METALLOPROTEINASE GENERATES SOLUBLE MHC CLASS I BY CELL SURFACE CLEAVAGE

Abstract

459 Soluble MHC class I antigen has been shown both in vitro and in vivo to regulate immune function, but little is known about how cells generate soluble MHC class I. Therefore the molecular and biochemical bases of soluble MHC class I synthesis were addressed in a rat model (ACI strain, RT1.Aa). Using RT-PCR and sequence analysis, an alternatively spliced non-classical class I mRNA was identified, lacking both exon 5 and 6. No truncated classical class I (RT1.Aa) mRNAs were found. Using allele-specific as well as pan-reactive anti-rat MHC class I antibodies, two distinctive soluble class I proteins were detected from the culture supernatants of the metabolically labeled splenocytes and hepatocytes. The soluble classical class I protein (RT1.Aa) with MW of 36K was precipitated by MN4.91.6 (or R3/13, or R2/15S, or OX18). The non-classical class I protein with MW of 39K was precipitated by OX18 only. Both soluble class I proteins were associated with β2m. The production of the 36K soluble RT1.Aa was inhibited by a hydroxamic acid-based inhibitor of metalloproteinase, RO 31-9790; but not by serine/thiol protease inhibitors. The production of the 39K non-classical class I protein was not affected by either type of protease inhibitor, consistent with our observation of the alternatively spliced non-classical class I transcript. Iodoacetamide, HgCl2 and zinc ionophore pyrithione which directly activate metalloprotease, upregulated the production of soluble RT1.Aa protein, as did PMA and calcium ionophore A23187, which indirectly activate metalloprotease. A zinc chelating agent, 1,10-phenanthrolin inhibited the production of soluble RT1.Aa protein. From surface biotin-labeled cells, the soluble RT1.Aa was detected in β2m-free form. However from the 4-hour metabolically labeled cells, the soluble RT1.Aa was associated with β2m. This indicates that RT1.Aa may be cleaved from the cell surface inβ2m-free form and re-associate with β2m in culture supernatant. Furthermore the metalloproteinase inhibitor, RO 31-9790 inhibited the production of both β2m-free and -associated soluble RT1.Aa proteins. The β2m-free soluble RT1.Aa protein lost its reactivities to anti-rat MHC class I antibodies R3/13, R2/15S, and OX18. When it re-associated with β2m, all the antibody reactivities recovered. In conclusion (1) alternative splicing does played a major role innon-classical but not classical soluble class I synthesis in the rat. (2) a zinc dependent, membrane-associated metalloproteinase(s) cleaved rat MHC class I protein from the cell surface and released a β2m-free form of classical soluble class I protein able to re-associate with β2m.