Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Stomach of Two Species of Fish, Chub Mackerel (<i>Scomber japonicus</i>) and Silver Croaker (<i>Pennahia argentata</i>)

Chitinolytic activities were measured in two fish species having different feeding habits, chub mackerel (Scomber japonicus) and silver croaker (Pennahia argentata). Chitinase (an endo-type chitinolytic enzyme) activity was measured using pNP-(GlcNAc)n (n = 2, 3) as substrates;its level was significantly high in the stomachs of both species, as well as in the gills, intestine, pyloric appendage, testis, and liver of chub mackerel and in the spleen, kidney, pyloric appendage, ovaries, heart, and liver of silver croaker. β-N-Acetylhexosaminidase (an exo-type chitinolytic enzyme) activity was measured using pNP-(GlcNAc) as a substrate;it was detected at high levels in many parts apart from the digestive tracts of both species. The optimum pH for chitinase activity was 3.0 - 5.0 in the stomachs of both species, 4.0 in the liver of chub mackerel, and 4.0 and 8.0 in the kidney of silver croaker. Full-length cDNAs encoding two chitinase isozymes were obtained from the stomachs of the two fish species: SjChi-1 (1604 bp) and SjChi-2 (1512 bp) from chub mackerel and PaChi-1 (1630 bp) and PaChi-2 (1606 bp) from silver croaker. Expression analysis of these genes in the organs of the two species revealed strong expression of SjChi-1 in the stomach of chub mackerel and that of PaChi-1 andPaChi-2 in the stomach of silver croaker. The difference in the expression pattern of these genes is likely attributed to the difference in the feeding habits of the two fish species. Our results suggested the presence of novel chitinases in the two species.

Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Liver of Golden Cuttlefish <i>Sepia esculenta</i>

The distribution of chitinolytic enzymes in eight organs of the golden cuttlefish Sepia esculenta was determined. Chitinase activity (activity of endo-type chitinolytic enzyme) was measured using pNP-(GlcNAc)n (n = 2, 3) as substrates, with high activity detected in the liver, posterior salivary gland, and stomach. β-N-acetylhexosaminidase (Hex) activity (activity of exo-type chitinolytic enzyme) was determined using pNP-(GlcNAc) as a substrate, and high activity was observed in six organs, including the liver, branchial heart, posterior salivary gland, and stomach. In addition, two chitin-binding proteins (CBP-A, CBP-B) were isolated from the liver using a chitin affinity column. Two full-length cDNAs (SeChi-1: 1484 bp;SeChi-2: 1748 bp) encoding chitinases were obtained from the liver of S. esculenta. SeChi-1 contained a 1377-bp open reading frame (ORF) encoding 459 amino acids, and SeChi-2 contained a 1656-bp ORF encoding 552 amino acids. Domain structures predicted from the deduced amino acid sequences of SeChi-1 and SeChi-2 (SeChi-1, SeChi-2) contained signal peptides, a GH Family 18 catalytic domain, one chitin binding domain (CBD) in SeChi-1, and two CBDs in SeChi-2. Proteome analysis revealed that 125 peptide residues of CBP-A were present in SeChi-1, and 116 peptide residues of CBP-B were present in SeChi-2. Organ expression analysis revealed that SeChi-1 and SeChi-2 were expressed only in the liver of S. esculenta. Phylogenetic analysis of SeChi-1, SeChi-2, and GH family 18 chitinases revealed that SeChi-2 belongs to a group of previously reported squid chitinases, while SeChi-1 does not belong to any previously reported group of mollusk chitinases.

Distribution of Chitinolytic Enzyme in the Organs and Molecular Cloning of a Novel Chitinase Gene from the Kidney of Marbled Rockfish <i>Sebastiscus marmoratus</i>

Actinopterygii express two types of chitinase (acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2)) that are active at acidic pHs and involved in digestion in the stomach. We proposed the existence of a new fish chitinase that has a non-digestive function. In this study, we used Sebastiscus marmoratus, for which characteristics and cDNA cloning of chitinase isozymes (SmChi-1, SmChi-2) in the stomach have been reported. Initially, we examined the distribution of chitinase and β-N-acetylhexosaminidase (Hex) in the body and then we tried to clone novel chitinase cDNA from the kidney. Chitinase and Hex activities were measured using pNP-(GlcNAc)n, (n = 2, 3) and pNP-GlcNAc as substrates, respectively. Total RNA was extracted from the kidney. RT-PCR was performed to obtain chitinase cDNA fragments using reverse transcriptase with an oligo dT primer. The RACE method was used to obtain sequences of the upstream and downstream regions of cDNA. The full-length chitinase cDNA was determined using PrimeSTAR&reg;?Max DNA polymerase with proofreading activity. High chitinase activity was observed in the stomach, as previously reported. In addition, relatively high activity was observed in the liver, spleen, kidney, and heart. In contrast, Hex activity was detected in all organs. This result is consistent with the report that Hex is related to body-wide metabolism. Full-length cDNA (SmChi-3) of the novel chitinase was obtained from the kidney, which contained 1440 bp open reading frames. The domain structure of SmChi-3 was assumed to be similar to those of SmChi-1 and SmChi-2. SmChi-1 and SmChi-2 have a serine and glycine-rich linker region, which is characteristic of AMCase. In contrast, SmChi-3 contained no apparent sequence in the linker region. Phylogenetic analysis revealed the existence of a new chitinase group, which was named fish chitinase-3 (FCase-3) and differed from AFCase-1 and AFCase-2.

Chondrichthyes Chitinase: Molecular Cloning, Distribution, and Phylogenetic Analysis

We have previously reported the presence of three types of chitinase (acidic fish chitinase-1: AFCase-1, acidic fish chitinase-2: AFCase-2, fish chitinase-3: FCase-3) in Actinopterygii. In the present research, we report the identification of the novel chitinase genes HjChi (ORF: 1380 bp) and DkChi (ORF: 1440 bp) from the stomach of Chondrichthyes, Japanese bullhead shark (Heterodontus japonicas) and Kwangtung skate (Dipturus kwangtungensis), respectively. Organ-specific expression analysis identified the stomach-specific expression of HjChi, whereas DkChi was expressed widely in all organs. Chitinase activity was measured using pNP-(GlcNAc)n (n = 2, 3) as a substrate and β-N-acetylhexosaminidase (Hex) activity was measured using pNPGlcNAc. Relatively high values of chitinase activity were observed in the stomach, spleen, and gonads of the Japanese bullhead shark, H. japonicas , compared with that observed in the stomach of the Kwangtung skate D. kwangtungensis . However, Hex activity was detected throughout the body of both species. The optimal pH of chitinase in both the Japanese bullhead shark, H. japonicas, and the Kwangtung skate, D. kwangtungensis, were 3.5 - 5.5 and 3.5 - 4.0, respectively, and 4.0 for Hex in both species. Phylogenetic analysis revealed that Chondrichthyes chitinase forms a unique group (Chondrichthyes chitinase). These results suggested that the possibility of the formation of chitinase groups for each class in the phylogenetic analysis based on the observation of class-specific chitinase.