The cDNA cloning of a 55-kilodalton protein from guinea pig seminal vesicle. Evidence that the protein is the precursor of a 25-kilodalton basic secretory protein.
The cDNA cloning of a 55-kilodalton protein from guinea pig seminal vesicle. Evidence that the protein is the precursor of a 25-kilodalton basic secretory protein.
复制标题
来自豚鼠精囊的 55 千道尔顿蛋白质的 cDNA 克隆。
DOI：
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发表时间：
1985
影响因子：
4.8
通讯作者：
C. Veneziale
中科院分区：
文献类型：
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作者：
J. Moore;M. Norvitch;C. Veneziale
豚鼠精囊上皮合成并分泌大量的四种分泌蛋白，包括一种称为SVP-1的碱性蛋白。后者通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳作为25 kDa的蛋白质迁移。当来自精囊上皮的总细胞RNA在体外由兔网织红细胞系统翻译时，SVP-1不能被鉴定为产物。相反，网织红细胞系统的主要产物是一种55 kDa的蛋白质，矛盾的是，它不能被鉴定为四种已知的分泌蛋白之一，也不能被鉴定为丰富的组织蛋白。我们分离了一个cDNA克隆，其对应于编码55-kDa蛋白的mRNA。mRNA为1800个碱基，丰度非常高;只有对应于主要分泌蛋白SVP-3和SVP-4的转录本同样明显。根据这些观察结果，我们决定测试55 kDa蛋白是否与SVP-1相关。发现55-kDa蛋白具有SVP-1的特性，包括碱性pI和通过抗SVP-1抗体的选择。在55-kDa蛋白质的蛋白水解消化后，一个片段与真实的SVP-1共迁移。此外，小于SVP-1的55-kDa蛋白质的可检测片段都与由SVP-1的蛋白水解消化产生的片段共迁移。最后，我们表明，55 kDa的蛋白质可以在体外加工的精囊腔提取物，以产生一个30 kDa的蛋白质和蛋白质的大小天然SVP-1。我们的研究结果的最好的解释是，55 kDa的蛋白代表的主要翻译产物和前体的SVP-1。我们的蛋白水解图谱和体外加工研究与55 kDa蛋白是两个SVP-1分子的串联重复的想法一致。我们利用该cDNA克隆研究了相应基因在雄激素耗竭和补充不同状态下的表达。
Guinea pig seminal vesicle epithelium synthesizes and secretes large amounts of four secretory proteins including a basic protein designated SVP-1. The latter migrates as a protein of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When total cellular RNA from seminal vesicle epithelium was translated in vitro by a rabbit reticulocyte system, SVP-1 was not identifiable as a product. In contrast, a major product of the reticulocyte system was a 55-kDa protein which paradoxically could not be identified as one of the four known secretory proteins or as an abundant tissue protein. We isolated a cDNA clone which corresponded to the mRNA which coded for the 55-kDa protein. The mRNA was 1800 bases and of very high abundance; only the transcripts corresponding to the major secretory proteins SVP-3 and SVP-4 were as conspicuous. From these observations, we decided to test whether the 55-kDa protein was related to SVP-1. The 55-kDa protein was found to share properties of SVP-1, including an alkaline pI and selection by antibody against SVP-1. After proteolytic digestion of the 55-kDa protein, one fragment co-migrated with authentic SVP-1. Furthermore, detectable fragments of the 55-kDa protein smaller than SVP-1 all co-migrated with fragments generated from the proteolytic digestion of SVP-1. Finally, we show that the 55-kDa protein could be processed in vitro by seminal vesicle lumenal extracts to yield a 30-kDa protein and a protein the size of native SVP-1. The best explanation of our results is that the 55-kDa protein represents the primary translation product and precursor of SVP-1. Our proteolytic mapping and in vitro processing studies are consistent with the idea that the 55-kDa protein is a tandem repeat of two SVP-1 molecules. We used the cDNA clone to study the expression of the corresponding gene in different states of androgen depletion and repletion.