Glycosylation Site-Specific Analysis of HIV Envelope Proteins (JR-FL and CON-S) Reveals Major Differences in Glycosylation Site Occupancy, Glycoform Profiles, and Antigenic Epitopesʼ Accessibility

Abstract

The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others. Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env's glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

“…Additionally, we explored whether deglycosylated gp120 allows selection of gp120-specific DARPins at higher frequency (Fig. 1B, selection IV), as sites vulnerable to neutralizing antibody attack are known to be efficiently shielded by the Env protein's heavy glycosylation (9)(10)(11).…”

“…The complex quaternary structure of the trimeric envelope spike, however, efficiently shields functionally important domains. Conserved domains are positioned facing inward in the trimeric complex, only to be exposed transiently upon receptor engagement (6)(7)(8), while the outer trimer surface is protected by flexible, variable loops and extensive glycosylation (7,(9)(10)(11). These strategies act in concert to shield the envelope complex from immune recognition and inhibitor attack (8,12).…”

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

“…There are between 18 and 33 (median, 25) canonical N-linked carbohydrate attachment sites (NX(S/T)) on gp120 proteins from different isolates (1,2). Although most of these sites are utilized, the glycosylation can be both isolatedependent and influenced by the producer cell type (3,4). The number of glycan sites present on Env, and the extent to which they are glycosylated, tend to increase during the course of HIV-1 infection, reflecting antibody-mediated selection pressures that drive resistance to autologous NAbs (5).…”

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis ofN-Linked Glycans

“…Their masses and deduced compositions are listed in Table 1 (columns 5 and 9-12). Structures have been determined earlier [58,[66][67][68] and those of the major neutral glycans are shown in Scheme 1 (the sialylated glycans are derivatives of these compounds and individual structures are not shown). Because MALDI produces mainly singly charged ions, separation of the glycan ions from the ions produced by background contamination was not as complete as that obtained with ESI but, nevertheless, most of the background was removed.…”