ADP-RIBOSYLATION CYCLES

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RT6 is a rat [T cell] alloantigen linked to the surface of T lymphocytes through a glycosylphosphatidylinositol (GPI) anchor. Two alleles of the rat RT6 gene have been reported. RT6.1 consists of a family of non-glycosylated and variably glycosylated proteins with molecular masses of 25 to 35 kDa and RT6.2 is a non glycosylated protein with molecular mass of 24-28 kDa. The two alloantigens possess both NAD glycohydrolase (NADase) and auto-ADP-ribosyltransferase activities. In the BB rat, a defect in RT6 expression has been shown to coincide with susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM). Treatment of diabetes-resistant (DR) rats with cytotoxic monoclonal antibody directed against RT6.1+ T cells (DS4.23) induces depletion of RT6+ T cells and leads to development of IDDM in two or four weeks. Following injection of antibody, NADase activity is increased in the plasma approximately 3.4-fold. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions followed by detergent extraction and quantification of enzyme activity, the size of the NADase was estimated to be approximately 30-36 kDa, in agreement with the expected size of a glycosylated form of RT6.1. To characterize the plasma distribution of released RT6, plasma was fractionated into very low/low density lipoprotein (VLDL/LDL), high density lipoprotein (HDL) and nonlipoprotein fraction (d>1.21 g/ml). After antibody treatment, a significant increase in NADase activity was found in the nonlipoprotein fraction whereas effects on the activities associated with HDL and VLDL/LDL were much less. The molecular size of this NADase was consistent with a protein of 30-36 kDa. After immunoprecipitation of VLDL/LDL, HDL, and nonlipoprotein fractions with DS4.23 antibody, an immunoreactive protein of 33 kDa was detected in nonlipoprotein fraction by the polyclonal rabbit anti-RT6 antiserum 1126. Effects of antibody on NADase activity in thymectomized and control rats were similar, suggesting that RT6 NADase activity released by antibody was not derived from peripheral lymphocyte population. Removal of the intestine abolished release of NADase activity, consistent with the hypothesis that RT6 NADase may derived from an intestinal source. Since fractionation by centrifugation in KBr might cause proteins to be released from HDL into nonliprotein fraction, separation was also done by FPLC, and which demonstrated that all the antibody-induced increase in NADase activity was associated with HDL.