Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression.

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5' end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.

Two juvenile hormone esterase-like carboxylesterase cDNAs from a Pandalus shrimp (Pandalopsis japonica): cloning, tissue expression, and effects of eyestalk ablation.

Methyl farnesoate (MF), a crustacean juvenile hormone (JH) analog, plays important roles in the regulation of a number of physiological processes such as molting, metamorphosis, and reproduction. Understanding its metabolic pathway is a key for various potential applications in crustacean aquaculture, including artificial seed production and enhancement of growth. Although the synthetic pathway of MF is well established, little is known about its degradation and recycling in crustaceans. In insects, juvenile hormone esterase (JHE), a carboxylesterase, is responsible for JH inactivation. Two cDNAs, encoding JHE-like carboxylesterases (CXEs) from the hepatopancreas and ovary of Pandalopsis japonica, were isolated by using a combination of in-silico data mining from an expressed sequence tag (EST) database and traditional PCR-based cloning. The full length Pj-CXE1 (2084bp) and Pj-CXE2 (1985bp) cDNAs encoded proteins composed of 584 and 581 amino acids, respectively. The active site sequence and domain organization of the Pj-CXEs were highly conserved, including the catalytic triad and other motifs, which suggested that both Pj-CXEs are biologically active carboxylesterases. Phylogenetic analysis of the deduced sequences of Pj-CXEs showed that both were most closely related to the JHEs from non-lepidopteran insects. End-point RT-PCR showed that Pj-CXE1 was expressed primarily in the gonad, whereas Pj-CXE2 was expressed in both the hepatopancreas and hindgut. Quantitative PCR showed that Pj-CXE1 was upregulated in the gonads by eyestalk ablation (ESA). In contrast, ESA had no significant effect on Pj-CXE2 expression in hepatopancreas or gonad. This is the first report of the cloning of two JHE-like CXE cDNAs in decapods and the upregulation of Pj-CXE1 by acute withdrawal of eyestalk neuropeptides. Further study is needed to understand the function of CXEs in MF metabolism and its regulation by eyestalk neuropeptides.

The enzyme and the cDNA sequence of a thermolabile and double-strand specific DNase from Northern shrimps (Pandalus borealis).

BACKGROUND: We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies. METHODOLOGY/PRINCIPAL FINDINGS: A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65 degrees C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases. CONCLUSIONS/SIGNIFICANCE: The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.

The heavy-light chain loop of human cathepsin-L modulates its activity and stability.

Differences evident in the sequence alignment of human cathepsin-L with shrimp cathepsin-L and silicatein-alpha suggest the indirect involvement of the heavy to light chain loop (E 286 to E 289) in the function of these enzymes. Deletion of the loop and adjacent residues S 290 to N 293, decreased specific protease activity by 81% and 63%, respectively; complete substitution for the corresponding silicatein-alpha loop decreased activity by 35%. In all cases the Km was largely unchanged. The conformational stability of human procathepsin-L was not altered by deletion of E 286 to E 289 but increased on deletion of S 290 to N 293. Therefore, shortening the loop does not change substrate affinity but does influence activity, in part via conformational change.

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.

Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp.

A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.