Yasueda H, Nakanishi K, Kumazawa Y, Nagase K, Motoki M, Matsui H
Food Research & Development Laboratories, Ajinomoto Co., Inc., Japan.
Eur J Biochem. 1995 Sep 1;232(2):411-9. doi: 10.1111/j.1432-1033.1995.tb20826.x.
A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from a cDNA library prepared from the liver of red sea bream (Pagrus major). The cDNA sequence had an open reading frame coding for a protein of 695 amino acids and showed 43% identity to the sequence of guinea pig liver TGase, revealing a relatively low overall similarity. However, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two species, and was also identical to the corresponding regions of human and bovine endothelial cell TGases. In addition, the critical residues (His332 and Asp355) thought to form the catalytic-center triad together with Cys272, were found in the highly conserved region. The red sea bream TGase had an extension of 11 amino acids in the C-terminal region and some differences in the N-terminal region when compared with guinea pig TGase. From the cloned cDNA, a semi-synthetic TGase gene suitable for overexpression in Escherichia coli was constructed (pTTG2-22). At a reduced temperature (28 degrees C), E. coli cells transformed with pTTG2-22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombinant red sea bream TGase induced gelation of actomyosin solutions, accompanied by a significant increase of epsilon-(gamma-glutamyl)lysine bonds, which are predominantly derived from the cross-linking of myosin heavy chains. These results indicate that this fish TGase should be useful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials.
从真鲷（Pagrus major）肝脏制备的cDNA文库中分离出一个编码组织型转谷氨酰胺酶（TGase）的cDNA克隆。该cDNA序列有一个编码695个氨基酸的开放阅读框，与豚鼠肝脏TGase的序列有43%的同一性，整体相似性相对较低。然而，包含该酶假定活性位点（Cys272）的25个氨基酸序列在这两个物种之间完全保守，并且与人及牛内皮细胞TGase的相应区域也相同。此外，被认为与Cys272一起形成催化中心三联体的关键残基（His332和Asp355）在高度保守区域中被发现。与豚鼠TGase相比，真鲷TGase在C末端区域有11个氨基酸的延伸，在N末端区域有一些差异。从克隆的cDNA构建了一个适合在大肠杆菌中过表达的半合成TGase基因（pTTG2 - 22）。在降低的温度（28摄氏度）下，用pTTG2 - 22转化的大肠杆菌细胞可以产生可溶性TGase，其在钙存在下表现出催化活性。含有重组真鲷TGase的大肠杆菌提取物诱导肌动球蛋白溶液凝胶化，伴随着ε-（γ-谷氨酰）赖氨酸键的显著增加，这些键主要来自肌球蛋白重链的交联。这些结果表明，这种鱼类TGase对于进一步分析TGase的结构/功能关系应该是有用的，并且它也可以用于增强蛋白质材料的粘弹性。
