Glycobiology Advance Access originally published online on May 25, 2005
Glycobiology 2005 15(10):994-1001; doi:10.1093/glycob/cwi083
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Concanavalin A binding to HIV envelope protein is less sensitive to mutations in glycosylation sites than monoclonal antibody 2G12
Present address: UAMS #824, 4301 Markham St., Little Rock, AR 72205
2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205; 3 Department of Chemistry, Ouachita Baptist University, Arkadelphia, AR 71998; and 4 U.S. Military HIV Research Program, Rockville, MD 20850
1 To whom correspondence should be addressed; e-mail: tke{at}uams.edu
Received on March 22, 2005; revised on May 19, 2005; accepted on May 20, 2005
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Abstract |
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Many mannose-binding proteins inhibit divergent strains of human immunodeficiency virus type 1 (HIV-1) in in vitro models of viral infectivity, suggesting that targeting mannose residues in vaccine applications might offset the strain restriction typically observed in antibody responses to HIV vaccine preparations. Concanavalin A (ConA) behaves like neutralizing antibodies that do not interfere with CD4 binding of gp120 but rather with later events in virus entry. The design of mannose-based vaccines, therefore, depends on understanding the mode of binding of ConA to the envelope protein in comparison with other mannose-binding proteins. Here, we further compare the binding affinity and fine specificity of ConA for the envelope protein to that of the human antibody 2G12. The 2G12 antibody is of unusual structure recognizing a cluster of 12 linked mannose residues associated with Man9GlcNAc2. Molecular structure comparison for Man9GlcNAc2 recognition by ConA and 2G12 indicates that 2G12 has a more restricted specificity to high mannose glycans of gp120 which correlates with kinetic analysis assessed by surface plasmon resonance (SPR) and ConA inhibits 2G12 binding to gp120 but 2G12 does not inhibit ConA binding to gp120. ConA binding to Env proteins from four different HIV strains proves significantly less sensitive to mutations in the glycosylation sites than 2G12 binding to the proteins. Thus, antibodies directed toward mannose epitopes reactive with ConA may prove to be more effective in the long run to thwart HIV infection and transmission.
Key words: 2G12 / carbohydrates / concanavalin A / HIV
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Introduction |
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The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein is a highly glycosylated and highly variable surface protein. Glycans expressed on the Env protein of HIV-1 influence its conformational properties (Bolmstedt et al., 1992)
, the infectivity (Turville et al., 2001; Appelmelk et al., 2003), and the immunogenicity of the virus (Pantophlet et al., 2003). An increasing number of proteins that bind high-mannose carbohydrates found on gp120 are known to interfere with the viral life cycle, providing a potential new way of controlling HIV infection (Botos and Wlodawer, 2005). Subsequently, moieties making up the glycan shield have become the subject of study as targets for drugs represented by Cyanovirin-N (CV-N) (Botos and Wlodawer, 2003) and for developing carbohydrate targeting vaccine design strategies (Agadjanyan et al., 1997; Singh et al., 2003; Dudkin et al., 2004, Geng et al., 2004; Li and Wang, 2004; Mandal et al., 2004; Monzavi-Karbassi et al., 2004).
An accurate assessment of the available antigenic epitopes, their distribution on the Env protein and potential fine specificity of vaccine-induced immune responses is important to successfully design an appropriate carbohydrate targeting vaccine. There is complexity in glycosylation within divergent strains of HIV Env. Not all potential glycosylation sites on the Env protein are fully occupied (Zhu et al., 2000). The protein context may not favor glycosylation at some potential glycosylation sites whereas others may be occluded sterically, thus preventing carbohydrate attachment.
High-mannose oligosaccharides also vary, having two to six mannose residues attached to a core. This organization of high mannose moieties implies that mannose-binding proteins will recognize different structural features or determinants on the glycan. This is evident from the crystal structure analysis of many mannose-binding proteins, including the anti-HIV antibody 2G12, C-type lectin dendritic cells (DC)-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) that has specificity for both Man9GlcNAc2 (Man-9) and the oligosaccharide Lewis x (Feinberg et al., 2001, Geijtenbeek et al., 2001), and concanavalin A (ConA) (Moothoo et al., 1999). The molecular characterization of the recognition properties of such molecules is a critical first step in carbohydrate-based vaccine design strategies (Lee and Lee, 2000; Adams et al., 2004; Dudkin et al., 2004).
Here, we further contrast the binding properties of ConA to Env with that of 2G12. The recognition by 2G12 relies on the Man1,2 linkage (Wang et al., 2004). In comparison, ConA, which also binds to Man1,2Man, has a 40 times higher affinity for the trisaccharide Man(1–3)[Man(1–6)] Man (Brewer and Bhattacharyya, 1986; Mandal et al., 1994). Furthermore, ConA displays enhanced affinity for Man9 relative to the trimannoside (Gupta et al., 1996). As this structure is associated with other high-mannose type N-glycans on gp120 that range from Man5 to Man9, it is anticipated that ConA will bind to numerous sites on Env from all HIV clades. In keeping with this recognition, model 2G12 binds non- or only partially overlapping sites associated with ConA binding to gp120. In this context, ConA is not affected by the diversity in the glycan shield of HIV isolates (Dacheux et al., 2004) suggesting that ConA binding is less sensitive to isolate mutations than is the monoclonal antibody 2G12.
Studies on carbohydrate directed human antibody responses imply that in vivo protection depends on the avidity of antibodies to pathogen (Usinger and Lucas, 1999) suggesting that low affinity antibodies may still have optimal avidity. Our surface plasmon resonance (SPR) analysis indicates that the dissociation constants for 2G12 and ConA on gp120 are similar despite the differences in their specificities and affinities. These studies, therefore, further imply that antibodies that mimic ConA binding tuned to an optimal avidity might be suitable for affecting HIV infection.
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Results |
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Fine specificity of ConA and 2G12 for mannose residues on Man9GlcNAc2
ConA is the most extensively studied legume lectin. The crystal structures of the complexes of ConA with a series of carbohydrates have been solved: methyl–D-mannopyranoside (Naismith et al., 1994)
, Man(1–3)[Man(1–6)]Man (which is the trimannoside ConA epitope of N-linked carbohydrates) (Loris et al., 1996), the pentasaccharide GlcNAc(1–2)Man(1–3)[GlcNAc(1–2)Man(1–6)]Man (Moothoo and Naismith, 1998), the dimannoside Man(1–2) Man(1-O)Me (Moothoo et al., 1999), and Man(1–6) Man(1-O)Me and Man(1–3)Man(1-O)Me, the two dimannoside parts of the highly specific ConA epitope Man(1–3) [Man(1–6)]Man(1-O)Me (Bouckaert et al., 1999).
Crystallographic analysis of 2G12 in complex with Man9 indicates that 2G12 contacts four sugars (3, 4, C, and D1) in the D1 arm of the Man9GlcNAc2 complex (Figure 1). The terminal 
-Man-(1–2)-Man disaccharide (D1) accounts for the majority of these contacts. ConA can also bind to -Man-(1–2)-Man disaccharide, forming complexes more tightly than do its 1–3 and 1–6 linked counterparts (Moothoo et al., 1999). As ConA in complex Man9 has not been reported crystallographically, we explored possible ways in which ConA may bind to Man9 by considering that ConA recognizes the pentasaccharide core (ß-GlcNAc-(1–2)--Man-(1–3)-[ß-GlcNAc-(1–2)--Man-(1–6)]-Man) (Moothoo and Naismith, 1998). The linkages for this pentasaccahride suggest that ConA can bind the Man9 structure involving D2, A, 4', B, and D3 (Figure 1). Superposition of the atoms defining the linkage atoms of the pentasaccharide with the Man9 structure in complex with 2G12 (Calarese et al., 2003) results in a root-mean-square (RMS) of 0.6 A, suggesting that the general positioning of the corresponding linked monomers in the Man9 structure are very similar defining the putative higher affinity ConA epitope on Man9 (Gupta et al., 1996) (Figure 1).
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Asymmetric inhibition of ConA and 2G12 binding to gp120
The high affinity-binding site for 2G12 consists of a set of 3 Man9 glycans in a particular conformation dictated by the glycosylation of the Env protein, defining a single conformational epitope (Calarese et al., 2003). In contrast, ConA recognizes any mannose rich glycan structure on gp120 contributing to its high avidity. The 2G12 epitope may, therefore, be considered a subset of the ConA binding sites on gp120. In addition, there may be multiple binding sites per single glycan. In this case, multiplicity in binding can compensate for weak affinity of individual sites, defining the avidity of ConA.
To demonstrate the relationship between the binding-sites on gp120 for ConA and the 2G12 epitope, we studied the two ligands in mutual inhibition assays. Before addition of 2G12, the gp120-coated plate was preincubated with ConA at concentrations ranging from 1 to 1,500 times the concentration of 2G12 (one which had previously been shown to yield sub half-maximal binding, data not shown) with specific binding of 2G12 detected as described above. Similarly, the gp120-coated plate was preincubated with 2G12 in the same concentration range before addition of biotinylated ConA, whose binding was detected as described above. ConA inhibited up to 80% the binding of 2G12 to a mixture of four different recombinant gp120 reaching saturation at an inhibitor : ligand ratio of 30 as analyzed by enzyme link immunosorbent assay (ELISA) (Figure 2). Consistent with the high specificity of its epitope, 2G12 could not inhibit ConA interaction with gp120 at the inverse ratios further indicating that there are more ConA reactive sites on gp120 than there is for 2G12. The inhibition curve did not reach saturation up to inhibitor : ligand ratio of 1000 (Figure 2).
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SPR analysis of ConA and 2G12 binding to gp120
The sensitivity of ConA and 2G12 epitopes to changes in the glycosylation profile of the Env protein both as mutations in N-glycosylation sites and as variations in the glycan structure was compared next. The binding to three gp120 recombinant glycoproteins from primary isolates of HIV and one from a laboratory isolate (BAL) was measured by SPR. Because the kinetics of the association phase were too complex to derive a meaningful association rate, only the dissociation rate was determined as a measure of the stability of complex formation. Biotinylated lectin (ConA) or biotinylated 2G12 was immobilized on an SA chip. The different HIV gp120 glycoproteins were injected over the surface at 81 and 28 nM using 20 µL/min flow rate (Figure 3). Although the number of tested gp120 proteins was very small, a clear-cut difference in the variation of the dissociation rates for 2G12 and ConA was observed (Figure 4). The chi-square value is a standard statistical measure of the closeness of fit. For good fitting to ideal data, chi2 is of the same order of magnitude as the noise in resonance units (RU; typically <2).
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The dissociation rate for 2G12 (1.4–10.4 x 10–4 s–1) varied over a five times wider range than that for ConA (1.49–2.22 x 10–4 s–1), suggesting that the avidity of ConA is not as sensitive to variation in the glycosylation pattern. The number and position of the potential glycosylation sites for 93TH975 and CM235 based upon sequence alignment of predicted glycosylation sites are very similar (Figure 5), which was reflected by their close dissociation rates for ConA. At the same time, CM235 dissociated 4.7 times faster than 93TH975 from 2G12. These gp120 have the same N-glycosylation sites when the 2G12 epitope is considered, but CM235 belongs to clade E, which is known to have low affinity for 2G12 because of an additional disulfide bond in the fourth variable loop (Figure 5, lower panel). The laboratory strain BAL has all five sites available and displays the most stable binding for both 2G12 and ConA. These data suggest that despite the expected enhanced affinity to the Env protein by 2G12 because of the clustered presentation of mannosyl residues reactive with 2G12, antibodies that display reactivity like ConA may still be capable of effectively interfering with viral infection or viral transmission.
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Discussion |
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There is strong evidence that lectins bind specifically to HIV-1 associated sugar epitopes (Bewley et al., 2004)
permitting a better understanding of the Env protein carbohydrate profiles which may be targeted in HIV vaccine design. Structural results indicate that the fine specificity of 2G12 is similar to that of cyanovirin (Botos and Wlodawer, 2003; Calarese et al., 2003; Adams et al., 2004). Crystallographic analyses indicate that ConA binds to the Mana1–6[Mana1–3] Man structure as does DC-SIGN (Botos and Wlodawer, 2005). ConA and DC-SIGN have been shown to have also a broader specificity. ConA can bind Man (1–2)Man -OMe (Moothoo et al., 1999) and methyl-3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (Loris et al., 1996) each in two different orientations and the nominal specificity—the pentasaccharide core (ß-GlcNAc-(1–2)--Man-(1–3)-[ß-GlcNAc-(1–2)--Man-(1–6)]-Man) contains GlcNAc residues that participate in the binding (Moothoo and Naismith, 1998). DC-SIGN was found to bind to mannan, fucose, and all fucosylated lactosamines excluding sLex and sLea (Appelmelk et al., 2003). In contrast to other C-type lectins, DC-SIGN binds internal trisaccharides of high-mannose oligosaccharides and does not bind terminal -Man-(1–2)-man residues (Feinberg et al., 2001).
The 2G12 epitope is known to be formed by N-linked oligomannose glycans on gp120 at three major glycosylation sites (N295 in the C2 region, N332 in the C3 region, and N392 in the V4 loop) and two additional nonessential sites (N339 in the C3 domain and N386 in the V4 loop) (Sanders et al., 2002; Scanlan et al., 2002; Dacheux et al., 2004). Thus, it may be more appropriate to consider DC-SIGN and ConA as examples of lectin type polyspecificity as opposed to the highly specific binding of 2G12. The latter is due not to its fine specificity (which is shared with cyanovirin and even partially with ConA) but rather to the recognition of particular subsets of the high mannose glycans on gp120 forming an array of glycans with a unique conformation.
N-linked glycosylation patterns in HIV Env are complicated by the fact that the host cells can affect glycosylation differently (Zhang et al., 2004). DC-SIGN preferentially binds to Env protein rich in high mannose oligosaccharides, which are typically produced by peripheral blood mononuclear cells and T cells, compared to macrophage-produced gp120, which contains more complex carbohydrates including lactosaminoglycans (Willey et al., 1996). The proteins expressed in the baculovirus system (including gp120 from CM235, 93TH975, BAL, and CN54 used in this study) are known to have a skewed glycosylation pattern because the insect cells trim oligomannose but do not build the complex type of glycans yielding paucimannose glycans (Altmann et al., 1999). The mannose rich glycans though are indistinguishable from those found on mammalian cells, thus insect cell expressed HIV Env protein probably still carries all of the glycans necessary for the formation of the 2G12 epitope.
The affinity (Kd) for the binding of mono- and oligosaccharides to most lectins, including ConA, is in the range 10–3 to 10–6 M. The binding to a dense cluster of oligomannose side chains, though, can reach much higher avidity which is apparent as ConA competes with 2G12 for gp120 binding (Figure 2). The incomplete inhibition (80%) could be due to the differences in fine specificity between ConA and 2G12 (Figure 1). Thus on a dense cluster of binding sites, the multimeric lectin may be functionally equivalent to a high affinity antibody. The typical anti-carbohydrate antibodies that resemble lectins (exemplified here by ConA) compensate low affinity for a single binding site by high avidity for the whole molecule. Increasing avidity can certainly augment even high affinity antibodies exemplified by recent results in which isotype switched 2G12IgM inhibited HIV-1 infection of peripheral blood mononuclear cell cultures up to 28-fold more efficiently than the corresponding IgG and neutralized all of the primary isolates tested (Wolbank et al., 2003).
Our results suggest that 2G12 binds to a subset of the ConA binding sites. At the inverse ratios of inhibitor : ligand, 2G12 only partially inhibited ConA although it has a higher affinity epitope. The apparently nonsaturable inhibition curve may be due to the existence of additional binding sites for ConA with varying but lower affinities. These results parallel data on mutual inhibition of 2G12 and CV-N (Dey et al., 2000) and on 2G12 and DC-SIGN (Sanders et al., 2002) although in the latter case inhibition of DC-SIGN by 2G12 was not tested. ConA binding to gp120 (like that of CV-N and DC-SIGN [Hong et al., 2002]) represents the lectin type of broad specificity, low affinity, high avidity interaction with multiple binding sites per Env molecule, whereas the 2G12 type of binding is of high affinity and high specificity with one primary binding site.
The binding profiles of ConA for gp120 isolates appear to be affected less than those of 2G12 by the glycan shield diversity for the isolates tested (Figure 4) as the dissociation rate ranged between 1.49 x 10–4 s–1 and 2.22 x 10–4 s–1 for ConA and from 1.4 x 10–4 s–1 to 10.4 x 10–4 s–1 for 2G12. The dissociation rate was measured rather than the affinity because of the complex binding in the association phase, which was difficult to interpret. On the other hand, the dissociation phase depends mostly on the binding energy. The laboratory BAL strain, which possesses a full set of glycosylation sites participating in the formation of the 2G12 epitope, showed the strongest binding to both 2G12 and ConA. The clade E isolate CM235 Env, which has an additional disulfide bond in the fourth variable loop forming a structural feature preventing the binding of 2G12 to gp120 (Trkola et al., 1995), forms the least stable complex with 2G12 but binds much better to ConA. These results suggest that lectins such as ConA and "lectin like" antibodies might be less susceptible to antigenic drift and glycan microheterogeneity. Mannose-binding antibodies are indeed effective in blocking HIV. Antibodies raised to Candida albicans mannan have been shown to inhibit HIV infection below micromolar concentrations (Muller et al., 1990; Muller et al., 1991).
The specificity in the "silent" face of HIV Env lies in the high density of glycans expressing terminal mannose residues (Weis et al., 1998), a subset of which produce the epitope accessible to 2G12. Its specificity, though, is due more to its conformational character than to its sugar composition. Yet another aspect of specificity is related to the avidity provided by the density of terminal mannose residues independent of their spatial arrangement. With varying levels of expression of an epitope, it may become visible to the immune system only on cells expressing levels above an antigen density threshold at least in some cases determined by the avidity of induced antibodies.
Typical self antigens like sTn (Lloyd, 1991), HER2/neu (Strasser et al., 2001), and MUC1(Gendler, 2001) are expressed at high levels on breast cancer cells in some patients and induce antibody responses in substantial proportion of them (von Mensdorff-Pouilly et al., 1996; Disis et al., 1997). Increasing the avidity of the induced antibodies above an optimal level may also be unnecessary. In anti-viral responses, Bachman et al. (1997) found that in vivo protective activity was governed by another threshold phenomenon—maximal avidity threshold. Analysis of affinities of carbohydrate reactive antibodies suggest that this threshold should be around 108M–1 (Sun et al., 2001, Usinger & Lucas 1999) which is far less than the target threshold set by affinity estimates based on 2G12.
Present strategies have evolved to synthesizing a full Man9 structure because of the number of Man1,2 constituents (Singh et al., 2003). However, the probability of generating conformation specific 2G12-like antibodies with its unusual VH–VH interface is much lower than the one of eliciting just antibodies to the intrinsic structure of Man9 containing Man1,2 constituents and concomitant di- and trisaccharides recognized by ConA (Figure 1). Therefore, immunization with Man9 will probably lead to ConA like antibodies and not 2G12 like antibodies. Multivalent forms of Man9 may be more relevant to the induction of spatially clustered epitopes (Dudkin et al., 2004) but this requires mimicking the conformational epitopes without creating neoepitopes because of the multivalent synthesis. Comparing ConA reactivity, which is functionally similar to neutralizing antibodies, with 2G12 indicates that a strategy to induce "lectin like" antibodies with avidity specificity based on physiological thresholds of antibody activity may be more effective in inhibiting a wide range of isolates as the availability of a high density of oligomannose glycans will be a constant feature on the silent face of the Env protein.
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Materials and methods |
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Reagents
ConA was obtained from Sigma Chemicals (St. Louis, MO) (Cat N: L 6397). Antibody 2G12 was kindly provided by Dr. H. Katinger. Recombinant gp120 was supplied by the AIDS Research and Reference Reagent Program (CM235, 93TH975, BAL, CN54). The primary isolate Env glycoproteins were expressed in insect cells whereas the laboratory isolate (BAL) was expressed in human cells (HEK293). Biotinylated ConA was purchased from Vector, Burlingame, CA. 2G12 was biotinylated by incubating 1 mg/mL with 2 mg/mL Biotin–N-hydroxysuccinimide ester for 1 h at room temperature followed by an extensive dialysis against phosphate buffered saline (PBS).
Biosensor experiments
In the gp120 binding experiments a biosensor chip coupled with streptavidin (Biacore SA chip, Biacore International SA, Neuchatel, Switzerland) was used. Biotinylated lectin or antibody was bound to the surface of the chip at ligand density 800RU. Then different gp120 (at concentrations 81 or 28 nM) were injected next at 20 µL/min flow rate in PBS, 0.5 M NaCl, 0.005% surfactant P20 for 4 min at flow rate of 20 µL/min, followed by 4 min of dissociation phase before regeneration. The surface was regenerated using 10 mM NaOH, 0.1% sodium dodecyl sulfate solution for 2 min. This treatment led to a partial stripping of the biotinylated ligand and denaturing of the remaining molecules which was verified by the loss of binding to the surface after the treatment. Intact ligand was next coupled to the chip surface for each individual experiment at ligand density of 800 RU before the next cycle of measurement. Data preparation for analysis and single exponential dissociation-rate fits of the direct binding data were performed using BIA Evaluation 3.1, Microsoft Excel 4.0 Macros.
Binding and competition ELISA
Immulon-2 microtitre plates were coated with 0.1 mg/mL gp120 in 0.05 M NaHCO3 (pH 8.5) overnight at 4°C. The four separate gp120 proteins were mixed together (at final concentration of 0.1 mg/mL or 0.025 mg/mL of each individual protein) before coating the microtiter plates. All binding and competition experiments were carried out at room temperature. The buffer used to block the nonspecific binding contained 0.5% BSA and 0.2% Tween 20 in PBS. The inhibitors were preincubated in the coated plate for 1 h before adding other ligand for incubation for an additional 2 h. All binding steps were carried out in the same blocking buffer. In the ConA experiment, biotinylated ConA was used. The 2G12 binding was demonstrated by an alkaline phosphatase conjugated anti-human IgG secondary antibody (Sigma), the ConA–biotin binding was detected by alkaline phosphatase conjugated streptavidin (Sigma), and 0.1% p-nitrophenyl phosphate in 0.1 M diethanolamine 10 mM MgCl2 was used as a chromogenic substrate, read at 405 nm.
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Acknowledgements |
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We thank Dr. Herman Katinger from The Institute of Applied Microbiology, Vienna, Austria for providing 2G12 antibody and Behjatolah Monzavi-Karbassi for critical reading of the manuscript. This work was supported by NIH grant R01AI049092, the Arkansas BRIN Program, and the Arkansas BioSciences Institute.
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Abbreviations |
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ConA, concanavalin A; CV-N, cyanovirin N; DC-SIGN, dendritic cells-specific intercellular adhesion molecule-3 grabbing nonintegrin; ELISA, enzyme link immunosorbent assay; Env, envelope; Man9, oligomannose 9; PBS, phosphate buffered saline; RMS, root-mean-square; RU, resonance units; SPR, surface plasmon resonance
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References |
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