Global N-Glycan Site Occupancy of HIV-1 gp120 by Metabolic Engineering and High-Resolution Intact Mass Spectrometry

Struwe, Weston B.; Stuckmann, Alexandra; Behrens, Anna‐Janina; Pagel, Kevin; Crispin, Max

doi:10.1021/acschembio.6b00854

Cited by 35 publications

(42 citation statements)

References 38 publications

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“…Glycans with a dramatic shift to complex-type included N88, N160, N276 and N411 (Figs 1d and 4a,d). Overall, the site occupancy of BG505 monomer gp120 was 86%, similar to that reported previously74. The results show that the glycosylation pattern of monomeric gp120 is dramatically different from that of the native-like Env trimer, which can be attributed in part to greater accessibility of glycans at the subunit interface by the glycosylation machinery2526.…”

Section: Resultssupporting

confidence: 87%

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

et al. 2017

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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.

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Section: Resultssupporting

confidence: 87%

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

et al. 2017

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“…2D and Supplementary Fig. 3E), consistent with recent observations of high occupancy for gp120 expressed with kifunensine (22). For Env expressed without kifunensine we recorded a lower mass of 315.3 ± 10.5 kDa.…”

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confidence: 91%

Quantitative mass imaging of single molecules in solution

Young

Hundt

Cole

et al. 2017

Preprint

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Abstract:The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo-and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

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“…However, the more appropriate methodology includes carrying out the reaction using heavy-oxygen water (O 18 -H 2 O), which discriminates deglycosylated sites from spontaneous Asn deamidation (Palmisano et al 2012;Cao et al 2017). It is also possible to evaluate the overall distribution of N-glycosite occupancy on intact glycoproteins by metabolically simplifying the glycans to homogeneous structures (Struwe et al 2017). For O-linked glycans, complete O-glycan removal does not result in chemical peptide modification and cannot be used for identification of glycosylated amino acid positions (Levery et al 2015).…”

Section: Glycoproteomics Of Virusesmentioning

confidence: 99%

Global aspects of viral glycosylation

2018

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Enveloped viruses encompass some of the most common human pathogens causing infections of different severity, ranging from no or very few symptoms to lethal disease as seen with the viral hemorrhagic fevers. All enveloped viruses possess an envelope membrane derived from the host cell, modified with often heavily glycosylated virally encoded glycoproteins important for infectivity, viral particle formation and immune evasion. While N-linked glycosylation of viral envelope proteins is well characterized with respect to location, structure and site occupancy, information on mucin-type O-glycosylation of these proteins is less comprehensive. Studies on viral glycosylation are often limited to analysis of recombinant proteins that in most cases are produced in cell lines with a glycosylation capacity different from the capacity of the host cells. The glycosylation pattern of the produced recombinant glycoproteins might therefore be different from the pattern on native viral proteins. In this review, we provide a historical perspective on analysis of viral glycosylation, and summarize known roles of glycans in the biology of enveloped human viruses. In addition, we describe how to overcome the analytical limitations by using a global approach based on mass spectrometry to identify viral O-glycosylation in virus-infected cell lysates using the complex enveloped virus herpes simplex virus type 1 as a model. We underscore that glycans often pay important contributions to overall protein structure, function and immune recognition, and that glycans represent a crucial determinant for vaccine design. High throughput analysis of glycosylation on relevant glycoprotein formulations, as well as data compilation and sharing is therefore important to identify consensus glycosylation patterns for translational applications.

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Global N-Glycan Site Occupancy of HIV-1 gp120 by Metabolic Engineering and High-Resolution Intact Mass Spectrometry

Cited by 35 publications

References 38 publications

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

Quantitative mass imaging of single molecules in solution

Global aspects of viral glycosylation

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