TISSUE-TYPE TRANSGLUTAMINASE FROM RED-SEA BREAM (PAGRUS-MAJOR) - SEQUENCE-ANALYSIS OF THE CDNA AND FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isola ted from a cDNA library prepared from the liver of red sea bream (Pagr us major). The cDNA sequence had an open reading frame coding for a pr otein of 695 amino acids and showed 43% identity to the sequence of gu inea pig liver TGase, revealing a relatively low overall similarity. H owever, the 25-amino-acid sequence containing the putative active site (Cys272) of the enzyme was completely conserved between the two speci es, and was also identical to the corresponding regions of human and b ovine endothelial cell TGases. In addition, the critical residues (His 332 and Asp355) thought to form the catalytic-center triad together wi th Cys272, were found in the highly conserved region. The red sea brea m TGase had an extension of 11 amino acids in the C-terminal region an d 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 p TTG2-22 could produce soluble TGase which exhibited catalytic activity in the presence of calcium. E. coli extracts containing the recombina nt red sea bream TGase induced gelation of actomyosin solutions, accom panied by a significant increase of epsilon-(gamma-glutamyl)lysine bon ds, which are predominantly derived from the cross-linking of myosin h eavy chains. These results indicate that this fish TGase should be use ful for further analysis of TGase structure/function relationships and that it could also be employed to enhance the viscoelastic properties of proteinaceous materials.