Monoclonal antibody that distinguishes between a phosphorylated, beta 2-microglobulin-associated, and a free, nonphosphorylated, chain of MHC class I.
We have shown that a mAb, 7.2.14, recognizes a conserved sequence in exon 7 of a number of murine MHC class I molecules. 7.2.14 binding is abolished when the molecule is phosphorylated, presumably at a serine residue in exon 7, whereas treatment of material in cell lysates with alkaline phosphatase increases the intensity of the binding. A genomic construct containing Dd was transfected into human fibroblasts and a clonal cell line expressing high levels of surface MHC was selected. Cell lysates were prepared from surface-iodinated cells and analyzed by using a panel of antibodies. An apparent size heterogeneity was detected in the MHC class I gene product precipitated by different anti-class I MHC antibodies, suggesting that more than one conformational species of Dd was present. This was further investigated regarding the molecules precipitated by antibodies 34-2-12, M1/42, and 7.2.14. After preclearing of surface-iodinated cell lysates by using one antibody, challenge with the others still precipitated a Dd molecule, confirming that there were three independent conformations of the Dd gene product. A similar complexity could be observed in the lysates of surface-labeled spleen cells from C57BL/6 mice. A major polypeptide at approximately 48 to 50 kDa, representing the MHC H chain, was seen, and one or two as yet unidentified but strongly associated polypeptides at 41 kDa and 56 kDa were also visible. Sequential clearing of surface-iodinated material with one antibody followed by precipitation with the other confirmed that the 7.2.14-reactive material was distinct from that which reacted with M1/42. We propose that the 7.2.14-reactive 50-kDa band is the nonphosphorylated form of class I MHC, which exists in a conformation different from that of the conventional 48-kDa, phosphorylated, beta 2-microglobulin-associated entity.
Thor
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Department of Biology, University of California, San Diego, La Jolla 92093-0063.
Sepulveda
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Chada
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Dutton
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000939:Epitopes; D005091:Exons; D005805:Genes, MHC Class I; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000276:immunology*; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D010768:Phosphoserine / Q000276:immunology; D011487:Protein Conformation; D001613:beta 2-Microglobulin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D046911:Macromolecular Substances; D010455:Peptides; D001613:beta 2-Microglobulin; D010768:Phosphoserine