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INTRODUCTION
The trimeric envelope (Env) glycoprotein spike of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in mediating host cell infection. Its exposed position on the virus surface also makes it the target for potent, broadly neutralizing antibodies (bNAbs) that are produced in a subset of infected individuals and that now influence the design of Env immunogens intended to induce similar antibody specificities. Env is extensively glycosylated, with glycans contributing to a significant proportion of the glycoprotein's mass (1). The heavily glycosylated surface of Env has been referred to as the “silent face,” with the glycans acting to shield the underlying viral protein surface from immune surveillance (2,–4). The glycan shield constantly evolves to protect the virus from newly produced neutralizing antibodies. However, compared to the highly diverse protein component of Env, many of the potential N-glycosylation sites (PNGSs) are well conserved; their total number has also remained relatively constant despite years of viral evolution (5, 6). While the glycans are derived from the host cell's glycosylation machinery, their high density leads to a large abundance of underprocessed, oligomannose-type glycans (Man5–9GlcNAc2) (7,–11). The glycan shield is therefore surprisingly homogenous given the extent of glycosylation, and it is itself a target for bNAbs that recognize exclusively glycan or mixed glycan/protein epitopes (12,–25).
The Env spike is a trimer of heterodimers composed of gp41 and gp120 subunits, which are generated when a protease, usually furin, cleaves the proprotein gp160 glycoprotein (26,–31). Furin most probably cleaves gp160 in the trans-Golgi network (TGN), but it is able to cycle between the TGN, the endosomal compartments, and the cell surface and is also believed to be able to act in the early secretory pathway (27, 32, 33). Furin is an important factor in Env immunogen production, as it is now commonly coexpressed to generate fully cleaved recombinant soluble trimers that structurally mimic native Env (34, 35). In contrast, structural and antigenic analyses of many uncleaved Env constructs show that they adopt nonnative conformations, with gp120 subunits dangling from a gp41 core, unless additional modifications are made to overcome the defect (36,–39). Cleavage is therefore important for the structural integrity of the trimer, which in turn greatly influences how the trimer is glycosylated (39, 40).
Several studies on monomeric gp120 proteins have demonstrated the presence of a cluster of oligomannose-type glycans, the “intrinsic mannose patch” (IMP), which arises because the glycan shield creates steric constraints that restrict the actions of endoplasmic reticulum (ER) and Golgi apparatus α-mannosidases (7, 8, 11, 41,–43). The IMP is localized at the outer domain of gp120 and comprises the epitopes of numerous glycan-influenced bNAbs (15, 21, 23, 24, 44,–47). The IMP is a characteristic feature of gp120 that is conserved across all HIV-1 clades (7, 8, 48) and longitudinally during infection (49). Analyses of recombinant soluble, native-like trimers (40, 50), membrane-associated trimers (51), and virion-derived Env (7,–9) all show that their oligomannose-type glycan contents are even higher than those of gp120 monomers, implying that a “trimer-associated mannose patch” (TAMP) exists (52). However, the glycan sites forming any such TAMP have not previously been defined.
Cleaved, soluble, native-like Env trimers are currently one focus of structure-guided immunogen design programs. A detailed site-specific N-glycosylation analysis of the prototype of this class of trimers, BG505 SOSIP.664, revealed fine structural details of the glycan shield (35, 50, 53). The gp120 subunit of this trimer is dominated by underprocessed Man8–9GlcNAc2 glycans, which form a mosaic of densely organized oligomannose clusters on the outer domain and the trimer interface (40, 50). In contrast, glycosylation sites with a mixed processing state (i.e., sites containing both highly processed, complex, and oligomannose-type glycans) are found at the trimer apex and near its N and C termini (50). Compact folding seems to be the major driver behind the oligomannose-dominated profile of the glycan shield on native trimers. For example, uncleaved, nonnative constructs that adopt a much more open and irregular conformation have a significantly reduced content of oligomannose-type glycans, because the now more accessible structures are efficiently processed (39, 40). We refer here to these uncleaved, nonnative glycoproteins as pseudotrimers, to reflect their lack of the 3-fold symmetry that is a characteristic feature of the structures of natively folded trimers (54, 55).
Here we reveal the detailed effects of cleavage-induced, native-like trimerization on N-glycan processing. Our quantitative, site-specific analysis of a set of comparably expressed and purified monomeric (i.e., gp120), uncleaved pseudotrimeric, and fully cleaved trimeric envelope glycoproteins of the same genotype (i.e., BG505) identifies the location of the cleavage-dependent TAMP. Thus, the glycans that form the TAMP are found in regions near the trimer apex and the protomer interface. We show that compared to those in pseudotrimers, these regions are even more highly processed on monomeric gp120, implying that the oligomerization events impose additional regional restrictions on glycan processing. This finding is echoed across the panel of glycoproteins in the analysis of O-linked glycosylation at T499, near the trimer base, which is present at almost unmeasurable levels on native-like trimers. We are also able to classify the glycan processing states of all N-glycans present on the various constructs, thus extending our earlier site-specific analysis of the BG505 SOSIP.664 trimer (50). Our findings emphasize how the glycan shield is influenced by the quaternary structure of the Env spike and highlight the relevance of native-like structures to Env vaccine development.
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RESULTS
Glycosylation processing on monomeric, uncleaved, and cleaved Env proteins.
To thoroughly investigate the influence of trimerization- and cleavage-induced structural integrity on Env glycosylation, we used comparable conditions to express and purify monomeric gp120, native-like SOSIP.664 trimers, and uncleaved, nonnative WT.SEKS gp140 pseudotrimers, all based on the BG505 genotype. The design and characteristics of these proteins have been described previously (35, 53, 54, 56,–59). The WT.SEKS construct differs from SOSIP.664 in that it lacks the stabilizing SOSIP mutations and contains an inactivated furin cleavage site (RRRRRR mutated to SEKS); the resulting uncleaved pseudotrimers have a nonnative conformation (36). The monomeric gp120 protein is isogenic with the gp120 subunit of the SOSIP.664 trimer. The designs of all three constructs are summarized in Fig. 1A. The three proteins were all produced in transiently transfected human embryonic kidney (HEK) 293F cells and purified using 2G12 affinity chromatography followed by size-exclusion chromatography (SEC).
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FIG 1
Design and glycosylation patterns of BG505 gp120, WT.SEKS, and SOSIP.664. (A) Schematic representation of the BG505 gp120, WT.SEKS, and SOSIP.664 constructs. Changes to the wild-type BG505 sequence are highlighted in blue. (B) HILIC-UPLC profiles of the enzymatically released N-linked glycans of the three constructs, transiently produced in HEK 293F cells and purified by 2G12 affinity chromatography followed by SEC. Oligomannose-type and hybrid glycans (green) were identified by their sensitivity to endo H digestion. Peaks corresponding to complex-type glycans are shown in pink. The peaks were integrated, and the pie charts summarize the quantification of the peak areas. Glycan symbols are as shown in Fig. 2.
N-linked glycans were enzymatically released from all three constructs, fluorescently labeled, and analyzed by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) (Fig. 1B). Using endo-β-N-acetylglucosaminidase H (endo H) to digest the oligomannose-type glycans and by integration of the resulting chromatograms, oligomannose-type glycans were shown to account for 36% (gp120 monomers), 35% (WT.SEKS gp140), and 68% (SOSIP.664 trimers) of the total glycan populations. This outcome is consistent with a previous report that complex-type glycans are significantly more abundant on uncleaved pseudotrimers than on native-like trimers (40). Although the analyses of the WT.SEKS and SOSIP.664 glycoproteins included contributions from the four glycans on gp41, the overall outcome was dominated by the 24 glycan sites on gp120. In summary, the total oligomannose contents of both the monomeric gp120 and uncleaved gp140 forms of BG505 Env are quite similar, with each protein having approximately half the oligomannose content of the native-like SOSIP.664 trimer.
Glycan databases reveal details of cell-directed glycan processing.
To facilitate the subsequent site-specific N-glycosylation analysis of the gp120, WT.SEKS, and SOSIP.664 glycoproteins and to probe their glycan structures in greater detail, we generated libraries of enzymatically released, unlabeled glycans by using ion mobility-electrospray ionization mass spectrometry (IM-ESI MS). The mobility-extracted singly charged glycans from the three glycoproteins are shown in Fig. 2. A significant population of oligomannose-type Man5–9GlcNAc2 glycans is present on all three glycoproteins and is most evident in the BG505 SOSIP.664 spectrum. This observation is consistent with the HILIC-UPLC data presented in Fig. 1. Ion mobility mass spectrometry using negative fragmentation mode allows the detailed assignment of isomeric glycan structures (60). The number and type of glycan structures identified for the gp120, WT.SEKS, and SOSIP.664 proteins, with about 90 identified isobaric structures, are highly similar, although the abundances vary (see Table S1 in the supplemental material). Fine structural details of the diversity of complex-type glycans present on all three constructs were revealed. The extremes of glycan processing, including the presence of terminal fucoses, sulfates, GlcNAcs, and GalNAcs, are directed by the producer cell's glycosylation machinery. In contrast, the oligomannose-type glycans represent an intrinsic structural feature of Env (40).
FIG 2
Ion mobility mass spectrometry analysis of BG505 Env glycoproteins. Mobility-extracted singly charged negative-ion electrospray spectra are shown for N-linked glycans found on the following BG505 Env proteins: gp120 monomers (A), WT.SEKS pseudotrimers (B), and SOSIP.664 trimers (C). The inset in panel B shows an example of a DriftScope image derived from gp120 monomers, with singly charged ions encircled with a yellow oval. The peaks of the oligomannose series Man5–9GlcNAc2 are highlighted in green. A list of identified glycans is given in Table S1 in the supplemental material.
Quantitative site-specific N-glycosylation analysis.
We performed a quantitative, site-specific N-glycosylation analysis using in-line liquid chromatography-ESI MS (LC-ESI MS) to thoroughly investigate how the formation of native-like, cleavage-induced trimers influences the glycan shield. We previously validated this method (50). We used a panel of different proteases to create peptides and glycopeptides from the gp120, WT.SEKS, and SOSIP.664 proteins, enriched glycopeptide populations, and then quantified the relative abundances of individual glycans by assessing the ion intensities over all charge states. The quantified and summarized data for the three Env constructs show that the processing states of distinct N-glycosylation sites in the glycan shields of gp120 monomers and pseudotrimers are very similar to each other but also quite different from those for native-like SOSIP.664 trimers (Fig. 3).
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FIG 3
Quantitative site-specific N-glycosylation of BG505 Env glycoproteins. Relative quantification is shown for the N-glycosylation sites on the gp120 subunit (A) and the gp41 subunit (B) of gp120 monomers (no gp41 present), WT.SEKS pseudotrimers, and SOSIP.664 trimers. The proteins were digested with trypsin, chymotrypsin, pronase, GluC, or GluC plus trypsin and then analyzed by LC-ESI MS. Quantifications are based on the peak lists given in Tables S2, S3, and S4 in the supplemental material. The percentages corresponding to this figure are shown in Table S5. Glycans are categorized as oligomannose series (M5 to M9; Man5GlcNAc2 to Man9GlcNAc2), hybrids (H), and fucosylated hybrids (FH), and also by the number of branching antennae (A) of complex-type glycans. An, number (n) of antennae; Gn, number (n) of galactose residues; F, presence of a core fucose (50). The bar graphs represent the means for two analytical replicates, and the quantification of oligomannose-type (green) and complex/hybrid glycans (pink) on individual sites is summarized in the pie charts. The processing states of sites for which no quantitative analysis could be performed were classified by qualitative analysis of exoglycosidase-treated glycopeptides, as summarized by colored squares (Table S6).
In the present study, the site-specific glycosylation profile of SOSIP.664 trimers derived from transiently transfected HEK 293F cells is highly similar to previously published data on the same trimers produced in a stable HEK 293T cell line (50). Quantitative site-specific glycan profiles of comparably expressed and purified BG505 gp120 and WT.SEKS gp140 proteins were not derived previously. Their availability now allows a comparison with SOSIP.664 trimers that reveals key aspects of how Env is glycosylated. In cases where quantitative site-specific data could not be obtained, the processing states of these sites could be classified broadly according to their susceptibility to sequential endo H and peptide-N-glycosidase F (PNGase F) digestions (Table S6). The resulting information is incorporated into Fig. 3, and the models are shown in Fig. 4. For reasons outlined in Materials and Methods, the endo H-plus-PNGase F digestion method is unsuitable for precisely quantifying the glycan compositions of specific sites, but it is sufficient for classifying glycans into broad processing states.
FIG 4
Models of a fully glycosylated BG505 gp120 monomer and a SOSIP.664 trimer. Models of the glycosylated gp120 monomer (A) and the glycosylated SOSIP.664 trimer (B) were derived from one previously described elsewhere (50). The monomer is oriented as it would appear in situ as a subunit of the SOSIP.664 trimer. The glycans on the models are colored according to their oligomannose content, as derived in the present study. N-glycan sites for which quantitative results of sufficient quality could not be obtained were classified by qualitative analysis according to their susceptibility to endo H and PNGase F digestion (Table S6).
The site-specific analysis of BG505 gp120 revealed numerous positions where exclusively or almost exclusively oligomannose-type glycans are present. These sites define the gp120 IMP (7). Note that the glycan repertoires are highly similar for the corresponding locations in the gp120 subunits of the uncleaved WT.SEKS pseudotrimer and the SOSIP.664 trimer (Fig. 3A). However, whereas Man9GlcNAc2 moieties dominate the sites forming the IMP on the SOSIP.664 trimer (e.g., N234, N363, N339, and N392) (Fig. 3A), there is additional but modest trimming of these sites toward smaller oligomannose glycans on the gp120 monomer and the pseudotrimer. Other sites on the gp120 monomer that contain a significant population of complex-type glycans are less processed on the native-like trimer (e.g., N137, N156, N160, N197, N276, and N355). The overall dominance of oligomannose-type glycosylation, however, is conserved among the three Env proteins, which supports the definition of the IMP as an intrinsic feature of the gp120 protein, whatever its quaternary context (7). The N295 site seems to be protected comparably from glycan processing enzymes on all three constructs; it is the only site where the quaternary context and cleavage status do not increase glycan processing. The highly conserved “supersite of vulnerability” on the uncleaved WT.SEKS glycoprotein, i.e., N332, carries a small population of complex-type glycans, whereas this site is entirely oligomannose on both the cleaved SOSIP.664 trimer and the gp120 monomer. A likely explanation is that the conformationally heterogeneous pseudotrimers include a subpopulation on which processing enzymes can access and modify this antigenically important site at the heart of the IMP. The site-specific glycosylation states of the gp41 subunits of the BG505 WT.SEKS pseudotrimer and the BG505.664 trimer (Fig. 3B) are largely similar in that complex-type glycosylation dominates. However, the native-like trimer has an elevated population of oligomannose glycans at N625 and N637, indicating steric inaccessibility in those regions.
To display the site-specific glycosylation analysis and to facilitate structural interpretation, we mapped the distribution of the different glycan processing states across the BG505 gp120 monomer (Fig. 4A) and the SOSIP.664 trimer (Fig. 4B), using a model of a fully glycosylated trimer derived from a cryo-electron microscopy structure (50, 61). All the glycosylation sites are displayed, with the sites classified as mainly oligomannose, mainly complex, or mixed.
Formation of native-like trimers results in interprotomer control of glycan processing.
Based on the BG505 SOSIP.664 glycosylation map, we created two heat maps displaying the site-specific increase in oligomannose-type glycans on the structure of the cleaved trimer relative to the gp120 monomer (Fig. 5A) and the pseudotrimer (Fig. 5B). The cleavage-dependent formation of native-like trimers has a significant effect on the processing state of several glycans, including those at the trimer apex and the protomer interface. The increase in oligomannose-type glycans on the native trimer relative to the monomer or pseudotrimer can be viewed as a decrease in enzymatic processing consistent with the structural constraints that limit processing. For example, the glycan at N276 on SOSIP.664, near the CD4 binding site (CD4bs), has an ∼75% oligomannose content, whereas the corresponding value for the same site on the gp120 monomer is ∼5%; the glycan at N276 is the gp120 glycan that differs the most in this regard (Fig. 5A). Other glycans affected similarly but less profoundly are N156 (∼60% oligomannose on monomers versus 100% on trimers, hence a 40 percentage point (pp) increase in Fig. 4), N160 (∼60 pp increase), and N197 (∼50 pp increase); each of these sites is located at the trimer apex or protomer interface and thus is in a different quaternary context on the SOSIP.664 trimer compared to the gp120 monomer. Similarly, the outer domain N355 glycan is highly processed on monomeric gp120 but is a mixed glycan site (∼40 pp increase in oligomannose) on the SOSIP.664 trimer. In this case, the model suggests that the presence (trimer) or absence (monomer) of proximal gp41 glycans may be the key influence (Fig. 4A).
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FIG 5
Heat maps on the surfaces of trimers, showing the increase in oligomannose glycans on BG505 SOSIP.664 trimers compared to gp120 monomers (A) and pseudotrimers (B). The increase in oligomannose content was calculated for sites for which quantitative data were available. To derive the heat map, a percentage point was calculated for each glycosylation site corresponding to an arithmetic difference of two percentage points; the percentage of oligomannose-type glycan for each of these sites on gp120 monomers or WT.SEKS pseudotrimers was subtracted from the corresponding percentage for SOSIP.664 trimers.
The formation of native-like trimers also has subtle influences on glycan trimming within the IMP. Thus, several IMP glycans are slightly less trimmed on the trimers, leading to an elevation in the abundance of Man9GlcNAc2 and an associated reduction in Man5–8GlcNAc2 (N234, N339, N363, N386, N392, and N448) and/or a slight decrease in complex-type glycans (N234, N262, and N363) (Fig. 3).
There is an additional mannose patch on native-like trimers but not pseudotrimers.
Broadly similar differences in glycan processing were also found when SOSIP.664 trimers were compared to the uncleaved, nonnative pseudotrimers (Fig. 4B). The differences were quantitatively smaller than those for comparison of gp120 monomers versus SOSIP.664 trimers, as is apparent from the color gradient of the heat map, but they were qualitatively similar (Fig. 5). Thus, there was an ∼35 pp increase in oligomannose for the N160 and N197 glycans at the apex of the native trimer compared to the pseudotrimer, whereas the corresponding increase for the N156 glycan was 20 pp. The biggest difference between the two proteins was seen for the N301 site, near the protomer interface; that glycan almost completely switched from highly processed on the pseudotrimer to oligomannose dominated on the SOSIP.664 trimer. We were not able to gain quantitative site-specific data for the N301 site on gp120 monomers, but the qualitative deglycosylation experiments did indicate that it had a mixed processing state (Table S6).
One notable glycan is the N262 glycan, which is known to play a critical role in gp120 folding (62, 63). While this site is exclusively dominated by oligomannose-type glycans on SOSIP.664, it has a mixed composition (∼50% complex glycans) on the pseudotrimer, although Man9GlcNAc2 still remains the most prominent oligomannose-type structure. One explanation may be that more than one folding state exists within the total population of pseudotrimers, for example, there may be a subpopulation in which aberrant disulfide bonds have formed (64). The gp41 glycan that differs most between the SOSIP.664 trimer and the pseudotrimer is N637, which resides near the protomer interface, very close to N276. Thus, there is an increase of more than 30 pp for oligomannose at the N637 site on the native trimer compared to the pseudotrimer (Fig. 4B). The remaining gp120 glycans near the IMP and the other gp41 glycans differ only subtly between the SOSIP.664 trimer and the pseudotrimer, similar to what was seen for the gp120 monomer comparison described above.
Overall, the results show that a glycan patch with increased mannose content is uniquely present on the fully cleaved, native-like SOSIP.664 trimer. This trimer-associated mannose patch (TAMP) wraps around the trimer apex and the protomer interface and includes, at minimum, the N156, N160, N197, N262, N276, N301, and N637 glycans.
Trimer formation constrains processing at a fractionally occupied O-glycosylation site.
Recombinant Env glycoproteins have been reported to contain O-linked glycans at gp120 position T499 (42, 51, 65, 66). We searched the LC-ESI MS spectra of the deglycosylated tryptic digests of the three Env constructs for exact mass matches of O-glycosylated peptides. We then confirmed them by the presence of the characteristic oxonium ions as well as by using higher-energy collisional dissociation (HCD) fragmentation data. We were thereby able to identify microheterogeneity of commonly known O-glycans on T499 of each BG505 Env protein, mainly of the mono- and disialyl core 1 types, but with highly different fractional occupancies (Fig. 6; Table S7). We quantified the relative abundances of individual O-glycan structures in the same way as that for the N-linked glycans, by adding up the ion intensities over all charge states for the individual glycopeptides. By including the nonglycosylated peptide, which contains an unmodified threonine, we could determine the fractional occupancy of the T499 site.
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FIG 6
O-glycosylation of recombinant Env glycoproteins. (A) The location of the T499 O-glycosylation site is highlighted in red on the BG505 SOSIP.664 trimer model. (B) Quantification of the O-glycans identified on the gp120, WT.SEKS, and SOSIP.664 Env proteins, based on the peak list shown in Table S7.
O-glycan occupancy of the T499 site was highest for monomeric gp120, at ∼7%, compared to ∼1% and ∼0.2% for the pseudotrimers and SOSIP.664 trimers, respectively (i.e., 7- to 35-fold reductions compared to the level for gp120). We therefore concluded that while T499 is indeed a potential site for O-glycosylation on gp120, it is only rarely occupied, and even less so on the pseudotrimer and the SOSIP.664 trimer. The T499 site is located close to the C terminus of gp120, so the nearby presence of gp41 may impose steric constraints on the glycosyltransferases. Furthermore, the structures of the native-like BG505 SOSIP.664 soluble trimer and the JR-FL membrane-associated trimer show that the T499 site is not surface exposed when gp41 is present (54, 55, 67). Indeed, in the native trimer context, the bulk of an O-glycan would not be accommodated within the gp120-gp41 interface, where T499 is located.
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DISCUSSION
An effective B cell-based HIV-1 vaccine may need to elicit bNAbs. Rational vaccine design has led to the development and evaluation of many different potential Env immunogens (68). An increasingly widespread opinion is that a native-like representation of Env where multiple bNAb epitopes are presented but nonneutralizing antibody epitopes are occluded is a highly relevant option (69, 70). As many bNAb epitopes include glycans, the integrity of the glycan shield is a key factor that needs to be considered in Env immunogen design. Glycan processing is not simply a function of the number of glycosylation sites on the Env protein backbone but is also related to the quaternary architecture of the native-like trimer (40). Nonnative conformations of Env are reflected in their overall glycan profile, with pseudotrimers generally bearing a much higher content of processed glycans (40). This observation applies to the uncleaved BG505 WT.SEKS pseudotrimers that serve as one point of comparison in the present study. These Env proteins, like others of the same basic design, adopt a variety of different shapes and conformations as observed by electron microscopy, contain aberrant, noncanonical disulfide bonds, and are atypically sensitive to proteases, and various of their trimer association domains are atypically accessible for hydrogen-deuterium exchange reactions (36, 39, 40, 64, 71). Consistent with their nonnative structure, the immunogenicity of BG505 WT.SEKS proteins in rabbits is markedly inferior to that of the corresponding SOSIP.664 trimers (53).
In this study, we used quantitative site-specific N-glycosylation analysis of isogenic monomeric, cleaved, and uncleaved soluble Env glycoproteins to show that the formation of native-like trimers markedly affects the integrity of the glycan shield by providing additional protection from α-mannosidase processing. The trimer-induced mannose patch (TAMP) is a previously postulated concept for how the glycan shield is remodeled upon oligomerization (52). We are now able to confirm its existence and to map its location on the trimer surface (Fig. 5). Furthermore, we show that the adoption of this characteristic glycosylation signature requires the formation of a compact, native-like trimer, not simply the presence of multiple gp120 subunits within the same Env protein. In other words, like gp120 monomers, pseudotrimers lack a TAMP. Both trimerization and protease cleavage need to occur early in the nascent Env glycoprotein's egress through the secretory pathway to provide steric protection of multiple oligomannose glycans from additional α-mannosidase processing (40).
The BG505 WT.SEKS pseudotrimers carry subpopulations of complex-type glycans on N-glycosylation sites (N234, N262, N332, and N363) where underprocessed, oligomannose-type Man8–9GlcNAc2 structures predominate on the native-like SOSIP.664 trimers. The presence of molecules with processing at these sites within the pseudotrimer preparation is consistent with nonnative folding and the impact of any aberrantly formed disulfide bonds. The heat maps in Fig. 5 show how the structural constraints that apply to the native trimer lead to an increase in oligomannose content. A converse perspective is that the loss of those constraints increases the processing of multiple glycans on the gp120 monomers and pseudotrimers. The outcome is generally an increase in the trimming of oligomannose-type glycans toward smaller forms and in the population of complex-type glycans (e.g., N355).
The detailed site-specific N-glycosylation profiles help us to understand how different Env immunogen designs drive differences in the processing of key glycans. For example, the gp120 monomers and WT.SEKS pseudotrimers bind the trimer-reactive, apex-recognizing bNAbs PG16 and PGT145 poorly or not at all (35, 36). The inability of the nonnative Env proteins to present these conformationally sensitive epitopes is also reflected in their aberrant profiles for the glycan sites near the trimer apex. For example, the N160 site on the native-like trimer is dominated by oligomannose-type glycans, whereas bi-, tri-, and tetra-antennary complex-type glycans predominate on the pseudotrimer. We also noted that the oligomannose content of the N276 glycan on the SOSIP.664 trimer is ∼70 pp higher than that at the same site on the gp120 monomer. Indeed, N276 was the gp120 glycan that differed the most between the trimer and the monomer in this regard. The N276 site was also more processed on the pseudotrimer than on the native-like trimer (35 pp lower oligomannose content). The N276 glycan is located near the CD4bs, where it plays a role in shielding key epitopes for bNAbs of the VRC01 class (72). An oligomannose-type glycan at this position forms part of the epitope of the CAP257-RH1 antibody, which was isolated from a donor infected with a clade C HIV-1 strain (73). The high-oligomannose status of the N276 glycan on native-like SOSIP.664 trimers may therefore reflect the composition of the same glycan on virion-associated Env (i.e., in the CAP257-RH1 donor). Inducing VRC01 class bNAbs is the goal of several Env vaccine programs based on targeting specific precursors of these antibodies in the human germ line repertoire followed by a boosting regimen intended to drive the maturation of any initially induced precursors. These programs use a variety of Env proteins, including those based on gp120 outer domains, gp120 monomers, or nonnative gp140s (74,–76). The glycoform compositions of these immunogens will need to be determined experimentally, but some sites (including N276, when present) may be processed more highly than those on native trimers, based on what we have seen with the BG505 gp120 monomers and uncleaved pseudotrimers. The implications for the design of germ line-targeting priming and boosting immunogens would need to be considered.
In summary, site-specific glycan composition data such as those we describe here should be valuable guides to the design and use of Env immunogens intended to induce bNAbs against virion-associated epitopes that either contain or are influenced by glycans of defined compositions.
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MATERIALS AND METHODS
Expression and purification of Env proteins.
The design of the BG505 SOSIP.644 and WT.SEKS constructs has been described elsewhere (35, 36). The BG505 gp120 monomer sequence was identical to that of the gp120 subunit of the SOSIP.664 trimer, including the T332N and A501C mutations. Each env gene was transiently expressed in human embryonic kidney (HEK) 293F cells by using the pPPI4 vector system as described elsewhere (35, 56). A furin expression plasmid was cotransfected with the SOSIP.664 construct to increase trimer cleavage efficiency (34, 56). Env proteins were isolated from culture supernatants by 2G12 affinity chromatography followed by size-exclusion chromatography (SEC), as described previously (35, 77).
Analysis of total glycan profiles by HILIC-UPLC.
N-linked glycans were enzymatically released from Env proteins by in-gel digestion with peptide-N-glycosidase F (PNGase F) and subsequently fluorescently labeled with 2-aminobenzoic acid (2-AA), as previously described (40, 78). The 2-AA-labeled glycans were subsequently analyzed by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) in a Waters Acquity UPLC instrument (40). The abundance of oligomannose-type glycans was assessed by digesting the released glycans with endo H (New England BioLabs) (40).
Site-specific N-glycosylation analysis.
Ion mobility MS using a Waters Synapt G2Si mass spectrometer was used to analyze the total pool of PNGase F-released glycans from the Env glycoproteins, as described previously (50). The results were used to create sample-specific glycan libraries for the subsequent site-specific N-glycosylation analyses. Env proteins (100 to 150 μg) were denatured and alkylated as described previously (50). The following proteases or combinations of proteases were used for digestion at a 1:30 (wt/wt) ratio for 12 h at 37°C, according to the manufacturers' instructions: trypsin, chymotrypsin, GluC (all mass spectrometry grade; Promega), and pronase (Sigma-Aldrich). We also performed a sequential digestion using GluC (4 h, 37°C) and then trypsin (12 h, 37°C). Protease-digested samples were enriched for glycopeptides by use of a ProteoExtract glycopeptide enrichment kit (Merck Millipore).
Additionally, deglycosylated Env samples were prepared by digestion of tryptic and chymotryptic (non-glycopeptide-enriched) peptides with endo H (4 h, 37°C), followed by digestion with PNGase F (4 h, 37°C), according to the manufacturer's instructions. The processing state of the glycan site was then classified according to its susceptibility to endo H and PNGase F. The basis of this assignment method is that endo H cleaves oligomannose-type and hybrid glycans but leaves a single GlcNAc moiety that is not removed by the subsequent PNGase F digestion (leads to deamidation at sites with complex-type glycans). Unoccupied PNGSs remain as unmodified asparagines. Deamidation of asparagines can also occur spontaneously as a nonenzymatic reaction in proteins and peptides (79), making this method unsuitable for exact quantifications. The information gained by this type of analysis is, however, sufficient for glycan classification into broad processing states.
All samples were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) on a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific), as described previously (50). Data interpretation and quantification were performed using Byonic and Byologic software (50).
Site-specific O-glycosylation analysis.
We assessed O-glycosylation by using Byonic and Byologic software to analyze LC-ESI MS spectra derived from tryptic-digested, deglycosylated Env proteins, searching for the most common human O-glycans. Any exact mass matches of O-glycosylated peptides were manually confirmed by seeking the characteristic oxonium ions, as well as peptide fragmentation ions, among the HCD fragmentation data. The relative abundances of individual O-glycoforms and the corresponding unmodified peptide were determined by summing the intensities of the extracted-ion chromatograms (XICs) over all charge states, as follows: relative abundance = intensity of one glycoform/intensities of all glycoforms identified on the same peptide.
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ACKNOWLEDGMENTS
We thank Raymond A. Dwek for his support and Rogier Sanders for critically reading the manuscript.
A.-J.B. is the recipient of the Chris Scanlan Memorial Scholarship from Corpus Christi College, Oxford, United Kingdom. M.C. is supported by Scripps CHAVI-ID (grant 1UM1AI100663) and by an International AIDS Vaccine Initiative Neutralizing Antibody Center CAVD grant (glycan characterization and outer domain glycoform design). J.P.M. is supported by NIH HIVRAD grant P01 AI110657. N.Z. and A.K. were funded by Unither Virology.
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FOOTNOTES
Supplemental material for this article may be found at https://doi.org/10.1128/JVI.01894-16.
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