Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells.
Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells.
复制标题
SV40 大肿瘤抗原与 SV40 转化小鼠细胞表面细胞蛋白的关联。
DOI：
10.1016/0042-6822(82)90002-2
复制
发表时间：
1982
期刊：
影响因子：
3.7
通讯作者：
Butel,JS
中科院分区：
文献类型：
--
作者：
Santos,M;Butel,JS
据报道，一种分子量约为 53,000 (53K) 的细胞蛋白和组织相容性抗原（小鼠 H-2 抗原）与转化细胞中的病毒特异性蛋白相关。我们研究了是否可以在 SV40 转化的小鼠细胞表面检测到这种关联。采用差异免疫沉淀技术，以便可以独立于细胞内抗原检测表面相关抗原。使用乳过氧化物酶催化反应，用 125 I-Na 对生长为单层的细胞进行酶标记，或用 [35S] 甲硫氨酸或 32Pi 进行代谢标记，然后与针对小鼠 H-2 抗原或 SV40 大 T 抗原 (T-ag) 的抗血清或针对小鼠 53K 非病毒 T 抗原 (nvT-ag) 的单克隆抗体一起孵育。然后用NP40溶液破碎细胞，通过离心澄清提取物，并用含有蛋白A的金黄色葡萄球菌吸附上清液中的免疫复合物。通过与抗血清的第二次孵育沉淀细胞裂解物中存在的内部抗原，并再次用免疫吸附剂收集抗原-抗体复合物。洗脱沉淀的蛋白质并通过SDS-聚丙烯酰胺凝胶电泳进行分析。重建实验证实，在提取过程中从细胞核释放的 T-ag 并未与外部反应中结合到细胞表面的抗体上的游离抗原结合位点结合，在提取过程中未结合的细胞核 T-ag 并未与结合的表面抗原进行交换，并且表面反应不是由于从死细胞释放核 T-ag 并非特异性吸附到活细胞表面。碘化94K T-ag在外部反应过程中被T抗体特异性免疫沉淀；共沉淀碘化 53K 多肽。相反，通过针对小鼠 53K nvT-ag 的单克隆抗体，从表面碘化的转化细胞中共沉淀标记的 T-ag 和 53K。因此，SV40 大 T-ag 和细胞 53K 蛋白似乎在 SV40 转化的小鼠细胞表面和内部相关。相反，在转化细胞上没有检测到 T-ag 和小鼠 H-2 抗原之间的洗涤剂稳定复合物。讨论了病毒和细胞编码蛋白之间的分子相互作用可能参与确定一些观察到的与转化相关的细胞表型变化的可能性。
A cellular protein with a molecular weight of about 53,000 (53K) and histocompatibility antigens (mouse H-2 antigens) have been reported to be associated with viral-specified proteins in transformed cells. We investigated whether such associations could be detected on the surface of SV40-transformed mouse cells. A differential immunoprecipitation technique was adapted so that surface-associated antigens could be detected independently from intracellular antigens. Cells grown as monolayers were enzymatically labeled with125I-Na using a lactoperoxidase-catalyzed reaction, or metabolically labeled with either [35S]methionine or32Pi, and were then incubated with antisera against mouse H-2 antigens or SV40 large T-antigen (T-ag) or with monoclonal antibodies against mouse 53K nonviral T-antigen (nvT-ag). The cells were then disrupted with an NP40 solution, the extracts were clarified by centrifugation, and the immune complexes in the supernatant fluids adsorbed with protein A-containingStaphylococcus aureus. Internal antigens, present in the cell lysates, were precipitated by a second incubation with antiserum and the antigen-antibody complexes collected again with immunoadsorbent. The precipitated proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Reconstruction experiments established that T-ag released from the nucleus during the extraction procedure was not combining with free antigen-binding sites on antibodies bound to the cell surface in the external reaction, that nuclear unbound T-ag was not exchanging with bound surface antigen during extraction, and that the surface reaction was not due to nuclear T-ag released from dead cells and nonspecifically adsorbed onto the surface of living cells. Iodinated 94K T-ag was specifically immunoprecipitated by T antibody during the external reaction; an iodinated 53K polypeptide was coprecipitated. Conversely, labeled T-ag and 53K were coprecipitated from surface-iodinated transformed cells by monoclonal antibodies against mouse 53K nvT-ag. Thus, it appears that SV40 large T-ag and cellular 53K protein are associated on the surface as well as within SV40-transformed mouse cells. In contrast, no detergent-stable complex between T-ag and mouse H-2 antigens was detected on the transformed cells. The possibility that molecular interactions between viral- and cell-coded proteins could be involved in determining some of the observed transformation-related cellular phenotypic changes is discussed.