The role of N-linked oligosaccharides of MHC class II antigens in T cell stimulation.
A specific increase in T cell extracellular acidification rate has been demonstrated recently when complexes of purified MHC class II molecules and antigenic peptides interact with T cell receptors (TCRs) on cloned T cells. The present study shows that such measurements of an increase in extracellular acidification rate can be used to evaluate the functional role of various N-linked oligosaccharides of MHC class II antigens. Affinity-purified murine IAk and IAs were deglycosylated in the presence of aspargine-amidase enzyme and were characterized by SDS-polyacrylamide gel electrophoresis. The complete removal of all three N-linked oligosaccharides from the alpha/beta heterodimer was confirmed by four different lectin-linked Western blot analyses. Similar to the native heterodimer, both deglycosylated IAk and deglycosylated IAs were fully capable of binding synthetic antigenic peptides derived from myelin basic protein (MBP). When equivalent amount of glycosylated and deglycosylated class II-peptide complexes were exposed to restricted cloned T cells, identical increases in T cell extracellular acidification rates were observed. The specificity of such increases in extracellular acidification rate was demonstrated by exposing cloned T cells to irrelevant complexes of glycosylated and deglycosylated class II and antigenic peptides. These results show how measurement of extracellular acidification rate can be used to study structure-function correlations of ligand-receptor interactions, and support an earlier observation that N-linked oligosaccharides of murine MHC class II molecules are not involved in either antigenic peptide binding or T cell recognition.
Nag
B
B
Anergen Inc., Redwood City, CA 94063.
Wada
H G
HG
Arimilli
S
S
Fok
K
K
Passmore
D
D
Sharma
S D
SD
McConnell
H M
HM
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D006031:Glycosylation; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000276:immunology; D009844:Oligosaccharides / Q000276:immunology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D013601:T-Lymphocytes / Q000276:immunology*
D000949:Histocompatibility Antigens Class II; D004676:Myelin Basic Protein; D009844:Oligosaccharides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase