Wolfert R L, Goodman M G, Weigle W O
J Immunol. 1985 Sep;135(3):1668-73.
Previously, we showed that murine B cell membrane proteins undergo rearrangements in the plasma membrane to form new molecular associations in response to mitogenic stimulation. These complexes were covalently stabilized by photoreactive cross-linking agents and were analyzed by SDS PAGE. We have now identified certain complexes that involve class II MHC products, the Ia antigens. Upon stimulation of B cells with LPS, Ia surface molecules (as identified by radioimmunoprecipitation with polyclonal anti-Ia antiserum) enter into a molecular complex with a 95-kd membrane-associated protein (p95) to form a 200-kd complex that may be stabilized by the cross-linking agent dithiobisphenylazide (DTPA). This molecular association is not observed upon stimulation with mitogenic anti-Ig reagents, nor with the polyclonal B cell activator 8-bromoguanosine. p95 is not a disulfide-linked molecule itself, and by separate immunoprecipitation experiments we have established that it is not a component of surface Ig, transferrin receptor, the B cell Fc receptor, or CR1, the receptor for complement component C3b. Further analysis of the association of Ia antigens with surface proteins, with the use of monoclonal antibodies directed against I-A or I-E, has demonstrated that each subregion gene product forms a unique molecular association. Precipitation of radiolabeled lysates from LPS-activated B cells with anti-I-A reveals the aforementioned association with p95. In contrast, the I-E antigen apparently forms complexes with a multimer of a 15-kd protein to give complexes of 45, 60, 75, and 90 kd. When analyzed by two-dimensional diagonal gels (nonreducing/reducing), only the I-E bands are revealed by autoradiography, indicating that the putative p15 that associates with I-E may not be accessible to surface labeling. The disparate molecular associations for I-A and I-E suggest that the formation of these distinct protein complexes may be functionally related to a different role in the process of cellular activation for each of these Ia subregion gene products.
此前，我们发现鼠B细胞膜蛋白在质膜中会发生重排，以响应促有丝分裂刺激形成新的分子关联。这些复合物通过光反应性交联剂共价稳定，并通过SDS-PAGE进行分析。我们现在已经鉴定出某些涉及II类MHC产物（即Ia抗原）的复合物。在用LPS刺激B细胞后，Ia表面分子（通过用多克隆抗Ia抗血清进行放射免疫沉淀鉴定）与一种95-kd的膜相关蛋白（p95）形成分子复合物，形成一个200-kd的复合物，该复合物可能被交联剂二硫代双苯叠氮（DTPA）稳定。在用促有丝分裂抗Ig试剂或多克隆B细胞激活剂8-溴鸟苷刺激时，未观察到这种分子关联。p95本身不是二硫键连接的分子，通过单独的免疫沉淀实验，我们确定它不是表面Ig、转铁蛋白受体、B细胞Fc受体或补体成分C3b的受体CR1的组成部分。使用针对I-A或I-E的单克隆抗体进一步分析Ia抗原与表面蛋白的关联，结果表明每个亚区基因产物都形成独特的分子关联。用抗I-A沉淀LPS激活的B细胞的放射性标记裂解物，揭示了上述与p95的关联。相比之下，I-E抗原显然与一种15-kd蛋白的多聚体形成复合物，产生45、60、75和90 kd的复合物。通过二维对角线凝胶（非还原/还原）分析时，放射自显影仅显示I-E条带，表明与I-E相关的假定p15可能无法进行表面标记。I-A和I-E不同的分子关联表明，这些不同蛋白质复合物的形成可能在功能上与这些Ia亚区基因产物在细胞激活过程中的不同作用相关。
