Wu X R, Manabe M, Yu J, Sun T T
Department of Dermatology, Kaplan Cancer Center, New York University School of Medicine, New York 10016.
J Biol Chem. 1990 Nov 5;265(31):19170-9.
The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.
哺乳动物尿路上皮的分化最终形成不对称单位膜（AUM）。我们通过梯度离心和去污剂洗涤，纯化出了毫克量的AUM，有趣的是，这些AUM包含三种主要蛋白质（15、27和47 kDa），它们似乎与我们之前描述的三种免疫亲和纯化的、推测与AUM相关的蛋白质相同（Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216）。肽图谱分析和免疫印迹表明这三种蛋白质是不同的分子。使用针对这三种蛋白质的单特异性抗体，我们发现它们都局限于尿路上皮表层细胞且与AUM相关。27 kDa和15 kDa的蛋白质仅在成熟顶端AUM的腔面被检测到。相反，47 kDa蛋白质的表位在顶端AUM的两侧都被检测到，这表明其具有跨膜结构。这些结果：（i）提供了迄今为止最有力的证据，表明AUM包含三种主要蛋白质（27 kDa的uroplakin I、15 kDa的uroplakin II和47 kDa的uroplakin III），它们形成一种极其不溶性的复合物；（ii）表明uroplakin II与uroplakin I一样（Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216），在AUM成熟过程中从膜的一侧转运到另一侧；（iii）表明uroplakin III在AUM形成中可能发挥与uroplakin I和II不同的结构作用；（iv）确立了这三种uroplakin作为尿路上皮分化晚期阶段的标志物。
