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.2015 Jun 24:6:7479.
doi: 10.1038/ncomms8479.

Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies

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Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies

Laura K Pritchard et al. Nat Commun..

Abstract

The envelope spike of HIV-1 employs a 'glycan shield' to protect itself from antibody-mediated neutralization. Paradoxically, however, potent broadly neutralizing antibodies (bnAbs) that target this shield have been isolated. The unusually high glycan density on the gp120 subunit limits processing during biosynthesis, leaving a region of under-processed oligomannose-type structures, which is a primary target of these bnAbs. Here we investigate the contribution of individual glycosylation sites in the formation of this so-called intrinsic mannose patch. Deletion of individual sites has a limited effect on the overall size of the intrinsic mannose patch but leads to changes in the processing of neighbouring glycans. These structural changes are largely tolerated by a panel of glycan-dependent bnAbs targeting these regions, indicating a degree of plasticity in their recognition. These results support the intrinsic mannose patch as a stable target for vaccine design.

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Figures

Figure 1
Figure 1. Contribution of individual glycans to the total glycosylation of gp120BaL
(a) HILIC-UPLC profile of N-linked glycans released from gp120BaL. The Man5-9GlcNAc2 series is labelled M5–M9 and highlighted in blue. Remaining peaks correspond to hybrid and complex-type glycans (b) Schematic showing the distribution of the 23 PNGSs of gp120BaL. Sites were allocated as putative complex-type or putative oligomannose-type based on previous reports,-. (c) Percentage conservation of the individual PNGSs across different clades, based on analysis of over 4000 aligned Env sequences from the Los Alamos HIV sequence database (http://www.hiv.lanl.gov/). (d) Effect of PNGS-deletion on the total abundance of oligomannose-type glycans, and the individual abundance of Man9GlcNAc2. Values were obtained by integration of corresponding HILIC-UPLC peaks (Supplementary Fig. 2), before and after Endoglycosidase H treatment, and represent the percentage change in abundance (relative to wild-type). Percentage change = [(% oligomannose in wild-type − % oligomannose in mutant)/(% oligomannose in wild-type)] × 100. Values are reported in Supplementary Table 2. The dashed line represents the predicted drop in oligomannose levels from the elimination of one site containing only oligomannose-type glycans.
Figure 2
Figure 2. Role of glycan clusters in maintaining the oligomannose population
(a) Model of a fully glycosylated BaL trimer based on the reported gp140 trimer crystal structure (PDB accession code: 4NCO). Modelled glycans are shown as sticks. The surface of the glycans and underlying protein are depicted in shades of gray. Man5GlcNAc2 glycans were modelled at sites of predicted complex-type glycans and Man8GlcNAc2 glycans were added at predicted oligomannose sites. (b) A close-up view of the gp120 monomer. Glycans are colored according to the effect of their elimination on total Man9GlcNAc2 levels or complex-type glycosylation according to Fig 1d. A change of over 5% is greater than the intrinsic variation of glycosylation (Supplementary Fig. 1; Supplementary Table 1). (c) Examples of changes in glycan composition upon deletion of individual glycan sites of the mannose patch. Residual plots were calculated by the subtraction of HILIC-UPLC spectra of PNGS-deletion mutants from that of the wild-type. Peaks corresponding to oligomannose-type glycans are highlighted. Man5-9GlcNAc2 (M5–9) are schematically labelled according to the combined nomenclature of Harveyet al and the Center of Functional Glycomics.
Figure 3
Figure 3. Recognition of PNGS-deletion mutants by conformation-dependent antibodies
Binding of the conformation-dependent monoclonal antibodies b12 and 17b was tested against the panel of gp120BaL PNGS-deletion mutants by capture ELISA. Error bars represent standard deviation. Each experiment was performed in duplicate and repeated three times.
Figure 4
Figure 4. Steric barriers to the processing of Env glycans
The α-mannosidase enzyme has impeded access to the terminal sugars of the mannose patch due to steric constraints imposed by both protein and glycan moieties. (a) The high-resolution structure of α1,2-mannosidase (PDB accession code: 2RI9) bound to a disaccharide substrate shows how the substrate binds in a deep, concave pocket. (b) When the α-1,2-mannosidase structure is docked onto the surface of the HIV-1 Env trimer (hybrid model of EMD-5779 and PDB 3TYG) and aligned on the terminal D1, D2, or D3 arms of the Man9GlcNAc2 moiety at position N332, there are significant clashes (c-e). In those three panels, the colored surfaces represent areas of the trimer that are within 2 Å of α-1,2-mannosidase after docking the terminal mannoses on Man9GlcNAc2 into the binding enzyme pocket, i.e., clashes occur that impede enzyme access to its glycan substrates and are therefore predicted to prevent glycan processing. The variable loops V4 and V1 account for a large proportion of the clashes seen in panels d and e, respectively.
Figure 5
Figure 5. The effect on processing at the N332 glycosylation site by the deletion of neighboring glycans
(a) Model of the N332 glycan (green sticks) on the outer domain surrounded by neighboring (dark blue sticks) and distant glycans (gray sticks). (b) Site-specific glycan analysis of the N332 site from a panel of mutants lacking the neighboring glycans (Panel A; dark blue glycans). A tryptic glycopeptide containing the N332 site (QAHCNLSR) was isolated and analyzed by MALDI-MS to determine the glycoforms present. The same glycopeptide was analyzed from gp120BaL mutants carrying PNGS-deletions proximal to N332. (c) Quantitation of the abundance of Man9GlcNAc2 on the wild-type and mutant glycopeptides as determined by MALDI-MS. Symbols as in Fig. 2.
Figure 6
Figure 6. Recognition of glycan site deletion mutants by N332-specific bnAbs
Binding of a panel of N332-specific bnAbs, including 2G12, PGT128, PGT135 and PGT121, was tested against the panel of gp120BaL glycan site deletion mutants by capture ELISA. Error bars represent standard deviation. Each experiment was performed in duplicate and repeated three times.
Figure 7
Figure 7. The effect of glycan site deletion on neutralization by N332-specific bnAbs
Neutralization of the BaL wild-type virus and mutant viruses by b12 and N332-specific bnAbs, including PGT121, PGT135, 2G12, PGT128 and PGT130. Error bars represent standard deviation. Each experiment was performed in duplicate and repeated three times.
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