Two 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 molecules are produced by different mechanisms. RT-PCR and sequencing of MHC class I transcripts identified an alternatively spliced nonclassical MHC class I gene product, lacking both exon 5 and 6, in both spleen and liver. Immunoprecipitation and SDS-PAGE identified two distinct soluble MHC class I proteins in both splenocyte- and hepatocyte-culture supernatants. The 36Kd classical soluble MHC class I protein (RT1.Aa) was precipitated by both allele-specific (MN4.91.6, R3/13, R2/15S) and pan-reactive (OX18) mAbs. The 39Kd non-RT1.A soluble MHC class I protein was precipitated only by OX18. The production of soluble RT1.Aa was inhibited by a metalloproteinase inhibitor, but not by serine/thiol protease inhibitors. None of these protease inhibitors interfered with the soluble non-RT1.A production, suggesting that it might be derived from an alternatively spliced MHC class I transcript. The soluble non-RT1.A was always associated with beta2m. However, soluble RT1.Aa molecule was cleaved in beta2m-free form and was reassociated with beta2m in culture supernatants. Thus two soluble MHC class I molecules, classical (36Kd RT1.Aa) and nonclassical (the alternatively spliced transcript), were produced from rat cells. Alternative splicing led to the nonclassical soluble MHC class I synthesis. Proteolytic cleavage by metalloproteinase led to the classical soluble MHC class I synthesis.
Department of Surgery, and Animal Health and Biomedical Science, University of Wisconsin-Madison, USA.
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Zhai
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Knechtle
D000483:Alleles; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006649:Histocompatibility Antigens / Q000302:isolation & purification / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011912:Rats, Inbred ACI; D011917:Rats, Inbred Lew; D012995:Solubility; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D014158:Transcription, Genetic; D001613:beta 2-Microglobulin / Q000378:metabolism
D006649:Histocompatibility Antigens; D015395:Histocompatibility Antigens Class I; D001613:beta 2-Microglobulin; C038353:histocompatibility antigens RT, rat; D008666:Metalloendopeptidases