title: Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers.
abstract: HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCEA protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.
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 Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Haitao
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Cancer Immunology & Virology, 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 Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Rajesh P
authors.initials: RP
authors.lastname: Ringe
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Nicely
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
authors.forename: David
authors.initials: D
authors.lastname: Hua
authors.affiliation: Department of Cancer Immunology & Virology, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Steinbock
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Golabek
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: James
authors.initials: J
authors.lastname: Alin
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: S Munir
authors.initials: SM
authors.lastname: Alam
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Cupo
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Barton F
authors.initials: BF
authors.lastname: Haynes
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 Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Moore
authors.affiliation: Department of Cancer Immunology & Virology, Dana-Farber Cancer Institute, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Joseph G
authors.initials: JG
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: D016915:AIDS Vaccines / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D019985:Benchmarking; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D015483:HIV Antibodies / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000276:immunology*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000276:immunology*; D015497:HIV-1 / Q000737:chemistry; D057131:Immune Evasion / Q000276:immunology; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000276:immunology*
chemical_list: D016915:AIDS Vaccines; D057134:Antibodies, Neutralizing; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1
keywords: Env; glycopeptides; human immunodeficiency virus