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Mutation at amino acid position 133 of H-2Dd prevents beta 2m association and immune recognition but not surface
R J Rubocki1, J M Connolly, T H Hansen
1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68105-1065.
Abstract:
The histocompatibility loss mutation H-2dm6 was derived from a mouse treated with the chemical mutagen ethylnitrosourea, and previous mapping studies implicated Dd as the affected locus. Southern blot analyses of DNA from H-2dm6 cells did not detect major deletions in the Ddm6 gene, suggesting that H-2dm6 was different from the previously characterized D region mutants H-2dm1 and H-2dm2. RNA blot analysis identified Ddm6 transcripts of appropriate size and a Ddm6 protein was immunoprecipitated from biosynthetically labeled H-2dm6 cells. Interestingly, the Ddm6 protein showed no beta 2m association and was only precipitated by a mAb to the alpha 3 domain. Furthermore, oligosaccharide maturation and low levels of surface expression of Ddm6 molecules were detected. However, the surface Ddm6 was nonfunctional as a target Ag in in vitro cytotoxicity assays, consistent with its original in vivo detection as a loss mutation. Sequence analyses of Ddm6 cDNA identified a single nucleotide base difference from wild-type, resulting in the substitution of a Trp to Arg at position 133. The significance of this substitution is discussed in the context of other class I expression variants.
Insights
A new mouse mutation, H-2dm6, affects the Dd locus. This mutation results in a nonfunctional Ddm6 protein lacking beta 2m association, impacting its role as a target antigen.
Area of Science:
- Immunogenetics
- Molecular immunology
- MHC Class I biology
Background:
- The H-2dm6 mutation is a chemically induced loss-of-function mutation affecting the mouse Dd locus.
- Previous studies characterized D region mutants H-2dm1 and H-2dm2, necessitating further investigation of H-2dm6.
Purpose of the Study:
- To characterize the molecular basis of the H-2dm6 histocompatibility loss mutation.
- To investigate the protein expression, assembly, and function of the Ddm6 molecule.
Main Methods:
- Southern blot and RNA blot analyses to assess gene integrity and transcript levels.
- Immunoprecipitation to analyze Ddm6 protein association with beta 2-microglobulin (beta 2m).
- DNA sequencing of Ddm6 cDNA to identify genetic alterations.
Main Results:
- H-2dm6 cells showed no major deletions in the Ddm6 gene.
- Ddm6 protein was detected but lacked beta 2m association and exhibited impaired oligosaccharide maturation.
- Surface-expressed Ddm6 molecules were nonfunctional as target antigens in cytotoxicity assays.
- A single nucleotide change resulted in a Trp to Arg substitution at position 133 of the Ddm6 protein.
Conclusions:
- The H-2dm6 mutation arises from a specific missense mutation in the Ddm6 gene, leading to a non-functional protein.
- The Trp133Arg substitution disrupts beta 2m association and surface expression, explaining the histocompatibility loss phenotype.
- This finding contributes to understanding structure-function relationships in MHC Class I molecules.