Structural properties of trypsin from cold-adapted fish, arabesque greenling (Pleurogrammus azonus)

A cDNA clone encoding trypsin (AG-T) was isolated from the pyloric ceca of cold-adapted fish, arabesque greenling (Pleurogrammus azonus). The cDNA was composed of 892 bp with an open reading frame of 729 bp at nucleotide positions 25-753. Similar to all the known trypsin, the AG-T seemed to be synthesized as preproenzyme that contains a hydrophobic signal peptide, an activation pentapeptide and a mature trypsin of 222 amino acid residues. The AG-T also completely conserved the major structural features common to trypsin such as the catalytic triad (His57, Asp102, and Ser195), the obligatory Asp189 and twelve Cys residues. On the other hand, the AG-T possessed the deletion of Tyr151 and substitution of Pro152 for Gly in the autolysis loop when aligned with the sequence of tropical-zone fish and bovine trypsins. In addition, Val75 concerned in a combination with calcium ion was exchanged for Ala in the AG-T, and the content of positively charged amino acid residues at the calcium-binding site of the AG-T was three times higher than those of tropical-zone fish trypsins. Moreover, the ratio between charged and hydrophobic amino acid residues in the N-terminal region of the AG-T was also higher than those of temperate-zone fish and tropical-zone fish trypsins. Such structural properties of the AG-T would contribute to its low thermostability

Cold-adapted structural properties of trypsins from walleye pollock (Theragra chalcogramma) and Arctic cod (Boreogadus saida)

Complementary DNA clones encoding trypsins were isolated from pyloric ceca of cold-adapted fish, walleye pollock (Theragra chalcogramma) (WP-T) and Arctic cod (Boreogadus saida) (AC-T). The isolated full-length cDNA clones of WP-T and AC-T were 852 bp and 860 bp, respectively, and both cDNAs were contained an open reading frame of 726 bp. WP-T and AC-T seemed to be synthesized as preproenzyme that contains a signal peptide, an activation peptide, and a mature trypsin. Although the amino acid sequence identities of WP-T and AC-T to that of bovine trypsin were 64% and 63%, respectively, they completely conserved the structural features for catalytic function of trypsin. On the other hand, WP-T and AC-T possessed the four Met residues (Met135, Met145, Met175 and Met242) in their molecules and the deletion of Tyr151 and substitution of Pro152 for Gly in their autolysis loops when aligned with the sequences of tropical-zone fish and bovine trypsins. In addition, the contents of charged amino acid residues at the N-terminal regions (positions 20-50) of WP-T and AC-T were extremely higher than those of other fish and bovine trypsins. Moreover, one amino acid (Asn72) and two amino acids (Asn72 and Val75) coordinating with Ca2+ in bovine trypsin were exchanged for another amino acids in WP-T (His) and AC-T (His and Glu), respectively, and the contents of negative charged amino acids at their Ca^[2+]-binding regions were lower than those of tropical-zone fish and bovine trypsins. Therefore, it was considered that these structural characteristics of WP-T and AC-T are closely related to their lower thermo stability