2 DISTINCT FORMS OF SOLUBLE MHC CLASS-I MOLECULES SYNTHESIZED BY DIFFERENT MECHANISMS IN NORMAL RAT-CELLS IN-VITRO

Rat soluble MHC class I synthesis was studied at both RNA and protein levels to determine whether multiple forms of soluble MHC class I mole cules are produced by different mechanisms. RT-PCR and sequencing of M HC class I transcripts identified an alternatively spliced nonclassica l MHC class I gene product, lacking both exon 5 and 6, in bot-h spleen and liver. Immunoprecipitation and SDS-PAGE identified two distinct s oluble MHC class I proteins in both splenocyte- and hepatocyte-culture supernatants. The 36Kd classical soluble MHC class I protein (RT1.A(a )) was precipitated by both allele-specific(MN4.91.6, R3/13, R2/15S) a nd pan-reactive (OX18) mAbs. The 39Kd non-RT1.A soluble MHC class I pr otein was precipitated only by OX18. The production of soluble RT1.A(a ) was inhibited by a metal loproteinase inhibitor, but not by serine/t hiol protease inhibitors. None of these protease inhibitors interfered with the soluble non-RT1.A production, suggesting that it might be de rived from an alternatively spliced MHC class I transcript. The solubl e non-RT1.A was always associated with beta 2m. However, soluble RT1.A (a) molecule was cleaved in beta 2m-free form and was reassociated wit h beta 2m in culture supernatants. Thus two soluble MHC class I molecu les, classical (36Kd RT1.A(a)) and nonclassical (the alternatively spl iced transcript), were produced from rat cells. Alternative splicing l ed to che nonclassical soluble MHC class I synthesis. Proteolytic clea vage by metallo-proteinase led to the classical soluble MHC class I sy nthesis. Human Immunology 59, 404-414 (1998). (C) American Society for Histocompatibility and Immunogenetics, 1998. Published by Elsevier Sc ience Inc.