Kershaw D B, Beck S G, Wharram B L, Wiggins J E, Goyal M, Thomas P E, Wiggins R C
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1997 Jun 20;272(25):15708-14. doi: 10.1074/jbc.272.25.15708.
Human renal cortex and heart cDNA libraries were screened for a human homolog of rabbit PCLP1 using the rabbit PCLP1 cDNA as a probe. Clones spanning 5869 base pairs with an open reading frame coding for a 528-amino acid peptide were obtained. The putative peptide contains a potential signal peptide and a single membrane-spanning region. The extracellular domain contains multiple potential sites for N- and O-linked glycosylation and 4 cysteines for potential disulfide bonding similar to rabbit PCLP1. On Northern blot a major transcript is seen at 5.9 kilobases. Antibodies to this protein show a doublet at 160/165 kDa on Western blots of human glomerular extract and a pattern of intense glomerular staining and vascular endothelial staining on immunofluorescence of human kidney sections. Comparison of the rabbit and human peptide sequences shows a high degree of identity in the transmembrane and intracellular domains (96%) with a lower degree of identity in the extracellular domain (36%). An antibody to the intracellular domain reacted across species (human, rabbit, and rat) and recognized both rabbit PCLP1 and rat podocalyxin. An interspecies Southern blot probed with a cDNA coding for the intracellular domain showed strong hybridization to all vertebrates tested in a pattern suggesting a single copy gene. We conclude that this cDNA and putative peptide represent the human homolog of rabbit PCLP1 and rat podocalyxin.
以兔PCLP1 cDNA为探针，对人肾皮质和心脏cDNA文库进行筛选，寻找兔PCLP1的人同源物。获得了一个跨越5869个碱基对的克隆，其开放阅读框编码一个528个氨基酸的肽段。推测的肽段包含一个潜在的信号肽和一个单一的跨膜区域。细胞外结构域含有多个潜在的N-和O-连接糖基化位点以及4个半胱氨酸，用于潜在的二硫键形成，类似于兔PCLP1。在Northern印迹上，在5.9千碱基处可见一个主要转录本。针对该蛋白的抗体在人肾小球提取物的Western印迹上显示出160/165 kDa的双峰，在人肾切片的免疫荧光上显示出强烈的肾小球染色和血管内皮染色模式。兔和人肽序列的比较显示，跨膜和细胞内结构域具有高度同源性（96%），而细胞外结构域的同源性较低（36%）。针对细胞内结构域的抗体可跨物种反应（人、兔和大鼠），并识别兔PCLP1和大鼠足突膜蛋白。用编码细胞内结构域的cDNA进行种间Southern印迹，结果显示与所有测试的脊椎动物都有强烈杂交，模式表明是单拷贝基因。我们得出结论，该cDNA和推测的肽段代表兔PCLP1和大鼠足突膜蛋白的人同源物。
