Prostaspanin: A novel member of tetraspanin superfamily specifically expressed in the prostate

我们利用基于PCR的消减杂交技术分离了在雄激素依赖的大鼠前列腺腹侧再生过程中特异表达的cDNA克隆。其中一个克隆AIEG322(暂定为prostaspanin)与Tetraspanin超家族成员的序列具有显著的同源性。在被检测的大鼠组织中，前列腺素mRNA在前列腺和精囊中检测到高水平的表达。为了研究前列腺素在前列腺癌中的生理功能，并评价血清前列腺素浓度作为前列腺癌标志物的可能性，我们将前列腺素抗体的制备放在首位。从大鼠和人的前列腺组织中分别扩增出前列腺素A(Prostaspanin)的cDNAs，并构建了带有GST和His标签的大鼠和人前列腺素的表达载体。重组蛋白在大肠杆菌中得到了表达。但是，由于包涵体的形成，表达的蛋白不能溶解。由于重组蛋白作为抗原的困难，我们开始生产抗寡肽抗体。用人工合成的前列腺素(Prostapanin，EC2)胞外区的寡肽FTQVWNTTM在兔体内产生高滴度的抗血清。免疫印迹分析表明，该抗血清可识别重组大鼠和人前列腺素S蛋白。然而，用大鼠组织提取物和膜组分进行的Western印迹分析表明，该抗血清不能与天然的Prostaspnin反应。Nativ前列腺素中天冬氨酸残基的糖基化可以解释这一观察结果。目前，我们正在尝试使用一种与蛋白质不同区域相对应的合成寡肽来制备抗前列腺素抗体。

We have isolated cDNA clones which are specifically expressed during androgen-dependent regrowth of rat ventral prostate by using PCR-based subtractive hybridization technique. One of the clones, AIEG322 (tentatively designated as prostaspanin), showed significant sequence homology with those of the members of tetraspanin superfamily. Among rat tissues examined, highlevels of prostaspanin mRNA expression was detected in the prostate and seminal vesicle. To investigate physiological function of prostaspanin in the prostate and to evaluate the possibility of utilization of the serum prostaspanin concentration as a marker of prostatic cancer, we set the highest priority for preparing anti-prostaspanin antibodies. Prostaspanin cDNAs were amplified from cDNAs derived from rat and human prostates, and the expression vectors for rat and human prostaspanin with a GST- and a hexahistidine-tag were prepared. The recombinant proteins were expressed in E. coli. However, the expressed proteins could not be solubilized, because of the formation of inclusion bodies. Due to the difficulties in using the recombinant proteins as antigens, we started to produce anti-oligopeptide antibodies. The use of an synthetic oligopeptide, FTQVWNTTM, corresponding to one of the extracellular domains of prostapanin (EC2), resulted in the production of high titer antiserum in rabbits. The antiserum recognized recombinant rat and human prostaspanin in Western blot analysis. Western blot analysis using rat tissue extracts and membrane fractions, however, showed that the antiserum could not react with native prostaspnin. Glycosylation at the aspargine residue in the nativ prostaspanin may explain the observation. Currently, we are trying to prepare anti-prostaspanin antibodies using a synthetic oligopeptide corresponding to different regions of the protein.