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Structure of the heavy chain of the H-2Kk histocompatibility antigen
Insights
Radiochemical techniques characterized the murine H-2Kk histocompatibility antigen heavy chain, revealing its six CNBr fragments and glycosylation sites. This analysis provides insights into H-2Kk structure and its relation to other MHC molecules.
Area of Science:
- Immunogenetics
- Protein Chemistry
- Biochemistry
Background:
- The murine H-2Kk histocompatibility antigen is crucial for immune responses.
- Understanding its heavy chain structure is key to elucidating its function.
Purpose of the Study:
- To characterize the murine H-2Kk histocompatibility antigen heavy chain using radiochemical techniques.
- To determine the order and composition of cyanogen bromide (CNBr) fragments.
- To identify glycosylation and phosphorylation sites.
Main Methods:
- Radiochemical sequencing of the H-2Kk heavy chain and its CNBr fragments.
- Analysis of tunicamycin-treated cells to assess carbohydrate contribution.
- CNBr cleavage of [3H]fucose-labeled H-2Kk heavy chains.
- 32PO4 labeling to identify phosphorylation sites.
Main Results:
- The H-2Kk heavy chain (Mr 46,000) was characterized by six CNBr fragments (I-VI).
- Carbohydrate moieties (approx. Mr 6000) are located on fragments II and IV.
- Phosphorylation occurs on the carboxyl-terminal fragment (VI) and beta 2-microglobulin.
- Sequence analysis identified 87 residues, showing high similarity to H-2Kb but distinct from HLA antigens.
Conclusions:
- The study elucidated the primary structure and post-translational modifications of the H-2Kk heavy chain.
- H-2Kk shares greater sequence homology with H-2Kb than with human HLA antigens.
- These findings contribute to understanding MHC molecule evolution and diversity.
Abstract:
We have used radiochemical techniques to characterize the heavy chain (Mr 46,000) of the murine H-2Kk histocompatibility antigen in terms of six fragments (I-VI) obtained after cleavage of the polypeptide chain with CNBr. The tentative order of the fragments, which account for more than 90% of the heavy chain, was assigned by radiochemical sequence analysis of the intact heavy chain and of each purified CNBr fragment and by analysis of the CNBr fragments obtained from the large papain fragment of the heavy chain. Treatment of cells with tunicamycin yielded H-2 molecules with heavy chains of molecular weight 40,000, suggesting that the carbohydrate moieties have a combined molecular weight of approximately 6000. CNBr cleavage of H-2Kk heavy chains labeled with [3H]fucose indicated that the carbohydrate moieties are located on fragments II and IV.. Incubation of cells with 32PO4 gave H-2 molecules with radioactive phosphoserine in the carboxyl-terminal CNBr fragment (VI) of the heavy chain and in the fraction containing beta 2-microglobulin. Sequence analysis of each CNBr fragment intrinsically labeled with 3H- and 35S-labeled amino acids identified a total of 87 residues in the H-2Kk heavy chain. The sequence closely resembles that of the H-2Kb molecule, and the 11 differences are scattered throughout the polypeptide chain. Comparison with HLA sequences indicates that the two allelic H-2 sequences are more closely related to each other (88% identity) than either is to the HLA-B7 or A2 antigens (approximately 70%). Similarly, the nonallelic HLA antigens are more closely related to each other (83%) than either is to the H-2Kk or H-2Kb molecules.