Tissue-Type Transglutaminase from Red Sea Bream (Pagrus major). Sequence Analysis of the cDNA and Functional Expression in Escherichia coli

Abstract

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.

Partial Purification and Characterization of Transglutaminase From Threadfin Bream (Nemipterus Sp.) Liver

“…1996). In addition, the cloned liver TGase from red sea bream showed the similar catalytic activity to the wild type (Yasueda et al. 1995).…”

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis

“…TGases are widely distributed in various organisms, including vertebrates (Chung and Folk, 1972; Wong et al ., 1990; Weraarchakul-Boonmark et al ., 1992; Grant et al ., 1994; Yasueda et al ., 1995), invertebrates (Tokunaga et al ., 1993; Signh and Metha, 1994), mollusks (Klein et al ., 1992), plants (Margosiak et al ., 1990) and microorganisms (Kanaji et al ., 1993). For example, human blood coagulation factor XIII, which forms fibrin clots in hemostasis and wound healing by catalyzing the cross-linking between fibrin molecules, has been well characterized (Bruner-Lorand et al ., 1966; Gladner and Nossal, 1983; Ichinose et al ., 1986; Hornyak et al ., 1989; Bishop et al ., 1990; Hornyak and Shafer, 1991), and its crystal structure has been determined (Pedersen et al ., 1994; Yee et al ., 1994, 1995; Weiss et al ., 1998).…”

Identification of mammalian‐type transglutaminase in Physarum polycephalum

“…Several gene structures responsible for enzymatic activity have been reported in various organisms. TGases with homologous primary structure have been cloned in fish and some invertebrates such as red sea bream [24], salmon [25], grasshoppers [27], and limuli [28]. In lower eukaryotes, however, homologous genes have not been reported so far.…”

“…3). The amino acid sequences of PpTGase were 40-50% identical to those of human TGase 2 [14] and TGases of red sea bream [24], ascidians [26], grasshopper [27], fruit fly, and limulus [28]. Ser, which is an essential amino acid residue for GTP binding, is also conserved (region A in Fig.…”