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

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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

CK Leonard, MW Spellman, L Riddle, RJ Harris, JN Thomas and TJ Gregory
Department of Medicinal Chemistry, Genentech, Inc., South San Francisco, California 94080.

This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.
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				J. Balzarini, K. Van Laethem, D. Daelemans, S. Hatse, A. Bugatti, M. Rusnati, Y. Igarashi, T. Oki, and D. Schols Pradimicin A, a Carbohydrate-Binding Nonpeptidic Lead Compound for Treatment of Infections with Viruses with Highly Glycosylated Envelopes, Such as Human Immunodeficiency Virus J. Virol., January 1, 2007; 81(1): 362 - 373. [Abstract] [Full Text] [PDF]

				J. Balzarini, K. Van Laethem, W. J. Peumans, E. J. M. Van Damme, A. Bolmstedt, F. Gago, and D. Schols Mutational Pathways, Resistance Profile, and Side Effects of Cyanovirin Relative to Human Immunodeficiency Virus Type 1 Strains with N-Glycan Deletions in Their gp120 Envelopes. J. Virol., September 1, 2006; 80(17): 8411 - 8421. [Abstract] [Full Text] [PDF]

				A. Duda, D. Luftenegger, T. Pietschmann, and D. Lindemann Characterization of the prototype foamy virus envelope glycoprotein receptor-binding domain. J. Virol., August 1, 2006; 80(16): 8158 - 8167. [Abstract] [Full Text] [PDF]

				U. Abdel-Motal, S. Wang, S. Lu, K. Wigglesworth, and U. Galili Increased Immunogenicity of Human Immunodeficiency Virus gp120 Engineered To Express Gal{alpha}1-3Gal{beta}1-4GlcNAc-R Epitopes J. Virol., July 15, 2006; 80(14): 6943 - 6951. [Abstract] [Full Text] [PDF]

				V. Briz, E. Poveda, and V. Soriano HIV entry inhibitors: mechanisms of action and resistance pathways J. Antimicrob. Chemother., April 1, 2006; 57(4): 619 - 627. [Abstract] [Full Text] [PDF]

				G. F. Doncel Exploiting common targets in human fertilization and HIV infection: development of novel contraceptive microbicides Hum. Reprod. Update, March 1, 2006; 12(2): 103 - 117. [Abstract] [Full Text] [PDF]

				W.M. Blay, S. Gnanakaran, B. Foley, N. A. Doria-Rose, B. T. Korber, and N. L. Haigwood Consistent Patterns of Change during the Divergence of Human Immunodeficiency Virus Type 1 Envelope from That of the Inoculated Virus in Simian/Human Immunodeficiency Virus-Infected Macaques J. Virol., January 15, 2006; 80(2): 999 - 1014. [Abstract] [Full Text] [PDF]

				J. Balzarini, K. Van Laethem, S. Hatse, M. Froeyen, W. Peumans, E. Van Damme, and D. Schols Carbohydrate-binding Agents Cause Deletions of Highly Conserved Glycosylation Sites in HIV GP120: A NEW THERAPEUTIC CONCEPT TO HIT THE ACHILLES HEEL OF HIV J. Biol. Chem., December 9, 2005; 280(49): 41005 - 41014. [Abstract] [Full Text] [PDF]

				J. Meschi, E. C. Crouch, P. Skolnik, K. Yahya, U. Holmskov, R. Leth-Larsen, I. Tornoe, T. Tecle, M. R. White, and K. L. Hartshorn Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication J. Gen. Virol., November 1, 2005; 86(11): 3097 - 3107. [Abstract] [Full Text] [PDF]

				C. Pikora, C. Wittish, and R. C. Desrosiers Identification of Two N-Linked Glycosylation Sites within the Core of the Simian Immunodeficiency Virus Glycoprotein Whose Removal Enhances Sensitivity to Soluble CD4 J. Virol., October 1, 2005; 79(19): 12575 - 12583. [Abstract] [Full Text] [PDF]

				D. C. Williams Jr., J. Y. Lee, M. Cai, C. A. Bewley, and G. M. Clore Crystal Structures of the HIV-1 Inhibitory Cyanobacterial Protein MVL Free and Bound to Man3GlcNAc2: STRUCTURAL BASIS FOR SPECIFICITY AND HIGH-AFFINITY BINDING TO THE CORE PENTASACCHARIDE FROM N-LINKED OLIGOMANNOSIDE J. Biol. Chem., August 12, 2005; 280(32): 29269 - 29276. [Abstract] [Full Text] [PDF]

				P. Rusert, H. Kuster, B. Joos, B. Misselwitz, C. Gujer, C. Leemann, M. Fischer, G. Stiegler, H. Katinger, W. C. Olson, et al. Virus Isolates during Acute and Chronic Human Immunodeficiency Virus Type 1 Infection Show Distinct Patterns of Sensitivity to Entry Inhibitors J. Virol., July 1, 2005; 79(13): 8454 - 8469. [Abstract] [Full Text] [PDF]

				D. Luftenegger, M. Picard-Maureau, N. Stanke, A. Rethwilm, and D. Lindemann Analysis and Function of Prototype Foamy Virus Envelope N Glycosylation J. Virol., June 15, 2005; 79(12): 7664 - 7672. [Abstract] [Full Text] [PDF]

				M. Witvrouw, V. Fikkert, A. Hantson, C. Pannecouque, B. R. O'Keefe, J. McMahon, L. Stamatatos, E. de Clercq, and A. Bolmstedt Resistance of Human Immunodeficiency Virus Type 1 to the High-Mannose Binding Agents Cyanovirin N and Concanavalin A J. Virol., June 15, 2005; 79(12): 7777 - 7784. [Abstract] [Full Text] [PDF]

				J. Martin-Garcia, S. Cocklin, I. M. Chaiken, and F. Gonzalez-Scarano Interaction with CD4 and Antibodies to CD4-Induced Epitopes of the Envelope gp120 from a Microglial Cell-Adapted Human Immunodeficiency Virus Type 1 Isolate J. Virol., June 1, 2005; 79(11): 6703 - 6713. [Abstract] [Full Text] [PDF]

				K. M. Copeland, A. J. Elliot, and R. S. Daniels Functional Chimeras of Human Immunodeficiency Virus Type 1 gp120 and Influenza A Virus (H3) Hemagglutinin J. Virol., May 15, 2005; 79(10): 6459 - 6471. [Abstract] [Full Text] [PDF]

				X. Ren, J. Sodroski, and X. Yang An Unrelated Monoclonal Antibody Neutralizes Human Immunodeficiency Virus Type 1 by Binding to an Artificial Epitope Engineered in a Functionally Neutral Region of the Viral Envelope Glycoproteins J. Virol., May 1, 2005; 79(9): 5616 - 5624. [Abstract] [Full Text] [PDF]

				G. A. Snyder, J. Ford, P. Torabi-Parizi, J. A. Arthos, P. Schuck, M. Colonna, and P. D. Sun Characterization of DC-SIGN/R Interaction with Human Immunodeficiency Virus Type 1 gp120 and ICAM Molecules Favors the Receptor's Role as an Antigen-Capturing Rather than an Adhesion Receptor J. Virol., April 15, 2005; 79(8): 4589 - 4598. [Abstract] [Full Text] [PDF]