ISOLATION OF THE CDNA FOR ERYTHROCYTE INTEGRAL MEMBRANE-PROTEIN OF 28-KILODALTONS - MEMBER OF AN ANCIENT CHANNEL FAMILY
ISOLATION OF THE CDNA FOR ERYTHROCYTE INTEGRAL MEMBRANE-PROTEIN OF 28-KILODALTONS - MEMBER OF AN ANCIENT CHANNEL FAMILY
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DOI：
10.1073/pnas.88.24.11110
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发表时间：
1991-12-01
影响因子：
11.1
通讯作者：
AGRE, P
中科院分区：
文献类型：
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作者：
PRESTON, GM;AGRE, P
CHIP 28是一种28-kDa的整合膜蛋白，与膜通道相似，存在于红细胞和肾小管中。通过三步聚合酶链反应（PCR）克隆策略，从人胎肝cDNA模板分离CHIP 28的cDNA，从对应于从纯化的CHIP 28蛋白确定的N-末端氨基酸序列的简并寡核苷酸引物开始。以第三步PCR产物为探针，从人骨髓cDNA文库中分离到一个重组体。PCR产物和骨髓cDNA的组合序列包含38个碱基对的5'非翻译核苷酸序列、807-bp的开放阅读框架和几乎等于2个碱基的含有多聚腺苷酸化信号的3'非翻译序列。这对应于通过RNA印迹杂交分析鉴定的3.1-脱氢酶转录物。通过表达和免疫印迹证实了CHIP 28蛋白C末端推导的氨基酸序列的真实性。推导的氨基酸序列分析表明，CHIP 28蛋白含有6个跨双层结构域，两个外表面潜在的N-糖基化位点，和细胞内的N和C末端。DNA序列数据库的搜索揭示了与牛透镜的主要内在蛋白的强同源性，牛晶状体是一个古老但最近被认可的膜通道家族的原型。这些蛋白质被认为形成可渗透水和其他可能的小分子的通道。CHIP 28与该通道家族的所有已知成员具有同源性，并且推测CHIP 28具有类似的功能。
CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and almost-equal-to 2 kilobases of 3' untranslated sequence containing a polyadenylylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.