Md. Nazmul Ahsan

Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus.

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.

Molecular cloning and characterization of cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis

We cloned a cDNA encoding cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis (NsCtB). Nucleotide sequence of the isolated clone encoded a preproenzyme of 328 amino acids, comprising a 15-residue putative signal peptide, a 60-residue propeptide and the 253-residue mature enzyme. The mature NsCtB was 53% identical to human cathepsin B and conserved all the structural features characteristic of cysteine protease. The presence of an occluding loop in the mature region, a unique feature of cathepsin B, suggested the shrimp protein to be cathepsin B. Northern blot analysis revealed expression of NsCtB transcripts exclusively in the hepatopancreas tissues, suggesting a possible digestive role of this enzyme. An interesting feature of NsCtB was its remarkably high negative charge in comparison with other cysteine proteases, which was predicted to effectively locate and guide the positively charged residues of a substrate into the binding cleft. We also observed a repertoire of cysteine protease activities in the acidic milieu of shrimp hepatopancreas using synthetic substrates specific to various cathepsins. The activity profile revealed cathepsin B as the single most dominant enzyme with a specific activity comparable to that attributable to combined activities of other cathepsins. This activity could be blocked by E-64, a cysteine protease inhibitor, but not by Z-Phe-Tyr (t-Bu)-CHN(2), a specific inhibitor of cathepsin L.

Molecular Cloning and Characterization of Two Isoforms of Trypsinogen from Anchovy Pyloric Ceca

Abstract: Complementary DNA clones encoding two isoforms of trypsinogen were isolated from the pyloric ceca of anchovy by rapid amplification of cDNA ends (RACE). Nucleotide sequences of isolated clones encoded, in addition to characteristic signal and activation peptides, two isoforms of trypsin containing 220 and 221 amino acid residues. Both enzymes contained the catalytic triad of a serine protease, together with the residues determining substrate specificity. The anchovy trypsins showed a high amino acid identity of about 80% to those of other fish species. Southern blot analysis with a probe cross-reactive to both isoforms showed a complex genomic pattern. Northern blot analysis with the same probe revealed the highest expression of meassenger RNA in the pyloric ceca. Structural parameters possibly involved in higher catalytic properties of fish trypsin were examined by three-dimensional modeling, which included deletion in the autolysis loop, lack of Tyr-151 at the entrance of the S1 pocket, and distribution of charged residues.

Molecular and enzymatic properties of a cathepsin L-like proteinase with distinct substrate specificity from northern shrimp (Pandalus borealis)

We purified a cathepsin L-like proteinase to homogeneity from the hepatopancreas of northern shrimp Pandalus borealis by several chromatographic procedures. The purified proteinase showed the highest specificity for leucine residue at P2, a specificity pattern similar to cathepsins S and K whereas proline and arginine residues were not suitable as P2 substrates. However, unlike these proteinases, it accepted valine almost equally to the phenylalanine residue at P2. The shrimp cathepsin was strongly inhibited by E-64, leupeptin and antipain, while benzyloxycarbonyl-Phe-Tyr(t-Bu)-CHN2, a specific inhibitor of cathepsin L, remained largely ineffective. Next, we determined the primary structure of the shrimp enzyme by molecular cloning and investigated the residues constituting the S2 subsite, which is possibly involved in its unusual substrate specificity. The deduced amino acid sequence of the shrimp proteinase shared the highest identity of 65% with a cathepsin L-like proteinase from lobster, but its identity to the well-characterized mammalian cathepsins S, L, and K fell within narrower ranges of 52–55%. However, the shrimp proteinase differed from these cathepsins in some key residues including, for example, the unique occurrence of cysteine and glutamine residues at the structurally important S2 subsite. Interestingly, transcripts of this proteinase were exclusively detected in the shrimp gut coinciding with its broad pH activity and stability profiles, which is also unusual as a cysteine proteinase. These results suggest that the shrimp enzyme is homologous to mammalian cathepsins S, L, and K, but is distinct from each of these proteinases in both enzymatic and structural properties.

Abalone collagens: immunological properties and seasonal changes of their mRNA levels.

The antisera were raised against pepsin-solubilized abalone collagen and its corresponding gelatin. The reactivity against abalone collagen was higher with the anti-collagen than anti-gelatin antiserum. The two antisera recognized all type I collagens from various vertebrates, whereas these had no reactivity against vertebrate type III and type V collagens. Furthermore, both antisera reacted with only alpha 2(I) chains from chicken, rat, and calf. The strong reactivity was observed against the two antisera in the case of invertebrate and protochordate collagens, especially for turban shell collagen. The seasonal changes of collagen mRNA levels were examined in relation to those of collagen content. Haliotis discus collagens (Hdcols) 1 alpha and 2 alpha coding for abalone collagen pro alpha-chains showed quite similar patterns. The highest mRNA levels in adductor and foot muscles for the two collagens were observed in December and January, in good agreement with the increase of collagen content. The mRNA levels decreased in July and August when collagen content decreased. These results indicate that collagen transcription levels are closely related to collagen contents.

CYR61 is a novel gene associated with temperature‐dependent changes in fish metabolism as revealed by cDNA microarray analysis on a medaka Oryzias latipes cell line

A microarray comprising 3,514 cDNAs was constructed from a medaka EST library to elucidate the transcriptional responses associated with temperature shift from 25 to 15°C in a medaka cell line. Microarray analysis revealed that the mRNA levels of 313 clones were significantly different in at least one combination of different incubation periods up to 7 days at a given incubation temperature or between 25 and 15°C at a given incubation period (P < 0.05). These genes are known to be associated with various biological processes including morphogenesis, cell proliferation and response to stress. A number of genes encoding proteins which localize in extracellular areas were apparently up‐regulated at 15°C, whereas those localizing in intracellular areas were down‐regulated at this temperature. In addition, while a number of genes represented long‐term expression changes, only a few responded to short‐term inductions. A typical example was CYR61, a multifunctional matricellular signaling modulator, the mRNA levels of which increased after temperature shift from 25 to 15°C in 3 h, and then decreased rapidly to near the original level within 12 h. Another series of analyses by quantitative reverse transcription‐PCR revealed that the mRNA levels of CYR61 at 5°C were significantly higher even at 24 h after temperature shift compared to those of the cells successively maintained at 25°C. These analyses suggest that remodeling and reorganizing of extracellular structure of cells are important to offset the low temperature effect and CYR61 is considered to be a novel gene associated with temperature response in poikilotherms. J. Cell. Biochem. 104: 1297–1310, 2008.