Primary structure of rat RT6.2, a nonglycosylated phosphatidylinositol-linked surface marker of postthymic T cells.

Abstract

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...

Increase in ADP‐ribosyltransferase activity of rat T lymphocyte alloantigen RT6.1 by a single amino acid mutation

“…2A). Based on the matured forms of RT6 molecules in lymphocytes [13,14], the hydrophobic regions of their amino termini (amino acids 1-25) and carboxy-terminal regions (amino acids 247-275) were truncated in the recombinant proteins.…”

Expression, purification, crystallization and preliminary X-ray analysis of rat ecto-ADP-ribosyltransferase 2 (ART2.2)

“…Expression of ART2.2 into the periplasm of E. coli NM522 cells secured the formation of the two potential disul®de bridges (Koch et al, 1990). The periplasmic fraction was isolated by osmolysis and the protein was puri®ed using cation-exchange and gelpermeation chromatography.…”

“…R. norvegicus ART2.2 displays NAD + glycohydrolase and auto-ADP-ribosylation activities (Haag et al, 1995;Maehama et al, 1995) and gains arginine transferase activity by the exchange Q187E (Hara et al, 1996;Karsten et al, 1997;Maehama & Katada, 1997). We focused on rat ART2.2 because it could be produced in the periplasm of E. coli and because it is the only non-glycosylated mammalian ecto-ART (Koch et al, 1990;Koch-Nolte & Haag, 1997). The structure of this ART will provide a new starting point for functional studies.…”

Analysis of the catalytic site of the actin ADP‐ribosylating Clostridium perfringens iota toxin

“…This list includes C3-1ike transferases [25 29], cholera toxin [30], the family of Rhodospirillum-like ADP-ribosyltransferases [31], the T2, T4, T6 bacteriophage transferases [32] and several recently identified eukaryotic glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferases (or NAD-ases) such as the rabbit muscle ADP-ribosyltransferase [33][34][35] and the family of RT6-1ike T-cell alloantigens [36][37][38].…”