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Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies

Nature Chemical Biology volume 9, pages 521526 (2013) | Download Citation


A new class of glycan-reactive HIV-neutralizing antibodies, including PG9 and PG16, has been recently discovered that seem to recognize previously uncharacterized glycopeptide epitopes on HIV-1 gp120. However, further characterization and reconstitution of the precise neutralizing epitopes are complicated by the heterogeneity of glycosylation. We report here the design, synthesis and antigenic evaluation of new cyclic V1V2 glycopeptides carrying defined N-linked glycans at the conserved glycosylation sites (Asn160 and Asn156 or Asn173) derived from gp120 of two HIV-1 isolates. Antibody binding studies confirmed the necessity of a Man5GlcNAc2 glycan at Asn160 for recognition by PG9 and PG16 and further revealed a critical role of a sialylated N-glycan at the secondary site (Asn156 or Asn173) in the context of glycopeptides for antibody binding. In addition to defining the glycan specificities of PG9 and PG16, the identified synthetic glycopeptides provide a valuable template for HIV-1 vaccine design.

  • Compound C38H63NO30


  • Compound C62H103NO50


  • Compound C76H123N5O56

    2-Methyl-{(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonic acid)-(2-6)-β-D-galactopyranosyl-(1-4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1-2)-α-D-mannopyranosyl-(1-6)-[(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonic acid)-(2-6)-β-D-galactopyranosyl-(1-4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1-2)-α-D-mannopyranosyl-(1-3)-]-β-D-mannopyranosyl-(1-4)-1,2-dideoxy-α-D-glucopyrano}-[2,1-d]-oxazoline

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  1. 1.

    et al. A blueprint for HIV vaccine discovery. Cell Host Microbe 12, 396–407 (2012).

  2. 2.

    , & Rational design of vaccines to elicit broadly neutralizing antibodies to HIV-1. Cold Spring Harb. Perspect. Med. 1, a007278 (2011).

  3. 3.

    & Rational antibody-based HIV-1 vaccine design: current approaches and future directions. Curr. Opin. Immunol. 22, 358–366 (2010).

  4. 4.

    Identifying epitopes of HIV-1 that induce protective antibodies. Nat. Rev. Immunol. 4, 199–210 (2004).

  5. 5.

    , & A role for carbohydrates in immune evasion in AIDS. Nat. Med. 4, 679–684 (1998).

  6. 6.

    et al. Antibody neutralization and escape by HIV-1. Nature 422, 307–312 (2003).

  7. 7.

    et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70, 1100–1108 (1996).

  8. 8.

    et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 2065–2071 (2003).

  9. 9.

    et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

  10. 10.

    & Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16. J. Virol. 84, 10510–10521 (2010).

  11. 11.

    et al. Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors. J. Virol. 85, 9998–10009 (2011).

  12. 12.

    et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466–470 (2011).

  13. 13.

    et al. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature 480, 336–343 (2011).

  14. 14.

    et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science 334, 1097–1103 (2011).

  15. 15.

    et al. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J. Biol. Chem. 265, 10373–10382 (1990).

  16. 16.

    , , & Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells. Biochemistry 39, 11194–11204 (2000).

  17. 17.

    et al. Glycosylation site–specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility. J. Proteome Res. 7, 1660–1674 (2008).

  18. 18.

    , & Glycoprotein synthesis: an update. Chem. Rev. 109, 131–163 (2009).

  19. 19.

    , & Enzymes in the synthesis of glycoconjugates. Chem. Rev. 111, 4259–4307 (2011).

  20. 20.

    Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation. Carbohydr. Res. 343, 1509–1522 (2008).

  21. 21.

    & Emerging technologies for making glycan-defined glycoproteins. ACS Chem. Biol. 7, 110–122 (2012).

  22. 22.

    et al. Chemoenzymatic synthesis of HIV-1 V3 glycopeptides carrying two N-glycans and effects of glycosylation on the peptide domain. J. Org. Chem. 70, 9990–9996 (2005).

  23. 23.

    , , , & Highly efficient endoglycosidase-catalyzed synthesis of glycopeptides using oligosaccharide oxazolines as donor substrates. J. Am. Chem. Soc. 127, 9692–9693 (2005).

  24. 24.

    , & Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands. J. Am. Chem. Soc. 130, 13790–13803 (2008).

  25. 25.

    et al. Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J. Biol. Chem. 283, 4469–4479 (2008).

  26. 26.

    et al. Glycosynthases enable a highly efficient chemoenzymatic synthesis of N-glycoproteins carrying intact natural N-glycans. J. Am. Chem. Soc. 131, 2214–2223 (2009).

  27. 27.

    , , , & Expeditious chemoenzymatic synthesis of CD52 glycopeptide antigens. Org. Biomol. Chem. 8, 5224–5233 (2010).

  28. 28.

    et al. Efficient glycosynthase mutant derived from Mucor hiemalis endo-β-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan. J. Biol. Chem. 285, 511–521 (2010).

  29. 29.

    et al. A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat. Chem. Biol. 6, 264–266 (2010).

  30. 30.

    et al. Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor. J. Am. Chem. Soc. 133, 18975–18991 (2011).

  31. 31.

    , , & Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control. J. Am. Chem. Soc. 133, 14404–14417 (2011).

  32. 32.

    , & Remarkable transglycosylation activity of glycosynthase mutants of Endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae. J. Biol. Chem. 287, 11272–11281 (2012).

  33. 33.

    , , , & Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 134, 12308–12318 (2012).

  34. 34.

    et al. Substrate specificities of recombinant murine Golgi α1, 2-mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing α1,2-mannosidases. Glycobiology 8, 981–995 (1998).

  35. 35.

    , , , & Efficient synthesis of sugar oxazolines from unprotected N-acetyl-2-amino sugars by using chloroformamidinium reagent in water. J. Org. Chem. 74, 2210–2212 (2009).

  36. 36.

    , , , & Arthrobacter endo-β-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C. ChemBioChem 11, 1350–1355 (2010).

  37. 37.

    , , & Introducing N-glycans into natural products through a chemoenzymatic approach. Carbohydr. Res. 343, 2903–2913 (2008).

  38. 38.

    Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43R–56R (2002).

  39. 39.

    et al. Structural basis for diverse N-glycan recognition and enhanced HIV-1 neutralization by V1/V2-directed antibodies. Nat. Struct. Mol. Biol. (2013).

  40. 40.

    et al. Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies. Proc. Natl. Acad. Sci. USA 109, E3268–E3277 (2012).

  41. 41.

    & Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787–796 (2010).

  42. 42.

    et al. Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9. Proc. Natl. Acad. Sci. USA 110, 4351–4356 (2013).

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We thank S. Fan (University of Maryland) for providing the recombinant Endo-D and K. Moremen and Y. Xiang (University of Georgia) for providing the recombinant mouse α-1,2-mannosidase. This work is supported in parts by grants from the National Institute of Allergy and Infectious Diseases (NIAID) (US National Institutes of Health (NIH) grant 1R21AI101035 to L.-X.W.), the International AIDS Vaccine Initiative's Neutralizing Antibody Consortium and by the Intramural Research Program of the Vaccine Research Center, NIAID-NIH.

Author information


  1. Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Mohammed N Amin
    • , Wei Huang
    • , Jared Orwenyo
    •  & Lai-Xi Wang
  2. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Mohammed N Amin
    • , Wei Huang
    • , Jared Orwenyo
    •  & Lai-Xi Wang
  3. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    • Jason S McLellan
    •  & Peter D Kwong
  4. Department of Immunology and Microbial Science, International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.

    • Dennis R Burton
  5. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA.

    • Dennis R Burton
  6. IAVI, New York, New York, USA.

    • Wayne C Koff


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M.N.A., J.S.M., W.H., P.D.K. and L.-X.W. designed the research and analyzed the data; M.N.A., J.S.M., W.H. and J.O. performed the research; L.-X.W. conceived the idea and supervised the research; D.R.B. and W.C.K. contributed PG9 and PG16 antibodies; L.-X.W. and M.N.A. wrote the manuscript; all of the authors contributed to revisions of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lai-Xi Wang.

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