Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules

B. Y. Wei, J. M. Buerstedde, M. Bell, C. Chase, A. Nilson, A. Browne, L. Pease, D. J. McKean

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

To evaluate the potential functional role of the α- and β-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II A(k/α) and A(k/β) genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The α1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The β1 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 (A(k/α)* or A(k/β)*) 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 A(k/β)- or A(k/α)-reactive mAb demonstrated that although the oligosaccharide-deleted A(k/α)A(k/β) molecules were serologically wild type, the A(d/α) serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 A(k/β)*A(d/α) molecules. Cloned cell lines expressing the T.19 or T.21 A(k/β)*A(k/α) molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 A(k/α)*A(k/β) 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.

Original languageEnglish (US)
Pages (from-to)2358-2366
Number of pages9
JournalJournal of Immunology
Volume146
Issue number7
StatePublished - Jan 1 1991

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Wei, B. Y., Buerstedde, J. M., Bell, M., Chase, C., Nilson, A., Browne, A., Pease, L., & McKean, D. J. (1991). Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules. Journal of Immunology, 146(7), 2358-2366.