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

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. How to cite this paper: Nishino, R., Kakizaki, H., Fukushima, H. and Matsumiya, M. (2017) Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Liver of Golden Cuttlefish Sepia esculenta. Advances in Bioscience and Biotechnology, 8, 361-377. https://doi.org/10.4236/abb.2017.810026 Received: September 7, 2017 Accepted: October 10, 2017 Published: October 13, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access

In marine animals, studies have reported the purification, properties, and cDNA cloning of chitinase isozymes mainly obtained from the fish stomach, which are involved in digestion [12]- [17]. Chitinases in the fish stomach are classified into two groups based on differences in their primary structure and the patterns of degradation of (GlcNAc) n : acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2) [16] [17] [18] [19]. Conversely, studies reporting the cDNA cloning and expression of chitinases and chitinase-like proteins from bivalves and gastropods, which are mollusks, have noted that these play roles in shell formation [20] [21] [22], immunity [23] [24] [25] [26], and digestion [27]. Chitinase isozymes have been purified and studied from the liver of Decembrachiata (squid and cuttlefish), and are involved in digestion [28] [29] [30] [31]. Furthermore, two chitinase isozymes have been reported in the liver of Japanese common squid, and identified based on differences in molecular weight and N-terminal amino acid sequences [29] [30], and two chitinase isozymes have been reported in the liver of spear squid, and identified based on expressed sequence tag (EST) analysis [32]. However, the full-length genes have not yet been determined. Conversely, chitinases have been obtained from the posterior salivary gland of octopus [33] and cuttlefish [34], and found to act as poison. A chitotriosidase gene, which is involved in the induction of luminescent bacteria, has been found in the light organ of the Hawaiian bobtail squid Euprym-  [35]. Thus, the roles of mollusk chitinases are not limited to digestion and range widely; thus, many isozymes exist to support these different roles.
Golden cuttlefish Sepia esculenta, used in the present study, belongs to Decembrachiata and is a type of mollusk that mainly ingests crustaceans, which contain chitinous substances, and fish [36]. We have previously reported the distribution of chitinase activity using glycolchitin as the substrate [37] and purification and properties of a chitinase obtained from the liver of S. esculenta [31]; however, no findings of enzyme proteins and genes corresponding with the chitinase isozymes have been reported. In this study, we first observed the distribution of chitinase activity using two kinds of chitinase specific substrates in the body of S. esculenta, and isolated two types of chitin-binding proteins (CBPs) from the liver that exhibited particularly high chitinase activity. Next, we cloned chitinase genes from the liver and obtained two types of full-length genes.
Furthermore, the organ expression of the genes was analyzed, domain structures were compared, and phylogenetic analyses was performed based on the deduced amino acid sequences. The relationship between the two types of CBPs was elucidated and the different chitinase genes obtained were examined by proteome analysis. In summary, this study is the first to discuss the distribution of chitinolytic enzymes in S. esculenta, the presence of chitinase isozymes and features of their domain structure, and the positioning of chitinase isozymes in phylogenetic analysis.

Materials
Fresh S. esculenta was purchased from Tsukiji Fish Market (body weight: 183 g, liver weight: 9.5 g).

Measurement of Chitinolytic Enzyme Activity
Organs were removed from S. esculenta for subsequent analysis. Each organ was homogenized in five volumes of 20 mM phosphate buffer (pH 7.2), and then the homogenate was centrifuged at 7000 ×g for 20 min. The supernatant was used as the crude enzyme solution. Chitinase and Hex activities were measured using p-nitrophenyl (GlcNAc) n , (pNP-(GlcNAc) n ) (n = 2, 3) (Seikagaku, Tokyo, Japan) and pNP-GlcNAc (Seikagaku) as substrates, respectively, according to the method described by Ohtakara [38], with slight modification. Briefly

Amino Acid Sequence of the CBPs Isolated from the Liver of S. esculenta
The chitinase-active fraction was subjected to sodium dodecyl sulfate-polyacry-

cDNA Cloning of Chitinases from the Liver of S. esculenta
Total RNA was extracted from the S. esculenta liver using ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Next, cDNA was synthesized using 1.0 μg of total RNA, a PrimeScript reverse transcriptase (Takara Bio, Shiga, Japan), and an oligo dT primer ( Table 1). The reaction conditions were 90˚C for 3 min, 42˚C for 60 min, and 70˚C for 10 min. The primers used are listed in Table 1, and the primer combinations are shown in Figure 1.

Organ Expression of SeChi-1 and SeChi-2
Total RNA was extracted from S. esculenta organs. cDNA was synthesized using 0.5 μg of total RNA obtained from each tissue and an oligo dT primer, and amplified using PCR with 1.0 μg of the synthesized cDNA, primers for SeChi-1, SeChi-2, and Decembrachiata β-actin amplification primers ( Table 1). The reaction conditions were: 30 cycles of 95˚C for 30 s, 55˚C for 1 min, and 72˚C for 2 min.

Phylogenetic Analysis of Chitinases
Phylogenetic analysis, based on the deduced amino acid sequences of the full-length SeChi-1 and SeChi-2 genes, was performed using chitinase genes obtained from multiple organisms. The analysis was performed using ClustalW (http://clustalw.ddbj.nig.ac.jp/) and Tree view.

Distribution of Chitinolytic Activities
Chitinolytic activity measurement using pNP-(GlcNAc) 2 and pNP-(GlcNAc) 3 as substrates showed that out of eight measured organs of S. esculenta, the liver and stomach, which are involved in digestion, and the posterior salivary gland, which contains chitinase genes that were also found to be present in other cephalopods [33] [34], exhibited high activity (Figure 2(a)). When using glycolchitin as substrate, chitinase activity was detected only liver, stomach, and caecum [37].
Moreover, Hex activity, which is characteristic of exo-type chitinolytic enzymes, was high in the following six organs: the liver, heart, branchial heart, posterior salivary gland, stomach, and caecum (Figure 2(b)).  In digestive organs, chitinases and Hex are involved in the degradation of chitinous substances following their intake as feed. This is consistent with the feeding habit of S. esculenta; that is, S. esculenta ingests organisms containing chitinous substances, such as shrimps and crabs [36], suggesting that S. esculenta degrades chitin from feed into GlcNAc using both endo-and exo-type enzymes. In addition, it is possible that chitinases in the posterior salivary gland act as a poison, as observed in other cephalopods [33] [34]. Furthermore, because blood chitinases in mollusks have important roles in immunity [23], chitinases in the heart and branchial heart, which are not involved in digestion, are involved in defense against organisms containing chitinous substances, such as parasitic crustaceans and nematodes.

Isolation of CBPs from the Liver of S. esculenta
Using a chitin affinity column, CBPs were separated from the enzyme solution   CBP-B was considered to be SeChi, which is a chitinase purified from the liver of S. esculenta [31], because the molecular weight of CBP-B (62 kDa) was consistent with that of SeChi. Two types of chitinase isozymes with molecular weights of 38 [28] and 42 kDa [30] have previously been purified from the liver of Japanese common squid. This suggests that the CBP-A newly detected in this study is an isozyme of chitinase in the liver of S. esculenta.

cDNA Cloning of Chitinases from the Liver of S. esculenta
The liver of S. esculenta was used as a sample, and internal sequences of chitinase cDNA were amplified by PCR using degenerate primers designed from conserved amino acid sequences of GH family 18 chitinases. As a result, amplified fragments approximately 550 bp in size were found, and two types of base sequences were obtained by base sequencing. NCBI Blast analysis revealed that these base sequences share homology with chitotriosidase of Hawaiian bobtail squid E. scolopes [35]. The upstream and downstream sequences of the afore-

Amino Acid Sequence of the Chitinases
Two CBPs (CBP-A and CBP-B) (Figure 3 and Figure 4) obtained from the liver of S. esculenta were fragmented into peptides by trypsin treatment and compared with the amino acid sequences of SeChi-1 and SeChi-2 via proteome analysis.

CGAGTTCTGGTGGACAAACGACGAAAAGGCACA -1
ATGCTTGCTGTGTCCCTGTTACTCTTACTGGCCGTTGGCGGAGTCAGCAGTGCTGGGTACCGCCGGGTATGTTATCATACCAACTGGTCTCAATACAGACCTTCTCCTGGAAAGTATTTC 120  Trypsin is reported to cleave the C-terminal side of lysine (K) and arginine (R) [39]. It was confirmed that trypsin worked adequately at all cleavage sites because all of the obtained peptides ended with K or R.

Organ Expression of SeChi-1 and SeChi-2
The expression of SeChi-1 and SeChi-2 was investigated in eight S. esculenta organs, with both genes found to be expressed only in the liver (Figure 9). Expression of SeChi-2 was stronger than that of SeChi-1 (Figure 9). This result was consistent with findings from SDS-PAGE of CBPs isolated from the liver, such that the CBP-B band encoded by SeChi-2 was thicker than that of CBP-A encoded by SeChi-1 (Figure 4). Because SeChi-1 and SeChi-2 were found to be expressed in the liver, where chitinolytic activity was found, SeChi-1 and Se-Chi-2 are suggested to encode enzymes involved in chitin degradation in this organ. Furthermore, although high chitinolytic activity was detected in the posterior salivary gland, as shown in Figure 2, neither SeChi-1 nor SeChi-2 were expressed there. The presence of chitinases acting as poison in the posterior salivary gland of other cephalopods has been reported [33] [34]. Additionally, the presence of chitinases isozymes, which differ from SeChi-1 and SeChi-2, is suggested in the posterior salivary glandof S. esculenta.

Phylogenetic Analysis of SeChi-1 and SeChi-2
On the basis of amino acid sequence homology, phylogenetic analysis was conducted using SeChi-1, SeChi-2, GH family 18 chitinases of other organisms, and a GH family 18 chitinase of Serratiamarcescens, as an outgroup. SeChi-2 formed a group with other Decembrachiata chitinases, whereas SeChi-1 did not form a group with any of the mollusk chitinases ( Figure 10). Considering that SeChi-1 is the first chitinase with one CBD to be identified in Decembrachiata ( Figure   8), it was considered to be a new-type chitinase.

Conclusion
In this study, the distribution of chitinolytic enzyme activity in S. esculenta was measured and high chitinolytic activity was found in digestion-related organs such as the liver. These chitinases can potentially degrade ingested chitinous substances. In addition, high chitinolytic activity was observed in the posterior salivary gland. Chitinases in the posterior salivary gland may act as a poison, as observed in other cephalopods. Chitinolytic activity in other organs suggests that chitinases are involved in defense against parasites and other activities. Two CBPs (CBP-A and CBP-B) with molecular weights of 52 and 62 kDa, respectively, were separated from the liver of S. esculenta. The molecular weight of CBP-B was consistent with that of SeChi, a chitinase previously purified from the liver of S. esculenta. CBP-A was suggested to be a chitinase isozyme obtained from the liver of S. esculenta. Full-length cDNAs (SeChi-1, SeChi-2) encoding two chitinases (SeChi-1, SeChi-2) were obtained from the liver of S. esculenta. The molecular weights of SeChi-1 and SeChi-2 calculated from their amino acid sequences were 51.2 and 61.0 kDa, respectively, and their isoelectric points were 8.87 and 8.79, respectively, indicating that they are basic proteins. SeChi-1 contained one CBD and SeChi-2 contained two CBDs. Peptide fragments of CBPs isolated from the liver of S. esculenta were analyzed and compared with the amino acid sequences of SeChi-1 and SeChi-2 by proteome analysis. A sequence obtained from peptide fragments of CBP-A was consistent with the amino acid sequence of SeChi-1 (27.23%) and a sequence obtained from peptide fragments of CBP-B was consistent with the amino acid sequence of SeChi-2 (21.01%).
Among the S. esculenta organs studied, SeChi-1 and SeChi-2 were only expressed in the liver. This suggests that the two chitinases are involved in chitin degradation in the liver. The two chitinase genes were not expressed in the posterior salivary gland, where high chitinolytic activity was detected. This suggests that other chitinase isozymes are present in the posterior salivary gland. Phylogenetic analysis revealed that SeChi-2 formed a group with other Decembrachiata chitinases, whereas SeChi-1 did not group with mollusk chitinases. Considering that SeChi-1 represents the first chitinase to possess one CBD in Decembrachiata, SeChi-1 is considered to be a new-type chitinase.