Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 11 (10), 1604-13

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

Affiliations

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers

Laura K Pritchard et al. Cell Rep.

Abstract

A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system, but paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles' heel that can be exploited for bNAb recognition and vaccine design.

Figures

Figure 1
Figure 1. Comparison of glycosylation patterns of BG505 SOSIP.664 trimers produced in CHO and 293T cells
HILIC-UPLC spectra of fluorescently labelled N-linked glycans isolated from (A) the entire trimeric gp120/gp41ECTO complex (gp140), and the (B) gp120 and (C) gp41ECTO subunits of BG505 SOSIP.664 trimers. The subunits analyzed in each case are illustrated on the left. Trimers were purified by 2G12-affinity chromatography followed by SEC. The gp140 band was extracted from a non-reducing SDS-PAGE gel, while the gp120 and gp41ECTO bands were resolved by SDS-PAGE under reducing conditions. The glycan contents of proteins extracted from the bands were analyzed. Peaks corresponding to oligomannose glycans (M5-M9; Man5-9GlcNAc2) are colored in shades of green; the remaining peaks (gray) correspond to complex and hybrid-type glycans. Peak areas (as a % of total glycans) are illustrated in the associated pie charts. Glycan structures are represented according to the color scheme established by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). The SOSIP.664 construct contains the following mutations: a T332N mutation to introduce the 332 glycosylation site; cysteines at 501 and 605 form a disulphide bridge to covalently link the gp120 and gp41ECTO subunits; replacement of the gp120 furin cleavage site (RXXR) with a hexa-arginine (R6) sequence to promote furin cleavage; an I559P mutation to stabilize the gp41ECTO subunits in the pre-fusion form; and deletion of the MPER region at residue 664 to reduce aggregation.
Figure 2
Figure 2. Glycan profiles of BG505 SOSIP.664 trimers following bNAb affinity purification
CHO cell-derived trimers were purified by bNAb affinity chromatography using 2G12, PGT145 or PGT151, followed by SEC. Glycans isolated from reducing SDS-PAGE gels bands containing cleaved gp120 subunits were analyzed by HILIC-UPLC. The various bNAb epitopes are colored light blue on an EM reconstruction on the left. Peaks corresponding to oligomannose glycans are colored in shades of green and the corresponding peak areas are illustrated in the associated pie charts, as in Figure 1.See also Figure S3.
Figure 3
Figure 3. Effect of proteolytic cleavage on Env glycan processing
His-tagged BG505 SOSIP.664 trimers were expressed in 293F cells with and without Furin co-expression and purified by Ni2+-NTA affinity chromatography. (A) Reducing SDS-PAGE of trimers expressed without furin, and the resulting HILIC-UPLC glycan profiles derived from gel bands corresponding to uncleaved gp140 (top panel) and cleaved gp120 (bottom panel). (B)Reducing SDS-PAGE of the same trimers but co-expressed with Furin, and the corresponding HILIC-UPLC glycan profiles derived from the band corresponding to cleaved gp120. Peaks corresponding to oligomannose glycans are colored in shades of green and the corresponding peak areas are illustrated in the associated pie charts, as in Figure 1. SDS-PAGE analysis in both panels reveals a minor population of low molecular weight contaminants.
Figure 4
Figure 4. Disordered configurations of uncleaved gp140s are associated with higher levels of glycan processing
HILIC-UPLC glycan data and 2D class averages from negative stain EM analysis are shown for: (A) BG505 SOSIP.664 trimers expressed in 293T cells and purified using 2G12 affinity chromatography followed by SEC. The UPLC chromatogram is reproduced from Figure 1A to facilitate data comparison. (B) Uncleaved BG505 WT.SEKS gp140 expressed in 293T cells and purified by 2G12 affinity chromatography followed by SEC. Quantitation of the complete datasets show that native-like, regular and compact trimers constitute >90% of the images of the BG505 SOSIP.664 proteins. In contrast, <5% of the uncleaved BG505 WT.SEKS proteins are in native-like form, where the predominant images represent disordered, splayed out trimers. The same is also true of: (C) uncleaved, His-tagged CZA97.012 gp140 proteins produced in 293T cells and purified by Ni2+-NTA affinity chromatography followed by SEC; (D) uncleaved UG37 gp140; (E) uncleaved CN54 gp140 proteins (the latter two purchased from Polymun Scientific, Vienna, Austria). Peaks corresponding to oligomannose glycans are colored in shades of green and the corresponding peak areas are illustrated in the associated pie charts, as in Figure 1.
Figure 5
Figure 5. Env cleavage induces highly homogenous glycoforms dominated by oligomannose glycans
Env is synthesized as a gp160 precursor in the ER and oligomannose-type glycans are incorporated at glycosylation sequons: Asn-Ser/Thr-X (where X is any amino acid except Pro). As glycoproteins pass through the secretory pathway they are exposed to α-mannosidases and other glycan processing enzymes, which normally convert oligomannose-type glycans (green) to complex-type glycans (gray). The quaternary structure of Env dictates how well processing enzymes can access the glycans on gp120. Cleaved Env exhibits a compact quaternary structure that prevents trimming by ER and Golgi α-mannosidases, and is thus secreted as an oligomannose-dominated glycoform. In contrast, uncleaved Env has a more open and irregular structure that facilitates easier access by processing enzymes, leading to conversion of the majority of the oligomannose glycans to more complex-type structures. However, high glycan density on the outer domain of gp120 creates a region that is largely protected from α-mannosidase trimming, known as the intrinsic mannose patch, which is present on both cleaved and uncleaved Env. Gp41 is more accessible to processing enzymes than gp120 and is dominated by complex-type glycosylation.

Similar articles

See all similar articles

Cited by 65 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback