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Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes¹

Hualin Li^*, Peter C. Chien, Jr.^*, Michael Tuen^*, Maria Luisa Visciano^*, Sandra Cohen^*, Steven Blais, Chong-Feng Xu, Hui-Tang Zhang and Catarina E. Hioe^2,*

^* Department of Veterans Affairs New York Harbor Healthcare System and Department of Pathology, Department of Pharmacology and Kimmel Center for Biology and Medicine at the Skirball Institute, and Department of Biochemistry, New York University School of Medicine, New York, NY 10016

The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by funds from a Merit Review Award and the Research Enhancement Award Program of the U.S. Department of Veterans Affairs, New York University Center for AIDS Research Immunology Core (AI-27742), the Training Program in TB and HIV Prevention and Treatment (D43 TWO1409), and by National Institutes of Health Grant AI48371. The mass spectrometry analysis was supported in part by National Institutes of Health Grants NS050276 and RR14662 to Dr. Thomas A. Neubert (Skirball Institute, New York University School of Medicine, New York, NY).

² Address correspondence and reprint requests to Dr. Catarina E. Hioe, Veterans Affairs Medical Center, 423 East 23rd Street, Room 18-124 North, New York, NY 10010. E-mail address: catarina.hioe{at}med.nyu.edu

³ Abbreviations used in this paper: MHC-II, MHC class II; CHO, Chinese hamster ovary; MS, mass spectrometry; tPA, tissue plasminogen activator; WT, wild type.