Chandrashekar R, Tsuji N, Morales T, Ozols V, Mehta K
Heska Corporation, Fort Collins, CO 80525, USA.
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):531-6. doi: 10.1073/pnas.95.2.531.
Transglutaminases (TGases; EC 2.3.2.13) are a family of enzymes that catalyze calcium-dependent covalent cross-linking of cellular proteins by establishing epsilon-(gamma-glutamyl)lysine isopeptide bonds. These covalent isopeptide bonds are of great physiological significance because they are highly resistant to proteolysis, denaturants, and reducing agents. Prior studies have demonstrated the presence of isopeptide bonds in the sheath and cuticle of filarial parasites, suggesting an important role for TGase-catalyzed reactions during the growth and development of filarial nematodes. Herein we report the identification and cloning of a cDNA encoding a TGase from the dog heartworm Dirofilaria immitis (DiTG). The DiTG expressed in Escherichia coli (recombinant DiTG) was able to catalyze calcium-dependent cross-linking reactions. The derived amino acid sequence of the DiTG cDNA (pDiTG) predicts a protein of 57.1 kDa and includes an N-terminal hydrophobic signal peptide. The pDiTG has no sequence similarity with any of the known TGases, but it has significant homology to protein disulfide isomerase (PDI) and, particularly, to the PDI-related endoplasmic reticulum protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. As predicted from the amino acid sequence homology, recombinant DiTG catalyzed the isomerization of intramolecular disulfide/sulfhydryl bonds in denatured RNase in vitro as effectively as did mammalian PDI. Conversely, purified PDI from bovine liver could catalyze protein cross-linking reactions in a Ca(2+)-dependent manner. This report describes the dual catalytic activity of TGase and PDI in post- and/or cotranslational modification of newly synthesized proteins. These TGase-catalyzed posttranslational modifications may play a pivotal role in the synthesis of new cuticle during the growth and maturation of filarial parasites.
转谷氨酰胺酶（TGases；EC 2.3.2.13）是一类酶，通过建立ε-（γ-谷氨酰基）赖氨酸异肽键来催化细胞蛋白的钙依赖性共价交联。这些共价异肽键具有重要的生理意义，因为它们对蛋白水解、变性剂和还原剂具有高度抗性。先前的研究已证明丝虫寄生虫的鞘和角质层中存在异肽键，这表明转谷氨酰胺酶催化的反应在丝虫线虫的生长和发育过程中起重要作用。在此，我们报告了从犬心丝虫恶丝虫（DiTG）中鉴定和克隆编码转谷氨酰胺酶的cDNA。在大肠杆菌中表达的DiTG（重组DiTG）能够催化钙依赖性交联反应。DiTG cDNA（pDiTG）推导的氨基酸序列预测其编码一个57.1 kDa的蛋白质，并包含一个N端疏水信号肽。pDiTG与任何已知的转谷氨酰胺酶均无序列相似性，但与蛋白质二硫键异构酶（PDI）具有显著同源性，特别是与内质网相关的内质网蛋白ERp60（在内质网腔中发现的一种PDI同工型）具有显著同源性。正如从氨基酸序列同源性预测的那样，重组DiTG在体外催化变性核糖核酸酶分子内二硫键/巯基的异构化，其效果与哺乳动物PDI相同。相反，从牛肝中纯化的PDI能够以Ca(2+)依赖性方式催化蛋白质交联反应。本报告描述了转谷氨酰胺酶和PDI在新合成蛋白质的翻译后和/或共翻译修饰中的双重催化活性。这些转谷氨酰胺酶催化的翻译后修饰可能在丝虫寄生虫生长和成熟过程中新角质层的合成中起关键作用。
