ADP-RIBOSYLATION CYCLES

ADP-核糖基化循环

• 批准号: 6162671
• 负责人: J MOSS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162671
• 关键词: ADP ribosylation; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; immunoprecipitation; integrins; laminin; molecular site; myoblasts; northern blottings; posttranslational modifications; protein sequence; protein structure function; receptor binding; tissue /cell culture; western blottings

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.

RT6是一种连接在T淋巴细胞表面的大鼠[T细胞]同种异体抗原 通过糖基磷脂酰肌醇(GPI)锚定。基因的两个等位基因 大鼠RT6基因已有报道。RT6.1由以下家族组成 非糖化和可变糖化蛋白质与分子 质量为25到35 kDa和RT6.2是一种非糖基化蛋白，具有 分子质量为24-28 kDa。这两种同种异体抗原都具有NAD 糖水解酶(NADase)和自身ADP-核糖基转移酶活性。在……里面 在BB大鼠中，RT6表达缺陷被证明与 易患自身免疫性胰岛素依赖型糖尿病(IDDM)。 细胞毒性单抗对糖尿病抵抗(DR)大鼠的治疗作用 针对RT6.1+T细胞的抗体(DS4.23)诱导T细胞耗竭 Rt6+T细胞，可在2~4周内发展为IDDM。 在注射抗体后，NADase的活性在 血浆大约是3.4倍。十二烷基硫酸钠-聚丙烯酰胺 非还原条件下的凝胶电泳，然后是洗涤剂 酶活性的提取和定量，NADase的大小 估计约为30-36 kDa，与 预期大小为糖基化形式的RT6.1。要刻画 释放的rt6在血浆中的分布，血浆被分成非常 低密度脂蛋白/低密度脂蛋白、高密度脂蛋白 非脂蛋白组分(d&gt；1.21g/ml)。在抗体治疗后， 非脂蛋白中NAD酶活性显著升高 而对与高密度脂蛋白相关的活动和 极低密度脂蛋白/低密度脂蛋白明显降低。该NADase的分子大小为 与30-36 kDa的蛋白质一致。在免疫沉淀后 极低密度脂蛋白/低密度脂蛋白、高密度脂蛋白和非脂蛋白组分与DS4.23抗体，以及 非脂蛋白组分中检测到33 kDa的免疫活性蛋白 用兔抗rt6多克隆抗血清1126。抗体的作用 去胸腺大鼠和正常大鼠的NAD酶活性相似， 提示抗体释放的RT6NADase活性不是来源于 来自外周血淋巴细胞群。切除肠管 取消了NADase活性的释放，与假设一致 该RT6NADase可能来源于肠道来源。自.以来 在KBR中通过离心分离可能会导致蛋白质 从高密度脂蛋白释放到非脂蛋白部分，也进行了分离 FPLC检测结果表明，所有抗体诱导的 在NADase中，活性与高密度脂蛋白有关。