Our experiments indicate that mannose residues in N-linked high-mannose and/or hybrid glycan chains are essential for 2G12 binding to recombinant gp120. Thus, exo-mannosidase treatment is sufficient to destroy the 2G12 epitope, without affecting the discontinuous epitope for the neutralizing MAb IgG1b12 (Fig. ). Endo F1 and Endo H treatment also abolished 2G12 binding to CHO cell-expressed gp120, whereas Endo F2 had no effect, indicating that the mannose residues of hybrid, rather than high-mannose, carbohydrates may be involved in the 2G12 epitope (Fig. ). This conclusion can be drawn because Endo F2 is able to remove mannose residues from high-mannose chains but not from hybrid chains (28
); moreover, Endo F2 does successfully digest gp120 (Fig. ) while leaving the 2G12 epitope intact (Fig. ). However, the efficiency of Endo F2 at cleaving high-mannose carbohydrates is at least 20-fold lower than its cleavage of complex chains (65
). It is therefore possible that, in addition to hybrid chain carbohydrates, high-mannose chains resistant to Endo F2 treatment could also be involved in 2G12 binding. Endo H and Endo D treatment of JR-FL gp120 produced from Drosophila melanogaster
cells has been shown to remove at most only 90% of the high-mannose carbohydrate (30
), although SIV gp160 has been reported to be completely sensitive to Endo H (14
). Thus, while we expect most high-mannose glycans on HIV-1 gp120 to be sensitive to Endo H, Endo F1, or Endo F2, the observed mobility change of only 10 kDa after Endo F1 and Endo H treatment suggests that part of the high-mannose and hybrid carbohydrates on HIV-1 JR-FL gp120 are not, in fact, accessible to endoglycosidases.
N-linked glycans are added onto proteins during synthesis as predominately mannose, preformed oligosaccharides; only through later modification in the Golgi apparatus do these oligosaccharides lose their terminal mannose sugars. The precise characterization of N-linked glycans has been carried out only on recombinant, monomeric gp120, so it is possible that the glycans on the native, trimeric Env complex might be modified differently. However, since 2G12 neutralizes HIV-1 virions derived from human cells (69
), the MAb must be able to recognize the native Env complex. The recognition of terminally linked mannose residues by 2G12 is not, therefore, an artifact of our use of recombinant gp120 expressed in CHO cells; the critical mannose residues must be exposed for 2G12 binding on the surfaces of both the monomeric gp120 molecule and the native, trimeric Env complex.
Mannosidase treatment reduces the infectivity of SIV virions (39
). This observation further confirms that terminal mannoses are present on the functionally relevant, trimeric Env complex. Indeed, the oligomerization of Env late in its biosynthesis may decrease the accessibility of gp120 to the glycan-modifying enzymes in the Golgi apparatus and thereby increase the retention of high-mannose glycans on the native Env complex. Consistent with this view, an analysis of N-linked oligosaccharides on gp120 derived from chronically virus-infected, human H9 cells showed that more than 80% of the gp120 glycans are of the high-mannose or hybrid variety (41
In contrast to the inhibitory effect of mannosidases, neuraminidase treatment increased the infectivity of SIV (39
). One explanation for this might be that the complex, sialic acid-containing carbohydrates of the variable loops are involved in shielding conserved functional regions of gp120 on the native Env complex (43
); another explanation is that alterations in the electrostatic properties of virions caused by neuraminidase treatment might increase their binding to the cell surface. Regardless of the precise explanation, the results obtained using neuraminidase show that it is not glycosidase treatment in general that decreases virion infectivity but rather the specific activity of the particular glycosidases that are used.
A mutagenesis study on LAI gp120 revealed that the 2G12 epitope is destroyed by amino acid substitutions that affect several different N-linked glycan residues in the C2, C3, C4, and V4 regions of gp120 (Fig. ) (69
). Most of these glycans consist of high-mannose and/or hybrid chains, not complex chains (35
). An exception is the complex glycan at residues 397, but our data indicate that this residue is probably not involved in 2G12 binding. In general, complex carbohydrates are present on the variable loops of gp120 and their positions often differ among HIV-1 isolates (12
). In contrast, gp120 glycans of a high-mannose or hybrid character that are located in the less-variable regions of the protein are usually conserved among divergent HIV-1 isolates and may play an important structural role by facilitating the correct folding of gp120 (12
The sequence analysis of isolates sensitive to 2G12 neutralization proved to be unexpectedly powerful in defining the 2G12 epitope. Such variational analysis works well in the context of the dense information provided by the highly variable HIV-1 genome. A similar but less detailed analysis helped to define some features of the epitope for the broadly neutralizing anti-gp41 MAb 2F5 (68
). The variational analysis of amino acids that are conserved in the 2G12-sensitive isolates, but highly variable in HIV-1 otherwise (31
), defines eight amino acids, including that at position 295. These residues are scattered across the surface, but only position 295 is a site of N-linked glycosylation. Thus, even in the absence of any substitutional mutagenesis data, the sensitivity of the 2G12 epitope to deglycosylation would have been sufficient for the variational analysis to locate this epitope on gp120. However, a limitation of the variational analysis method of epitope definition is that there needs to be significant natural sequence variation within the epitope; it is thus most useful for defining the less-conserved gp120 epitopes.
Our analysis identifies N-linked glycans at positions 295, 332, 392, 386, and 448 as making up the 2G12 epitope. Clearly, some of these glycans (e.g., those at positions 386 and 448) can be modified and not completely eliminate 2G12 binding. It is generally true of all protein-protein interactions that modification of the interactive surface can often be tolerated except at special hot spots of thermodynamic energy (16
). However, N-linked glycans differ in several respects from the typical amino acid side chain, and these differences should be noted. Thus, N-linked glycans are larger, with an average molecular weight more than 20 times that of a typical amino acid side chain. They also contain more structure and they affect a greater volume of their surrounding environment. Our surface analysis showed that 2G12 must bind to only a small portion of the N-linked glycans that we identified as contributing to the epitope—the total surface area of a single pentasaccharide is roughly the same size as a typical antibody footprint. In our analysis, we did not specify the precise details of how 2G12 recognizes glycans. Rather, we identified glycans on the surface of gp120 that form a mannose-rich structure which 2G12 recognizes. This structure may be affected by alterations of any of the glycan sites we have identified, but compensatory changes may also occur to restore the epitope. Thus, in the context of natural HIV-1 sequences, some of the glycans that we have identified here could perhaps be absent without necessarily eliminating 2G12 binding. Moreover, other, 2G12-like antibodies might exist that recognize broadly similar glycan-dependent epitopes without necessarily competing for 2G12 binding to gp120; we suspect, however, that such glycan-dependent antibodies will be rare.
Although 2G12 is broadly reactive with many HIV-1 isolates, it is not pan-reactive (68
). For example, analysis of 2G12 neutralization resistance identified subtype C viruses that were 2G12 resistant (9
). We analyzed the subset of those resistant viruses for which sequence information was available (isolates DU151, DU179, and DU422) (9
). All of them lacked glycan 295. While the diversity represented by database sequences probably does not reflect the frequency of viral populations, it nonetheless shows that many HIV-1 isolates will naturally lack some of the N-linked glycans required for the formation of the 2G12 epitope (12
). Indeed, glycan 295 is poorly conserved among subtype C strains (Table ), suggesting that most subtype C isolates will be resistant to 2G12 neutralization.
The 2G12 epitope that we have identified on gp120 contains, or is directly proximal to, seven of the eight high-mannose or hybrid sites that are conserved between the JR-FL and HXBc2 isolates. Indeed, the site is the only conserved, exposed surface on the gp120 trimer that does not interact with the known cellular receptors, CD4 and a chemokine receptor (33
). Nonetheless, 2G12 is able to interfere with the binding of gp120 to CCR5 (67
) and with the attachment of HIV-1 virions to cells (70
). The positioning of its epitope on the gp120 moieties of the native Env trimer suggests that this inhibition is an indirect, steric effect manifested by the sheer bulk of an antibody molecule located physically close to the receptor-binding sites. Such interference is particularly relevant in the context of the physically crowded virion-receptor complex on the cell surface.
Terminal mannose residues are rarely found on mammalian cell surface or serum glycoproteins (reviewed in references 66
, and 73
). Indeed, the presence of a terminal mannose results in binding of proteins to hepatic lectin receptors and their rapid clearance from the plasma (34
). Virions expressing HIV-1 envelope glycoproteins are very rapidly removed from plasma after their infusion into macaques, with a half-life measured in minutes (27
). Thus, the presence of terminal high-mannose residues on gp120 glycans represents a paradox: these residues appear to be highly conserved and so presumably have a relevant function, yet their presence should be detrimental to the viral life cycle by accelerating the rate of virion clearance. No doubt, overall, HIV-1 finds the terminal mannose residues to be advantageous in its battle with the human immune system.
What could be an evolutionarily conserved function for the terminal mannose residues of gp120? One explanation is that HIV-1 is known to use the mannose components of its gp120 N-linked glycans to bind to the cell surface receptors DC-SIGN and DC-SIGNR (3
). These dendritic cell and macrophage receptors augment the efficiency of both vertical and horizontal HIV-1 transmission by enhancing the presentation of virions to both macrophages and T cells (reviewed in references 3
). Structural and biochemical analyses of DC-SIGN and DC-SIGNR show that these proteins bind to the central, protein-proximal mannose residues of high-mannose glycans (23
). The rate-limiting step for retroviral infection is known to be the initial stage of virus-cell attachment (15
), so the use of DC-SIGN as a high-affinity attachment site provides a significant advantage to HIV-1 (3
). The high-mannose and/or hybrid sugars that form and surround the 2G12 epitope are a possible component of the binding site for DC-SIGN and related proteins. Of note is that the location of these high-mannose sugars on a surface distal from the viral membrane (Fig. ), facing outwards from the virus, is optimal for cell surface binding.
There is precedent for the glycan residues of gp120 being the target of an antiviral compound. The cyanobacterial protein CV-N is an inhibitor of HIV-1 entry that acts by binding to gp120 (8
). The binding site for CV-N on gp120 comprises exclusively mannose residues on N-linked glycans, specifically Manα1-2Manα moieties presented on Man8
high-mannose structures (4
). CV-N has high-affinity and low-affinity binding sites, each of which recognizes the mannose moieties of a single N-linked glycan (4
). CV-N can block 2G12 binding to gp120, but it does not inhibit the gp120 binding of other neutralizing or nonneutralizing MAbs (22
). The converse competition does not occur, however, in that 2G12 does not inhibit CV-N binding to gp120, most probably because there are multiple binding sites for CV-N, only some of which are occluded by 2G12 (Table ) (4
). Overall, however, there are clear similarities in the gp120 binding sites of 2G12 and CV-N and probably also in the mechanisms of action of these infection inhibitors. However, 2G12 does not inhibit DC-SIGN binding to gp120 (Table ). This is, again, probably a result of the relatively promiscuous binding of DC-SIGN to gp120, in that DC-SIGN can probably recognize any of the exposed high-mannose glycans, whereas 2G12 is more selective in its interactions. The converse competition between DC-SIGN and 2G12 has not been reported (Table ). Any partial overlap that does occur between the DC-SIGN and 2G12 binding sites could help explain why unusually low concentrations of 2G12 are able to protect some macaques from vaginal challenge with SHIV-89.6P, albeit inconsistently (38
). Much greater concentrations of other anti-Env MAbs are required to achieve the same degree of protection (58
Competition between reagents that bind mannose residues on gp120
Our principal conclusion is that the 2G12 MAb recognizes an epitope that is dependent on the presence of mannose residues on N-linked glycans. In all probability, the epitope is completely composed of sugars, with no involvement of the gp120 peptide backbone. This will need to be confirmed by crystallographic analysis of the 2G12-gp120 complex. However, for several reasons, we believe that 2G12 is not a conventional anti-carbohydrate antibody (26
). Firstly, 2G12 is specific for HIV-1 gp120 and does not, for example, recognize SIV gp120 expressed in the same cells (unpublished data). Secondly, denaturation of gp120 with SDS and DTT causes at least a 500-fold reduction in 2G12 binding, yet the mannose residues are still present on denatured gp120. Thirdly, the N-linked moieties that 2G12 recognizes are present on many extracellular, host proteins. For 2G12 to avoid being self-reactive, it cannot bind with high affinity to just a single N-linked moiety. Furthermore, given the flexibility of N-linked attachments, a binding site involving even two or three moieties would probably not provide enough specificity. Hence, we think that 2G12 recognizes a discontinuous structure that comprises the mannose elements of several individual glycan chains, folded into proximity. Based on our analyses, up to five individual N-linked glycans could be involved in forming the 2G12 epitope.
If we are correct that the 2G12 epitope is a discontinuous structure comprising only carbohydrate residues, it may be very difficult to exploit this information for HIV-1 vaccine development. Common N-linked glycans are rarely immunogenic, and sera from HIV-1-infected individuals do not compete with 2G12 binding to gp120 (65
). In addition, raising anticarbohydrate antibodies of broad specificity could cause problems from the perspective of autoimmunity. On the other hand, if the 2G12 epitope is indeed a discontinuous structure unique to gp120, perhaps that structure could be appropriately immunogenic in the context of a vaccine antigen if it can be further defined and then appropriately presented. After all, the structure was immunogenic in the individual whose immune system made 2G12, and the resulting antibody does recognize and neutralize a broad range of HIV-1 isolates (68
There are very few conserved neutralization epitopes on gp120, yet there is a great need to exploit these limited weaknesses in the otherwise efficient defenses present on gp120 (47
). HIV-1 sequence analysis demonstrates that gp120 glycans are often conserved. Moreover, a loss of five glycosylation sites when SIV was cultured in vitro was reversed when the virus replicated in macaques (21
). This confirms the functional requirement to preserve gp120 glycans, probably to help resist the humoral immune response (54
). Unusual approaches to raising 2G12-like antibodies that focus on carbohydrate chemistry should, therefore, now be explored. For example, a synthetic structure containing clustered mannosyl structures on a peptide scaffold, resembling the recently synthesized trimeric Lewis(y) conjugate (29
), could be considered. Alternatively, peptide mimeotopes of carbohydrate antigens might be a useful technique (1
). Our results also suggest that vaccine design strategies intended to deglycosylate gp120, and thereby uncover hidden neutralization epitopes, should focus on the complex carbohydrates and perhaps leave the high-mannose-containing structures intact, in the hope that 2G12-like antibodies might somehow be induced.