Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

Abstract

SummaryThe HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

“…The recombinant JR-FL trimer used in that study, a membrane-anchored gp150 protein purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity, had multiple high-mannose glycans at specific sites, consistent with the original glycan-based virion analyses (43,44). The major finding of the study by Go et al, that Env trimers contain multiple sites with highmannose glycans, was replicated more recently in two analyses of the glycosylation pattern on the soluble, recombinant BG505 (clade A) SOSIP.664 gp140 trimer, purified with a 2G12 column (46,47). In one BG505 analysis, data on 20 of 28 glycan sites were reported, with about 160 unique glycopeptides detected (46).…”

“…The major finding of the study by Go et al, that Env trimers contain multiple sites with highmannose glycans, was replicated more recently in two analyses of the glycosylation pattern on the soluble, recombinant BG505 (clade A) SOSIP.664 gp140 trimer, purified with a 2G12 column (46,47). In one BG505 analysis, data on 20 of 28 glycan sites were reported, with about 160 unique glycopeptides detected (46). Many of the characterized sites were exclusively occupied by high-mannose glycans (46).…”

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

“…A core fucose was sometimes visible in one structure but not in the other. Glycans at some individual PNGSs were therefore interpreted with different compositions in the two structures; this type of heterogeneity is consistent with multiple glycoforms at single PNGSs in preparations of the BG505 SOSIP.664 protein (Behrens et al, 2016). Although the composite-annealed OMIT maps displayed additional density near PNGSs compared with the maps calculated with model phases, some of the extra densities were not interpretable.…”

“…Glycans were assigned as complextype if there was density for a core fucose and/or based on mass-spectrometry assignments (Behrens et al, 2016;Go et al, 2011). A core fucose was sometimes visible in one structure but not in the other.…”

“…Because of steric constraints that limit the activities of endoplasmic reticulum and Golgi carbohydrateprocessing enzymes, the HIV-1 Env glycoprotein includes regions of under-processed N-glycans in oligomannose forms GlcNAc 2 ), especially in the intrinsic mannose patch on gp120, which forms portions of the epitopes for many characterized HIV-1 broadly neutralizing antibodies (bNAbs; Doores, 2015). Although oligomannose glycans dominate parts of HIV-1 Env such as the N332 gp120 glycan-associated region on gp120, processed complex-type N-glycans predominate at N-linked glycosylation sites on gp41 and gp41-proximal regions of gp120 (Behrens et al, 2016) and are thought to protect the host receptor (CD4) binding site (CD4bs) and the V3 loop of gp120 (Binley et al, 2010).…”