Introduction of H-2D(d) determinants into the H-2L(d) antigen by site-directed mutagenesis

David Koeller, R. Lieberman, J. I. Miyazaki, E. Appella, K. Ozato, D. W. Mann, J. Forman

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2D(d) antigen forms s-allodeterminants, the H-2L(d) gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2D(d) amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2D(d) antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2D(d)-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2D(d) s-epitopes. Together, more than half of all the relevant H-2D(d) s-epitopes were mapped into amino acid position 63-70 of the H-2D(d) molecule, which was expressed in the mutant H-2L(d) gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few D(d) s-epitopes acquired by the preceding mutations were lost. All of the H-2L(d)-specific s-determinants were retained in the mutant molecules, as were H-2D(d) s-determinants specific for the α-2 or α-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2D(d) CTL cultures and an anti-H-2D(d) CTL clone recognized the mutant H-2L(d) molecules, M66 and M70. Some CTL clones generated against the Q10(d) molecule, which has an identical sequence to H-2D(d) between residues 61 and 73, failed to recognize native H-2D(d) or L(d) but did crossreact with mutant L(d). While bulk-cultured anti-H-2L(d) CTL cultures reacted strongly against M73, bulk-cultured H-2L(d) restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2L(d) CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the α-1 domain controls major s- and c-antigenic sites on the H-2D(d) antigen and c-sites on H-2L(d) antigen.

Original languageEnglish (US)
Pages (from-to)744-760
Number of pages17
JournalJournal of Experimental Medicine
Volume166
Issue number3
StatePublished - 1987
Externally publishedYes

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Histocompatibility Antigen H-2D
Site-Directed Mutagenesis
Epitopes
Amino Acids
Clone Cells
Antigens
Dilatation and Curettage
Histocompatibility Antigens Class I
Vesicular Stomatitis
Mutation
Codon
Genes
Amino Acid Sequence
Viruses

ASJC Scopus subject areas

  • Immunology

Cite this

Koeller, D., Lieberman, R., Miyazaki, J. I., Appella, E., Ozato, K., Mann, D. W., & Forman, J. (1987). Introduction of H-2D(d) determinants into the H-2L(d) antigen by site-directed mutagenesis. Journal of Experimental Medicine, 166(3), 744-760.

Introduction of H-2D(d) determinants into the H-2L(d) antigen by site-directed mutagenesis. / Koeller, David; Lieberman, R.; Miyazaki, J. I.; Appella, E.; Ozato, K.; Mann, D. W.; Forman, J.

In: Journal of Experimental Medicine, Vol. 166, No. 3, 1987, p. 744-760.

Research output: Contribution to journalArticle

Koeller, D, Lieberman, R, Miyazaki, JI, Appella, E, Ozato, K, Mann, DW & Forman, J 1987, 'Introduction of H-2D(d) determinants into the H-2L(d) antigen by site-directed mutagenesis', Journal of Experimental Medicine, vol. 166, no. 3, pp. 744-760.
Koeller, David ; Lieberman, R. ; Miyazaki, J. I. ; Appella, E. ; Ozato, K. ; Mann, D. W. ; Forman, J. / Introduction of H-2D(d) determinants into the H-2L(d) antigen by site-directed mutagenesis. In: Journal of Experimental Medicine. 1987 ; Vol. 166, No. 3. pp. 744-760.
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abstract = "We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2D(d) antigen forms s-allodeterminants, the H-2L(d) gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2D(d) amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2D(d) antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2D(d)-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2D(d) s-epitopes. Together, more than half of all the relevant H-2D(d) s-epitopes were mapped into amino acid position 63-70 of the H-2D(d) molecule, which was expressed in the mutant H-2L(d) gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few D(d) s-epitopes acquired by the preceding mutations were lost. All of the H-2L(d)-specific s-determinants were retained in the mutant molecules, as were H-2D(d) s-determinants specific for the α-2 or α-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2D(d) CTL cultures and an anti-H-2D(d) CTL clone recognized the mutant H-2L(d) molecules, M66 and M70. Some CTL clones generated against the Q10(d) molecule, which has an identical sequence to H-2D(d) between residues 61 and 73, failed to recognize native H-2D(d) or L(d) but did crossreact with mutant L(d). While bulk-cultured anti-H-2L(d) CTL cultures reacted strongly against M73, bulk-cultured H-2L(d) restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2L(d) CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the α-1 domain controls major s- and c-antigenic sites on the H-2D(d) antigen and c-sites on H-2L(d) antigen.",
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AU - Koeller, David

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N2 - We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2D(d) antigen forms s-allodeterminants, the H-2L(d) gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2D(d) amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2D(d) antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2D(d)-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2D(d) s-epitopes. Together, more than half of all the relevant H-2D(d) s-epitopes were mapped into amino acid position 63-70 of the H-2D(d) molecule, which was expressed in the mutant H-2L(d) gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few D(d) s-epitopes acquired by the preceding mutations were lost. All of the H-2L(d)-specific s-determinants were retained in the mutant molecules, as were H-2D(d) s-determinants specific for the α-2 or α-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2D(d) CTL cultures and an anti-H-2D(d) CTL clone recognized the mutant H-2L(d) molecules, M66 and M70. Some CTL clones generated against the Q10(d) molecule, which has an identical sequence to H-2D(d) between residues 61 and 73, failed to recognize native H-2D(d) or L(d) but did crossreact with mutant L(d). While bulk-cultured anti-H-2L(d) CTL cultures reacted strongly against M73, bulk-cultured H-2L(d) restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2L(d) CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the α-1 domain controls major s- and c-antigenic sites on the H-2D(d) antigen and c-sites on H-2L(d) antigen.

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