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RT6 is an unusual cell membrane protein that is expressed exclusively by postthymic T cells. The inherent defect in its expression has been correlated to lymphopenia and genetically determined susceptibility for insulin-dependent diabetes mellitus in the rat. We report here the primary structure of the RT6.2 alloantigen as deduced from the cDNA sequence. The predicted amino acid sequence of RT6.2 begins with a conventional leader of 20 amino acids and ends in a hydrophobic C-terminal extension peptide of 29 amino acids as is common for phosphatidylinositol-anchored proteins. Native RT6.2 is predicted to comprise 226 amino acids, with a calculated M, of 26,036. Four cysteine residues account for two intrachain disulfide bonds. The sequence lacks potential Nglycosylation sites and contains an excess of positively charged residues. Homology searches in protein sequence data banks suggest that RT6.2 is not encoded by a member of the immunoglobulin supergene family. Moreover, these analyses did not reveal any close homologies of RT6.2 to known proteins: the highest homology found was 21.2% identity in a 52-amino acid overlap to the torpedo acetylcholinesterase precursor. Southern blot analyses indicate that RT6.2 is the product of a single-copy gene and provide evidence for closely related genes in the mouse and other species. The corresponding gene products remain to be identified.The rat T-cell alloantigenic system RT6 was originally discovered by several groups because of its potent immunogenicity in the allogeneic context (1-7). Two allelic gene products-designated RT6.1 and RT6.2-of Mr 25-30 x 103 could be discriminated by allotype-specific antibodies (8, 9). The corresponding alleles, RT6a and RT6b, were mapped to linkage group I between the loci for albinism and hemoglobin ,8 chain complex (Hbb) (10), a region of high-linkage-synteny homologies to mouse chromosome 7 and human chromosome 11 (11,12). Intriguingly, no comparable proteins have yet been described in mouse or man.The RT6 antigenic system is of interest for studies of postthymic T-cell development (13) RT6 is also of interest with respect to genetically determined insulin-dependent diabetes mellitus (IDDM) (15,(17)(18)(19). Thus, the diabetes-prone Bio-Breeding (dpBB) rat, an animal model for human autoimmune type I IDDM, has an inherent defect in the generation of RT6+ cells (15,18), and this defect has been associated with the pathogenesis of IDDM in these animals (18,19).Biochemically, the RT6 antigens represent unusual membrane proteins in that they occur in nonglycosylated (RT6.2) or in nonglycosylated and differently glycosylated (RT6.1) variants (8, 9). All forms of RT6 are anchored in the cell membrane by covalent linkage to a phosphatidylinositol moiety (9,20). Treatment of T cells with RT6-specific antisera reportedly is mitogenic (21), similar to the case of other phosphatidylinositol-anchored T-cell surface proteins (22).We report here the molecular cloning of RT6.2-specific cDNAs and the entire coding sequence for RT6.2.t The pr...

The rat T cell alloantigen RT6.2 shows a slow rate of synthesis in isolated T cells which hampers studies on the metabolism of RT6.2 in these cells (1). In order to facilitate further molecular and functional characterization of this molecule we have established a T-T hybridoma cell line which stably expresses RT6.2. Only 1 of 102 T cell hybridomas obtained upon fusion of the rat thymoma C58NT with DA rat lymph node cells expressed this antigen. This clone--EpD3--initially showed a relatively slow rate of cell division and a highly unstable pattern of expression of RT6.2. Thus, the relative number of RT6.2 bearing cells consistently decreased during cultivation of EpD3 and of EpD3-derived subclones. Cell separation studies suggest that the switch in RT6.2 phenotype of EpD3 cultures was due to the appearance of variant cells of RT6.2- phenotype with a higher proliferative capacity and was not induced by factors in the culture medium. By repeated panning and subcloning procedures to select for RT6.2 expressing cells, a subline of EpD3--EpD3/87--was obtained which stably expresses high levels of RT6.2. Metabolic labeling studies of RT6.2 in EpD3 show that it is synthesized very efficiently in these cells and support our previous suggestion that RT6.2 does not bear any classic oligosaccharide side chains. Moreover, RT6.2 can be released almost completely from EpD3 cells by phosphatidylinositol-specific phospholipase C (PI-PLC).

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