N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness.
To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.
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Anergen Inc., Redwood City, California 94063.
Passmore
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D
Kendrick
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Bhayani
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H
Sharma
S D
SD
D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D000941:Antigens / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D004676:Myelin Basic Protein / Q000276:immunology / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology*
D000941:Antigens; D006023:Glycoproteins; D000949:Histocompatibility Antigens Class II; D004676:Myelin Basic Protein; D010455:Peptides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase