Abstract
2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N → A substitution produced significant decreases in 2G12 binding affinity to gp120JR.CSF. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Manal → 2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by other hexoses. The data presented here suggests that the most likely epitope for 2G12 is formed from a specific cluster of mannose residues on the outer face of gp120, with the other glycans playing an indirect role in maintaining epitope conformation. [Journal of Virology, July 2002, pp. 7306–7321, Vol. 76, No. 14.]
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Similar content being viewed by others
References
Burton, D.R., 1997, A vaccine for HIV type 1: the antibody perspective, Proc Natl Acad Sci USA. 94:10018–10023.
Burton, D.R. and Montefiori, D.C., 1997, The antibody response in HIV-1 infection, AIDS. 11Suppl A:S87–98.
Burton, D.R., Pyati, J., Koduri, R., Sharp, S.J., Thornton, G.B., Parren, P.W., Sawyer, L.S., Hendry, R.M., Dunlop, N., Nara, P.L. et al. 1994, Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody, Science. 266:1024–1027.
Conley, A.J., Kessler, J.A., 2nd, Boots, L.J., Tung, J.S., Arnold, B.A., Keller, P.M., Shaw, A.R., and Emini, E.A., 1994, Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody, Proc Natl Acad Sci USA. 91:3348–3352.
Connor, R.I., Korber, B.T., Graham, B.S., Hahn, B.H., Ho, D.D., Walker, B.D., Neumann, A.U., Vermund, S.H., Mestecky, J., Jackson, S., Fenamore, E., Cao, Y., Gao, F., Kalams, S., Kunstman, K.J., McDonald, D., McWilliams, N., Trkola, A., Moore, J.P., and Wolinsky, S.M., 1998, Immunological and virological analyses of persons infected by human immunodeficiency virus type 1 while participating in trials of recombinant gp120 subunit vaccines, J Virol. 72:1552–1576.
Cygler, M., Rose, D.R., and Bundle, D.R., 1991, Recognition of a cell-surface oligosaccharide of pathogenic Salmonella by an antibody Fab fragment, Science. 253:442–445.
D’Souza, M.P., Geyer, S.J., Hanson, C.V., Hendry, R.M., and Milman, G., 1994, Evaluation of monoclonal antibodies to HIV-1 envelope by neutralization and binding assays: an international collaboration, AIDS 8:169–181.
Garcia, K.C., Degano, M., Stanfield, R.L., Brunmark, A., Jackson, M.R., Peterson, P.A., Teyton, L., and Wilson, I.A., 1996, An alphabeta T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex, Science. 274:209–219.
Kostrikis, L.G., Cao, Y., Ngai, H., Moore, J.P., and Ho, D.D., 1996, Quantitative analysis of serum neutralization of human immunodeficiency virus type 1 from subtypes A, B, C, D, E, F, and I: lack of direct correlation between neutralization serotypes and genetic subtypes and evidence for prevalent serum-dependent infectivity enhancement, J Virol. 70:445–458.
Kunert, R., Ruker, F., and Katinger, H., 1998, Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5, AIDS Res Hum Retroviruses. 14:1115–1128.
Kwong, P.D., Wyatt, R., Majeed, S., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A., 2000, Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates, Struct Fold Des. 8:1329–1339.
Kwong, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A., 1998, Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody, Nature. 393:648–659.
Kwong, P.D., Wyatt, R., Sattentau, Q.J., Sodroski, J., and Hendrickson, W.A., 2000, Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus, J Virol. 74:1961–1972.
Lee, R.T. and Lee, Y.C., 2000, Affinity enhancement by multivalent lectin-carbohydrate interaction, Glycoconj J. 17:543–551.
Mascola, J.R., Stiegler, G., VanCott, T.C., Katinger, H., Carpenter, C.B., Hanson, C.E., Beary, H., Hayes, D., Frankel, S.S., Birx, D.L., and Lewis, M.G., 2000, Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies, Nat Med. 6:207–210.
Moog, C., Fleury, H.J., Pellegrin, I., Kirn, A., and Aubertin, A.M., 1997, Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals, J Virol. 71:3734–3741.
Moore, J.P., Cao, Y., Leu, J., Qin, L., Korber, B., and Ho, D.D., 1996, Inter-and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes, J Virol. 70:427–444.
Moore, J.P. and Sodroski, J., 1996, Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein, J Virol. 70:1863–1872.
Moulard, M., Phogat, S.K., Shu, Y., Labrijn, A.F., Xiao, X., Binley, J.M., Zhang, M.Y., Sidorov, I.A., Broder, C.C., Robinson, J., Parren, P.W., Burton, D.R., and Dimitrov, D.S., 2002, Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4-CCR5 complexes, Proc Natl Acad Sci USA. 99:6913–6918.
Myszka, D.G., Sweet, R.W., Hensley, P., Brigham-Burke, M., Kwong, P.D., Hendrickson, W.A., Wyatt, R., Sodroski, I, and Doyle, M.L., 2000, Energetics of the HIV gp120-CD4 binding reaction, Proc Natl Acad Sci USA. 97:9026–9031.
Neth, O., Jack, D.L., Dodds, A.W., Holzel, H., Klein, N.J., and Turner, M.W., 2000, Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition, Infect Immun. 68:688–693.
Parekh, R.B., Dwek, R.A., Thomas, J.R., Opdenakker, G., Rademacher, T.W., Wittwer, A.J., Howard, S.C., Nelson, R., Siegel, N.R., Jennings, M.G. et al. 1989, Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator, Biochemistry. 28:7644–7662.
Parekh, R.B., Tse, A.G., Dwek, R.A., Williams, A.F., and Rademacher, T.W., 1987, Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1, Embo J. 6:1233–1244.
Parker, C.E., Deterding, L.J., Hager-Braun, C., Binley, J.M., Schulke, N., Katinger, H., Moore, J.P., and Tomer, K.B., 2001, Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5, J Virol. 75:10906–10911.
Parren, P.W., Moore, J.P., Burton, D.R., and Sattentau, Q.J., 1999, The neutralizing antibody response to HIV-1: viral evasion and escape from humoral immunity, AIDS. 13Suppl A:S137–S162.
Perkins, S.J., Johnson, L.N., Phillips, D.C., and Dwek, R.A., 1981, The binding of monosaccharide inhibitors to hen egg-white lysozyme by proton magnetic resonance at 270 MHz and analysis by ring-current calculations, Biochem J. 193:553–572.
Poignard, P., Sabbe, R., Picchio, G.R., Wang, M., Gulizia, R.J., Katinger, H., Parren, P.W., Mosier, D.E., and Burton, D.R., 1999, Neutralizing antibodies have limited effects on the control of established HIV-1 infection in vivo, Immunity. 10:431–438.
Roben, P., Moore, J.P., Thali, M., Sodroski, J., Barbas, C.F., 3rd, and Burton, D.R., 1994, Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1, J Virol. 68:4821–4828.
Rudd, P.M. and Dwek, R.A., 1997, Glycosylation: heterogeneity and the 3D structure of proteins, Crit Rev Biochem Mol Biol. 32:1–100.
Sanders, R.W., Venturi, M., Schiffner, L., Kalyanaraman, R., Katinger, H., Lloyd, K.O., Kwong, P.D., and Moore, J.P., 2002, The Mannose-Dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120, J. Virol. 76:7293–7305.
Scanlan, C.N., Pantophlet, R., Wormald, M.R., Ollmann Saphire, E., Stanfield, R., Wilson, I.A., Katinger, H., Dwek, R.A., Rudd, P.M., and Burton, D.R., 2002, The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha 1 → 2 mannose residues on the outer face of gp120, J Virol. 76:7306–7321.
Stiegler, G., Kunert, R., Purtscher, M., Wolbank, S., Voglauer, R., Steindl, F., and Katinger, H., 2001, A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1, AIDS Res Hum Retroviruses. 17:1757–1765.
Taylor, M.E. and Drickamer, K., 1993, Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor, J Biol Chem. 268:399–404.
Thomas, R., Patenaude, S.I., MacKenzie, C.R., To, R., Hirama, T., Young, N.M., and Evans, S.V., 2002, Structure of an anti-blood group A Fv and improvement of its binding affinity without loss of specificity, J Biol Chem. 277:2059–2064.
Trkola, A., Pomales, A.B., Yuan, H., Korber, B., Maddon, P.J., Allaway, G.P., Katinger, H., Barbas, C.F., 3rd, Burton, D.R., Ho, D.D. et al. 1995, Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG, J Virol. 69:6609–6617.
Trkola, A., Purtscher, M., Muster, T., Ballaun, C., Buchacher, A., Sullivan, N., Srinivasan, K., Sodroski, J., Moore, J.P., and Katinger, H., 1996, Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1, J Virol. 70:1100–1108.
Weis, W.I., Taylor, M.E., and Drickamer, K., 1998, The C-type lectin superfamily in the immune system, Immunol Rev. 163:19–34.
Wilson, I.A., Skehel, J.J., and Wiley, D.C., 1981, Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution, Nature. 289:366–373.
Wilson, I.A. and Stanfield, R.L., 1995, A Trojan horse with a sweet tooth, Nat Struct Biol. 2:433–436.
Woods, R.J., Edge, C.J., and Dwek, R.A., 1994, Protein surface oligosaccharides and protein function, Nat Struct Biol. 1:499–501.
Wormald, M.R. and Dwek, R.A., 1999, Glycoproteins: glycan presentation and protein-fold stability, Struct Fold Des. 7:155–160.
Wyatt, R., Kwong, P.D., Desjardins, E., Sweet, R.W., Robinson, J., Hendrickson, W.A., and Sodroski, J.G., 1998, The antigenic structure of the HIV gp120 envelope glycoprotein, Nature. 393:705–711.
Wyatt, R., Sullivan, N., Thali, M., Repke, H., Ho, D., Robinson, I, Posner, M., and Sodroski, J., 1993, Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions, J Virol 67:4557–4565.
Wyss, D.F., Choi, J.S., Li, J., Knoppers, M.H., Willis, K.J., Arulanandam, A.R., Smolyar, A., Reinherz, E.L., and Wagner, G., 1995, Conformation and function of the N-linked glycan in the adhesion domain of human CD2, Science. 269:1273–1278.
Zhu, X., Borchers, C., Bienstock, R.J., and Tomer, K.B., 2000, Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells, Biochemistry. 39:11194–11204.
Zwick, M.B., Labrijn, A.F., Wang, M., Spenlehauer, C., Saphire, E.O., Binley, J.M., Moore, J.P., Stiegler, G., Katinger, H., Burton, D.R., and Parren, P.W., 2001, Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41, J Virol. 75:10892–10905.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Springer Science+Business Media New York
About this paper
Cite this paper
Scanlan, C.N. et al. (2003). The Carbohydrate Epitope of the Neutralizing Anti-HIV-1 Antibody 2G12. In: Axford, J.S. (eds) Glycobiology and Medicine. Advances in Experimental Medicine and Biology, vol 535. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-0065-0_13
Download citation
DOI: https://doi.org/10.1007/978-1-4615-0065-0_13
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4613-4908-2
Online ISBN: 978-1-4615-0065-0
eBook Packages: Springer Book Archive