Rindler M J, Naik S S, Li N, Hoops T C, Peraldi M N
Department of Cell Biology, New York University Medical Center, New York 10016.
J Biol Chem. 1990 Dec 5;265(34):20784-9.
Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.
尿调节蛋白最初被鉴定为孕妇尿液中的一种免疫抑制糖蛋白，此前已证明它与人Tamm-Horsfall糖蛋白（THP）相同。THP由肾脏合成，定位于肾髓袢升支粗段和远曲小管起始部。它大量释放到尿液中，因此是体内上皮细胞顶端质膜以极化方式分泌的一种蛋白质的潜在候选物。然而，通过转染将编码尿调节蛋白/THP的全长cDNA导入HeLa、Caco-2和Madin-Darby犬肾细胞后，经放射性标记细胞后的免疫沉淀测定，所表达的糖蛋白几乎完全与细胞相关。通过免疫荧光，THP定位于转染细胞的质膜。在转染细胞提取物中，在Triton X-114分配试验中，THP也主要保留在去污剂相中，表明它具有疏水性，这与其从人尿液中分离后的行为相反。用磷脂酰肌醇特异性磷脂酶C处理细胞提取物后，与Triton X-114去污剂相关的THP重新分布到水相中。用磷脂酰肌醇特异性磷脂酶C处理完整的转染HeLa细胞也导致THP释放到培养基中，表明它是一种糖基磷脂酰肌醇（GPI）连接的膜蛋白。与其他已知的GPI连接蛋白类似，尿调节蛋白/THP在其极端羧基末端含有一段16个疏水氨基酸，可作为GPI添加信号，并显示能用[3H]乙醇胺标记。结果表明，THP是这类脂连接膜蛋白的成员，在其疏水锚丢失后可能通过磷脂酶或蛋白酶的作用释放到尿液中。
