0022-1 767/93/1511-211$02.0O/O The Journal of Immunology Copyright @ 1993 by The American Association of Immunologists Vol 151, 211-224, No. 1, July 1, 1993 Printed in U.S.A. Monoclonal Antibody that Distinguishes between a Phosphorylated, P,-Microglobulin-Associated, and a Free, Nonphosphorylated, Chain of M H C Class I' Gautam Thor,2* Homero Sepulveda,* Sunil Chada,' and Richard W. Dutton* *Department of Biology and the Cancer Center, University of California, San Diego, La Jolla, CA 92093-0063, and 'Viagene, San Diego, CA 92121 ABSTRACT. We have shown that a mAb, 7.2.1 4, 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 conformationalspecies of Dd was present. This was further investigated regarding the molecules precipitated by antibodies 34-2-1 2, 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 C57BV6 mice. A major polypeptide at approximately 48 to 50 kDa, representing the MHC H chain, wasseen, 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, ~2-microglobulin-associated entity. lournal of Immunology, 1993, 151 : 211. T he a 1 and a2 domains of the class I MHC H chain form a peptide binding site (1) for peptides derived from endogenous proteins, which can then be rec- ognized by CD8+ T cells bearing the appropriatereceptor. The a3 domain is complexed with the Ig-like Lchain P2m.3 Received for publication August 14, 1992. Accepted for publication March 30, 1993. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. A108795 and A123287. ' This work wassupported, in part,by National Institutes of Health Grants * Address correspondence and reprint requests to Dr. Gautam Thor, Depart- ment of Biology and Cancer Center, University of California, San Diego, 9500 Cilman Dr., 0063, La Jolla, CA 92093-0063. The transmembrane domain spans the membrane once and is followed by ashort cytoplasmic tail (2, 3). The cyto- plasmic tail is encoded by three short exons (about 10 amino acids each) in the K gene products, but in the D gene products the last exon, exon 8, codes for only a single amino acid. The sequence in these exons is highly conserved be- tween alleles and, for exons 6 and 7, between K and D (4). Peptides derived from intracellular pathogens provide the targets for cytolytic T cells (reviewed in Reference 5), in which case their role is clear. Peptides can also be derived Abbreviations used in this paper: p2m, &microglobulin; CIAP, calf intesti- nal alkaline phosphatase;7.2.14, FS 23 7.2.14; M1/42, M1/423; PFB, phos- phate-free buffer. 211