Nara K, Ito S, Ito T, Suzuki Y, Ghoneim M A, Tachibana S, Hirose S
Department of Biological Sciences, Tokyo Institute of Technology.
J Biochem. 1994 Mar;115(3):441-8. doi: 10.1093/oxfordjournals.jbchem.a124357.
Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)
弹性蛋白酶抑制因子被证明是一种具有锚定序列的新型蛋白酶抑制剂。人弹性蛋白酶抑制因子是一种对弹性蛋白酶和蛋白酶3具有特异性的强效抑制剂，其前肽段具有独特的重复序列，富含谷氨酰胺和赖氨酸残基。该前肽段被称为“黏结蛋白”，与精囊凝块蛋白的重复元件具有高度同源性，精囊凝块蛋白是前列腺转谷氨酰胺酶的良好底物。组织转谷氨酰胺酶对黏结蛋白的交联表明，黏结蛋白部分确实是转谷氨酰胺酶的优选底物。此外，在体外观察到转谷氨酰胺酶介导的黏结蛋白与层粘连蛋白之间的交联，表明黏结蛋白具有与其他细胞外基质蛋白共价连接的能力。为了确定这种通过黏结蛋白部分对弹性蛋白酶抑制因子进行的共价黏合在体内是否发生，我们通过蛋白质印迹法测定了气管黏液上皮中黏结蛋白-弹性蛋白酶抑制因子的分子大小；这种方法的基本原理是：（i）如下所示，气管是黏结蛋白-弹性蛋白酶抑制因子最丰富的来源，（ii）如果黏结蛋白-弹性蛋白酶抑制因子与其他蛋白质共价结合，它在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上应迁移为分子量更高的物种；在对应于50 kDa的位置确实检测到了黏结蛋白-弹性蛋白酶抑制因子的免疫反应性，该值远高于其单体形式。核糖核酸酶保护分析和免疫组织化学染色显示，黏结蛋白-弹性蛋白酶抑制因子在皮肤和气管中密集分布，在胃、十二指肠和小肠中分布适中。这些定位部位与弹性纤维丰富的位置一致。（摘要截断于250字）
