Dataset Information

Impact of glycan positioning on HIV-1 Env glycan shield density, function, and antibody recognition

ABSTRACT: N-glycans, which represent >50% mass of the HIV-1 envelope (Env) trimer, play important roles for virus-cell entry and immune evasion. How each glycan unit interacts to shape the Env protein-sugar complex and affects Env function is not well understood. Here, high-resolution glycomics analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation-site not only affected the Env structure at distant locations, but also had a ripple effect on Env-wide glycan processing, virus infectivity, and antibody recognition and virus neutralization. Specifically, the N262 glycan, although not located in the CD4-binding site, controlled Env binding to the CD4 receptor, affected the recognition of Env by several glycan-dependent broadly neutralizing antibodies, and altered heterogeneity of glycosylation at several sites, with N156, N160, and N448 displaying limited glycan processing. Molecular dynamic simulations visualized how specific oligosaccharide positions can move to compensate for loss of a glycan. This study demonstrates how changes in individual glycan units can alter molecular dynamics and processing of the Env-glycan shield and, consequently, Env function.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Hiv-1 M:g_pt919

DISEASE(S): Human Immunodeficiency Virus Infectious Disease

SUBMITTER: Matthew Renfrow

LAB HEAD: Matthew Renfrow

PROVIDER: PXD017941 | Pride | 2021-09-09

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	2020-03-07WeiHargettgp120N-glycosylaitonlabelfreedata.xlsx	Xlsx
	ah_20140509_ORBI_QW6_gp120_3M_CID.raw	Raw
	ah_20140509_ORBI_QW6_gp120_3M_ETD.raw	Raw
	ah_20140509_ORBI_QW6_gp120_3M_HCD.raw	Raw
	ah_20140509_ORBI_QW6_gp120_3_CID.raw	Raw

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Publications

Glycan Positioning Impacts HIV-1 Env Glycan-Shield Density, Function, and Recognition by Antibodies.

Wei Qing Q Hargett Audra A AA Knoppova Barbora B Duverger Alexandra A Rawi Reda R Shen Chen-Hsiang CH Farney S Katie SK Hall Stacy S Brown Rhubell R Keele Brandon F BF Heath Sonya L SL Saag Michael S MS Kutsch Olaf O Chuang Gwo-Yu GY Kwong Peter D PD Moldoveanu Zina Z Raska Milan M Renfrow Matthew B MB Novak Jan J

iScience 20201021 11

HIV-1 envelope (Env) N-glycosylation impact virus-cell entry and immune evasion. How each glycan interacts to shape the Env-protein-sugar complex and affects Env function is not well understood. Here, analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation sites affected the Env structure at distant locations and had a ripple effect on Env-wide glycan processing, virus infectivi ...[more]

PMID: 33205023

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Project description:Recombinant proteins are of great interest in glycobiology and proteomics, known especially for their reproducibility and accessibility. However, variation in glycosylation among recombinant glycoproteins is not well understood and may depend on numerous conditions in the biomanufacturing process. In order to confidently assess variation in glycosylation measurements, it is vital to both optimize the measurement of, and determine the degree of variation between, distributions of glycosylation on specific sites of glycoproteins. This is especially important for glycoproteins that are known to have rapid sequence changes, such as with different influenza strains. In this study, eight strains of recombinant influenza hemagglutinin and neuraminidase produced from HEK293 cell line were obtained from four vendors and digestion was conducted using a series of complex multi-enzymatic methods designed to isolate glycopeptide sequons. Site-specific glycosylation profiles of intact glycopeptides were produced using mass spectrometric evaluation on an orbitrap system and compared using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 1000), whereas replicates resulted in low variation (similarity score = 957 out of 1000). Glycan variation was also measured based on differences between vendors, lots, batches, protease digestion, and intra-protein site. The most abundant glycans in all of these influenza glycoproteins were monofucosylated and complex, as reported by other laboratories. However, it was found that different vendors can produce very different glycan distributions for the same glycosylation site. Notably, it is demonstrated that glycan distributions are similar for conserved regions of influenza glycoproteins. Overall, these methods present a potential use in developing reproducible measurements of glycosylated biologics for quality control or making more informed decisions in biomanufacturing.