title: Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140.
abstract: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140.
  
  
  OBJECTIVE
  A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
authors.forename: Eden P
authors.initials: EP
authors.lastname: Go
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Alon
authors.initials: A
authors.lastname: Herschhorn
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gu
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Castillo-Menendez
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Shijian
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Youdong
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Haitao
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Wakefield
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
authors.forename: David
authors.initials: D
authors.lastname: Hua
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Hua-Xin
authors.initials: HX
authors.lastname: Liao
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, Alabama, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Kappes
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Sodroski
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA hdesaire@ku.edu.
authors.forename: Heather
authors.initials: H
authors.lastname: Desaire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D015699:HIV Envelope Protein gp120; D054299:env Gene Products, Human Immunodeficiency Virus; C514793:gp120 protein, Human immunodeficiency virus 1; C097729:gp140 envelope protein, Human immunodeficiency virus 1