Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules.
To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.
Wei
B Y
BY
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
Buerstedde
J M
JM
Bell
M
M
Chase
C
C
Nilson
A
A
Browne
A
A
Pease
L
L
McKean
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D006031:Glycosylation; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology*; D014415:Tunicamycin / Q000494:pharmacology
D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; D011948:Receptors, Antigen, T-Cell; D014415:Tunicamycin