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 e xon 7 of a number of murine MHC class I molecules. 7.2.14 binding is a bolished when the molecule is phosphorylated, presumably at a serine r esidue in exon 7, whereas treatment of material in cell lysates with a lkaline phosphatase increases the intensity of the binding. A genomic construct containing D(d) 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 b y using a panel of antibodies. An apparent size heterogeneity was dete cted in the MHC class I gene product precipitated by different anti-cl ass I MHC antibodies, suggesting that more than one conformational spe cies of D(d) 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 D(d) molecule, confirmi ng that there were three independent conformations of the D(d) gene pr oduct. A similar complexity could be observed in the lysates of surfac e-labeled spleen cells from C57BL/6 mice. A major polypeptide at appro ximately 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-iodi nated material with one antibody followed by precipitation with the ot her 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 c onformation different from that of the conventional 48-kDa, phosphoryl ated, beta2-microglobulin-associated entity.