These antibodies target glycan epitopes on viral envelope spikes, and yet the positions and degree of occupancy of glycosylation sites is diverse. Therefore, there is a need to understand glycosylation occupancy on recombinant immunogens. The sheer number of potential glycosylation sites and degree of chemical heterogeneity impedes assessing the global sequon occupancy of gp120 glycoforms. Here, we trap the glycan processing of recombinant gp120 to generate homogeneous glycoforms, facilitating occupancy assessment by intact mass spectrometry. We show that gp120 monomers of the BG505 strain contain either fully occupied sequons or missing the equivalent of one and sometimes two glycans across the molecule. This biosynthetic engineering approach enables the analysis of therapeutically important glycoproteins otherwise recalcitrant to analysis by native mass spectrometry.
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford , Oxford, OX1 3QU, United Kingdom.
authors.forename: Weston B
authors.identifier: 0000-0003-0594-226X
authors.initials: WB
authors.lastname: Struwe
authors.affiliation: Free University Berlin , Department of Chemistry and Biochemistry, Takustrasse 3, 14195 Berlin, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Stuckmann
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford , Oxford, OX1 3QU, United Kingdom.
authors.forename: Anna-Janina
authors.initials: AJ
authors.lastname: Behrens
authors.affiliation: Free University Berlin , Department of Chemistry and Biochemistry, Takustrasse 3, 14195 Berlin, Germany.
authors.forename: Kevin
authors.initials: K
authors.lastname: Pagel
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford , Oxford, OX1 3QU, United Kingdom.|Department of Immunology and Microbial Science, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Max
authors.initials: M
authors.lastname: Crispin
mesh_terms: D001665:Binding Sites; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D060847:Metabolic Engineering; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D015699:HIV Envelope Protein gp120; D011134:Polysaccharides