Journal of Experimental Medicine, Vol 166, 1329-1350, Copyright © 1987 by Rockefeller University Press

ARTICLES

Site-directed mutagenesis of class I HLA genes. Role of glycosylation in surface expression and functional recognition

JA Barbosa, J Santos-Aguado, SJ Mentzer, JL Strominger, SJ Burakoff and PA Biro
Department of Tumor Virology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.

We have investigated the role of the carbohydrate moiety on the HLA-B7 molecule in mAb and CTL recognition using oligonucleotide-directed mutagenesis and gene transfer techniques. A conservative substitution of asparagine to glutamine at amino acid 86 in HLA-B7 was created to abolish the unique glycosylation site present on all HLA molecules. A second mutant B7 molecule was made by substituting asparagine-aspartic acid-threonine for the resident lysine-aspartic acid/lysine tripeptide at amino acids 176-178, thus creating an N-linked glycan at amino acid 176, which is additionally present on all known murine H-2 class I antigens. Upon gene transfer into mouse and human cell recipients, the HLA-B7M176+ mutant and normal HLA-B7 expressed identical levels of surface protein. However, the binding of two mAbs (MB40.2 and MB40.3) thought to recognize different epitopes of the HLA-B7 molecule was completely eliminated. In contrast, the HLA-B7M86- mutant displayed no surface expression (mouse L cells) or minimal surface expression (human RD cells or mouse L cells coexpressing human beta 2 microglobulin [beta 2m]) after indirect immunofluorescence (IIF) and flow cytometric analysis with a panel of 12 HLA-B7 mAb reactive with monomorphic and polymorphic determinants. Immunoprecipitation analysis demonstrated that intracellular denatured mutant protein was present. Tunicamycin treatment did not rescue the expression of HLA-B7M86- antigens to the cell surface; while interferon did induce higher levels of surface expression. Tunicamycin treatment also did not allow binding of the mAbs MB40.2 or MB40.3 to HLA-B7M176+ mutant antigens, suggesting that the carbohydrate moiety itself was not directly involved in the recognition or conformation of these mAb epitopes. Further mutation of the B7M86- molecule to create a glycan moiety at amino acid position 176 (B7M86-/176+) did not rescue normal levels of surface expression. Finally, neither mutation was seen to affect recognition by a panel of 12 allospecific CTL clones. The low expression of HLA-B7M86- on the surface of human cell transfectants was sufficient to achieve lysis, albeit at a reduced efficiency, and lysis could be increased by interferon induction of higher levels of expression. Thus, the carbohydrate moiety on HLA antigens plays a minimal or nonexistent role in recognition by available mAb and allospecific CTL clones.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

• Martayan, A., Sibilio, L., Setini, A., Monaco, E. L., Tremante, E., Fruci, D., Colonna, M., Giacomini, P. (2008). N-Linked Glycosylation Selectively Regulates the Generic Folding of HLA-Cw1. J. Biol. Chem. 283: 16469-16476 [Abstract] [Full Text]  
• Duprat, E., Lefranc, M.-P., Gascuel, O. (2006). A simple method to predict protein-binding from aligned sequences--application to MHC superfamily and {beta}2-microglobulin. Bioinformatics 22: 453-459 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search

TABLE OF CONTENTS