Altin J G, Pagler E B, Parish C R
Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra City.
Immunology. 1994 Nov;83(3):420-9.
In human T cells CD45 is reported to associate with both cell surface and intracellular molecules including CD2, CD4/CD8, CD5, p56lck and p59fyn. In this study the association of molecules with CD45 in murine T lymphocytes was explored using biotinylation, chemical cross-linking, immunoprecipitation and 32P-labelling. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of CD45 monoclonal antibody (mAb) (S-450-15.2) immunoprecipitates from Triton X-100 lysates of murine thymocytes that were surface biotinylated and treated with the chemical cross-linker 3,3'-dithio-bis(sulpho-succinimidylpropionate) (DTSSP) showed that CD45 can be chemically linked to molecules of 25,000-32,000, 42,000 and 60,000-70,000 MW. The CD45 mAb also co-precipitated a prominent 32,000 MW molecule from digitonin lysates of surface biotinylated murine thymocytes, splenocytes and D10 cells, but a weaker association was also detected on splenic B cells and on the murine B-cell lymphoma line A20. The results suggest that in these cells CD45 is associated with a 32,000 MW molecule which is exposed extracellularly. Experiments in which thymocytes were biotinylated after permeabilization with lysolecithin showed that additional molecules of 33,000, 55,000, 60,000 and 90,000 MW, presumably localized intracellularly, also co-precipitated with CD45. Labelling of murine thymocytes or D10 cells with H3(32)PO4 in vivo, and of CD45 immunoprecipitates by in vitro kinase reaction, revealed that the 32,000-33,000 MW molecules are phosphoproteins. The relationship of these molecules with the 30,000-34,000 MW molecules previously reported to associate with CD45 in human T cells is not clear as a number of differences were observed. Firstly, the molecular weight of the CD45-associated 32,000-33,000 MW molecule(s) on murine T cells and B cells is slightly lower than that observed in the human T-cell line Jurkat (34,000 MW). Secondly, phosphoamino acid analysis after in vitro kinase labelling of CD45 immunoprecipitates showed that the murine 32,000-33,000 MW molecules are phosphorylated exclusively on serines. Thirdly, although in vitro phosphorylation of the 32,000-33,000 MW molecules was inhibited by preincubation with either GTP-gamma-S or GDP-beta-S, the 32,000-33,000 MW CD45-associated molecules did not bind 32P-GTP, GDP-agarose, or react with antisera to a consensus sequence of G proteins. The crucial role of CD45 for proper function of the T-cell receptor (TCR), suggests that the CD45-associated 32,000-33,000 MW molecules and kinases also may play a role in the signalling events leading to T-cell activation.
据报道，在人类T细胞中，CD45与细胞表面及细胞内分子相关联，这些分子包括CD2、CD4/CD8、CD5、p56lck和p59fyn。在本研究中，运用生物素化、化学交联、免疫沉淀及³²P标记技术，对鼠T淋巴细胞中与CD45相关的分子进行了探究。对经表面生物素化处理并用化学交联剂3,3'-二硫代双（磺基琥珀酰亚胺丙酸酯）（DTSSP）处理的鼠胸腺细胞的Triton X-100裂解物进行CD45单克隆抗体（mAb）（S-450-15.2）免疫沉淀，然后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，结果显示CD45可通过化学方式与分子量为25,000 - 32,000、42,000及60,000 - 70,000的分子相连。该CD45 mAb还从经表面生物素化处理的鼠胸腺细胞、脾细胞及D10细胞的洋地黄皂苷裂解物中共同沉淀出一个显著的分子量为32,000的分子，但在脾B细胞及鼠B细胞淋巴瘤细胞系A20上也检测到较弱的关联。结果表明，在这些细胞中，CD45与一个细胞外暴露的分子量为32,000的分子相关联。用溶血卵磷脂通透处理后对胸腺细胞进行生物素化的实验表明，另外一些分子量分别为33,000、55,000、60,000及90,000的分子（推测定位于细胞内）也与CD45共同沉淀。在体内用H³(³²)PO₄标记鼠胸腺细胞或D10细胞，并通过体外激酶反应标记CD45免疫沉淀产物，结果显示分子量为32,000 - 33,000的分子为磷蛋白。这些分子与先前报道的在人类T细胞中与CD45相关联的分子量为30,000 - 34,000的分子之间的关系尚不清楚，因为观察到了一些差异。首先，鼠T细胞和B细胞上与CD45相关的分子量为32,000 - 33,000的分子的分子量略低于在人类T细胞系Jurkat中观察到的分子量（34,000）。其次，对CD45免疫沉淀产物进行体外激酶标记后的磷酸氨基酸分析表明，鼠的分子量为32,000 - 33,000的分子仅在丝氨酸上被磷酸化。第三，尽管用GTP-γ-S或GDP-β-S预孵育可抑制分子量为32,000 - 33,000的分子的体外磷酸化，但与CD45相关的分子量为32,000 - 33,000的分子不结合³²P-GTP、GDP-琼脂糖，也不与针对G蛋白共有序列的抗血清发生反应。CD45对T细胞受体（TCR）正常功能的关键作用表明，与CD45相关的分子量为32,000 - 33,000的分子及激酶可能也在导致T细胞活化的信号传导事件中发挥作用。
