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Title
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Tissue-type transglutaminase from red sea bream (Pagrus major). Sequence analysis of the cDNA and functional expression in Escherichia coli.
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Authors
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H.Yasueda,
K.Nakanishi,
Y.Kumazawa,
K.Nagase,
M.Motoki,
H.Matsui.
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Ref.
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Eur J Biochem, 1995,
232,
411-419.
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PubMed id
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Abstract
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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.
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