Department of Experimental Medicine, University of Milano-Bicocca, Monza, Italy.
Proteomics Clin Appl. 2007 Jun;1(6):588-97. doi: 10.1002/prca.200601048. Epub 2007 May 11.
Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex.
人水通道蛋白-1（AQP1）是水通道家族中研究最多的成员，作为分子水通道。它也被认为是近端肾小管细胞的分化标志物，透明细胞肾细胞癌（RCC）起源于此，在尿液形成中发挥重要作用。因此，我们在蛋白质组水平上研究了 RCC 和正常组织中的 AQP1 表达，主要集中在 AQP1 高度集中的微域富集膜上。通过差速离心制备亚细胞级分，并用 1%Triton X-100 处理从富含质膜的级分中纯化富含微域的级分，然后在蔗糖梯度中超离心。用针对 AQP1 的抗体进行 SDS-PAGE 和 Western blot 分析后，与正常肾组织的级分相比，在 RCC 的每个亚细胞级分中观察到 AQP1 同工型的表达水平较低。通过 MALDI-TOF-MS 和 LC-ESI-MS/MS 分析验证了免疫反应带中 AQP1 的存在。还使用 N-糖苷酶 F 研究了 AQP1 的糖基化，证实了 AQP1 的 N-糖基化同工型存在于 35-45-kDa 区域。这些结果突出表明与相邻正常皮质相比，RCC 组织匀浆和亚细胞级分中 AQP1 蛋白及其糖基化同工型的表达下调。
