Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structures of the designed V1V2 glycopeptides derived from HIV-1 ZM109 and CAP45 strains.
Figure 2: Chemoenzymatic synthesis of ZM109 V1V2 glycopeptides carrying defined N-glycans at the Asn160 site.
Figure 3: Controlled glycosylation and HPLC separation of monoglycosylated and doubly glycosylated ZM glycopeptides.
Figure 4: Chemoenzymatic synthesis and ESI-MS characterization of the doubly glycosylated ZM glycopeptides.
Figure 5: SPR analysis of the binding of synthetic V1V2 glycopeptides to PG9 or PG16 Fabs.

Similar content being viewed by others

References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kwong, P.D., Mascola, J.R. & Nabel, G.J. Rational design of vaccines to elicit broadly neutralizing antibodies to HIV-1. Cold Spring Harb. Perspect. Med. 1, a007278 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Walker, L.M. & Burton, D.R. Rational antibody-based HIV-1 vaccine design: current approaches and future directions. Curr. Opin. Immunol. 22, 358–366 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Reitter, J.N., Means, R.E. & Desrosiers, R.C. A role for carbohydrates in immune evasion in AIDS. Nat. Med. 4, 679–684 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Trkola, A. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bonsignori, M. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Leonard, C.K. 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).

    CAS  PubMed  Google Scholar 

  16. Zhu, X., Borchers, C., Bienstock, R.J. & Tomer, K.B. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells. Biochemistry 39, 11194–11204 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Go, E.P. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gamblin, D.P., Scanlan, E.M. & Davis, B.G. Glycoprotein synthesis: an update. Chem. Rev. 109, 131–163 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Schmaltz, R.M., Hanson, S.R. & Wong, C.H. Enzymes in the synthesis of glycoconjugates. Chem. Rev. 111, 4259–4307 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang, L.X. & Lomino, J.V. Emerging technologies for making glycan-defined glycoproteins. ACS Chem. Biol. 7, 110–122 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Li, H. 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).

    Article  CAS  PubMed  Google Scholar 

  23. Li, B., Zeng, Y., Hauser, S., Song, H. & Wang, L.X. Highly efficient endoglycosidase-catalyzed synthesis of glycopeptides using oligosaccharide oxazolines as donor substrates. J. Am. Chem. Soc. 127, 9692–9693 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Ochiai, H., Huang, W. & Wang, L.X. Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands. J. Am. Chem. Soc. 130, 13790–13803 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Huang, W. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huang, W., Zhang, X., Ju, T., Cummings, R.D. & Wang, L.X. Expeditious chemoenzymatic synthesis of CD52 glycopeptide antigens. Org. Biomol. Chem. 8, 5224–5233 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Umekawa, M. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zou, G. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Amin, M.N., Huang, W., Mizanur, R.M. & Wang, L.X. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fan, S.Q., Huang, W. & Wang, L.X. Remarkable transglycosylation activity of glycosynthase mutants of Endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae. J. Biol. Chem. 287, 11272–11281 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Huang, W., Giddens, J., Fan, S.Q., Toonstra, C. & Wang, L.X. Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 134, 12308–12318 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lal, A. 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).

    Article  CAS  PubMed  Google Scholar 

  35. Noguchi, M., Tanaka, T., Gyakushi, H., Kobayashi, A. & Shoda, S.I. 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).

    Article  CAS  PubMed  Google Scholar 

  36. Huang, W., Yang, Q., Umekawa, M., Yamamoto, K. & Wang, L.X. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Huang, W., Ochiai, H., Zhang, X. & Wang, L.X. Introducing N-glycans into natural products through a chemoenzymatic approach. Carbohydr. Res. 343, 2903–2913 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Pancera, M. et al. Structural basis for diverse N-glycan recognition and enhanced HIV-1 neutralization by V1/V2-directed antibodies. Nat. Struct. Mol. Biol. http://dx.doi.org/10.1038/nsmb.2600 (2013).

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bachmann, M.F. & Jennings, G.T. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787–796 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Lai-Xi Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures, Tables and Note

Supplementary Results: Supplementary Figures 1–12, Supplementary Table 1, and Supplementary Notes (PDF 1249 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Amin, M., McLellan, J., Huang, W. et al. Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies. Nat Chem Biol 9, 521–526 (2013). https://doi.org/10.1038/nchembio.1288

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1288

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research