Kuroki J, Hosoya T, Itakura M, Hirose S, Tamechika I, Yoshimoto T, Ghoneim M A, Nara K, Kato A, Suzuki Y, Furukawa M, Tachibana S
Tsukuba Research Laboratories, Eisai Co., Ltd, Japan.
J Biol Chem. 1995 Sep 22;270(38):22428-33. doi: 10.1074/jbc.270.38.22428.
The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a polypeptide of 187 amino acids, which is composed of three domains: a hydrophobic presequence of 21 amino acids, a prosegment of 105 amino acids ending with Asp126, and the mature SPAI-2 sequence of 61 amino acids beginning with Pro127. The prosegment contained 16 repeats of a hexapeptide that is highly homologous to the repetitive sequence found in the transglutaminase domain of the human elafin, an elastase-specific inhibitor that also belongs to the WAP superfamily. The repetitive sequence was demonstrated to be a good substrate of transglutaminase using a recombinant preparation produced in Escherichia coli. A porcine genomic library was then screened for the SPAI gene. Characterization and sequencing of positive clones indicated that the gene is similar to the elafin gene, having 3 exons encoding the 5'-untranslated region and signal sequence, proSPAI, and 3'-untranslated region, respectively. Northern blot analysis revealed intestine-specific expression of SPAI mRNA; the message was especially abundant in the small intestine. ProSPAI was also found in the circulation. The similarity of proSPAI to elafin in the domain structure, the acid-labile nature of the cleavage site (Asp126-Pro127), and the fact that the major form of SPAI in the plasma is proSPAI strongly suggest that proSPAI is not the precursor but rather it is the native form of SPAI. Like elafin, therefore, SPAI appears to be a new type of biologically active substance with a transglutaminase substrate domain that acts as an anchoring sequence.
猪SPAI - 2是一种属于WAP蛋白超家族且具有钠钾ATP酶抑制活性的蛋白质，通过分子克隆和Northern分析确定了其初级结构、基因结构及组织分布。从猪十二指肠cDNA文库中分离出一个全长cDNA克隆。该cDNA插入片段编码一个由187个氨基酸组成的多肽，其由三个结构域组成：一个21个氨基酸的疏水前导序列、一个以Asp126结尾的105个氨基酸的前体片段，以及一个从Pro127开始的61个氨基酸的成熟SPAI - 2序列。前体片段包含一个六肽的16个重复序列，该序列与人弹性蛋白酶特异性抑制剂elafin（也属于WAP超家族）转谷氨酰胺酶结构域中的重复序列高度同源。使用在大肠杆菌中产生的重组制剂证明该重复序列是转谷氨酰胺酶的良好底物。随后筛选猪基因组文库以寻找SPAI基因。阳性克隆的鉴定和测序表明该基因与elafin基因相似，分别有3个外显子编码5' - 非翻译区和信号序列、proSPAI以及3' - 非翻译区。Northern印迹分析显示SPAI mRNA在肠道特异性表达；该信息在小肠中特别丰富。ProSPAI也存在于循环中。ProSPAI在结构域结构上与elafin的相似性、切割位点（Asp126 - Pro127）的酸不稳定性质，以及血浆中SPAI的主要形式是ProSPAI这一事实强烈表明ProSPAI不是前体，而是SPAI的天然形式。因此，与elafin一样，SPAI似乎是一种新型的生物活性物质，具有作为锚定序列的转谷氨酰胺酶底物结构域。
