Open Journal of Marine Science, 2018, 8, 136-151 http://www.scirp.org/journal/ojms

ISSN Online: 2161-7392 ISSN Print: 2161-7384

DOI: 10.4236/ojms.2018.81007 Jan. 31, 2018 136 Open Journal of Marine Science

Chondrichthyes Chitinase: Molecular Cloning, Distribution, and Phylogenetic Analysis

Miku Watanabe1, Hiromi Kakizaki1, Momo Kanai1, Satoshi Kawashima1, Kaneyuki Hamaguchi1, Hiroki Mizuno1, Tsubasa Ueno1, Chiaki Yasukawa1, Ryuji Agata1, Mana Ikeda1, Hideto Fukushima1, Mitsuhiro Ueda2, Masahiro Matsumiya1*

1College of Bioresource Sciences, Nihon University, Fujisawa, Japan

2Laboratory of Biocycle Engineering, Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Japan

Abstract 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 identifica-tion of the novel chitinase genes HjChi (ORF: 1380 bp) and DkChi (ORF: 1440 bp) from the stomach of Chondrichthyes, Japanese bullhead shark (He-terodontus japonicas) and Kwangtung skate (Dipturus kwangtungensis), re-spectively. Organ-specific expression analysis identified the stomach-specific expression of HjChi, whereas DkChi was expressed widely in all organs. Chi-tinase activity was measured using pNP-(GlcNAc)n (n = 2, 3) as a substrate and β-N-acetylhexosaminidase (Hex) activity was measured using pNP- GlcNAc. Relatively high values of chitinase activity were observed in the sto-mach, spleen, and gonads of the Japanese bullhead shark, H. japonicas, com-pared with that observed in the stomach of the Kwangtung skate D. kwang-tungensis. 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 (Chondrich-thyes 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.

Keywords Chitinase, Distribution, Chondrichthyes, cDNA Cloning, Phylogenetic Analysis

How to cite this paper: Watanabe, M., Kakizaki, H., Kanai, M., Kawashima, S., Hamaguchi, K., Mizuno, H., Ueno, T., Yasukawa, C., Agata, R., Ikeda, M., Fuku-shima, H., Ueda, M. and Matsumiya, M. (2018) Chondrichthyes Chitinase: Molecu-lar Cloning, Distribution, and Phylogenetic Analysis. Open Journal of Marine Science, 8, 136-151. https://doi.org/10.4236/ojms.2018.81007 Received: December 29, 2017 Accepted: January 28, 2018 Published: January 31, 2018 Copyright © 2018 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

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 137 Open Journal of Marine Science

1. Introduction

Chitinase (EC 3.2.1.14) is an endo-chitinolytic enzyme that randomly hydrolyzes the β-1,4 glycosidic linkages of chitin to produce N-acetyl chitooligosaccharides ((GlcNAc)n) [1]. β-N-acetylhexosaminidase (Hex, EC 3.2.1.52) is an exo-type chitin-decomposing enzyme that sequentially hydrolyzes terminal residues, re-sulting in the formation of N-acetyl-D-glucosamine (GlcNAc) [2]. After cellu-lose, chitin is the second most abundant biopolymer and is found in the exoske-leton of arthropods, the cell walls of fungi, and the epithelium of nematodes [3]. (GlcNAc)n, a degradation product of chitin, displays various physiological prop-erties based on the length of its carbohydrate chain; similarly, GlcNAc also exhi-bits useful physiological activity [4]. The aforementioned points are a clear indi-cation of the potential for the application of chitin in a wide range of fields and the research on chitinase enzymes was invaluable to their efficient use. A wide range of functions is suggested by the distribution of chitinase throughout a wide range of organisms. For example, the enzyme has been found to play a physiological role in digestion in mammals and fish [5] [6], plant and mamma-lian host defense [7] [8], and arthropod morphogenesis [9] [10].

It is reported that chitinase from marine animals displays characteristics that are unlike chitinase from other organisms. In particular, we made very interest-ing findings regarding chitinase in the stomach of fish that feed on organisms containing a chitin exoskeleton. For example, two or more kinds of chitinase isozymes have been discovered in the stomach of Actinopterygii, one of the most successful vertebrates on earth. The activity of these chitinase isozymes is de-pendent on an acidic pH and the crystalline α-chitin decomposition activity of these enzymes has been demonstrated to be superior to that of other organisms [6]. Two varieties of the chitinase genes were obtained from the stomach of the fish and subjected to phylogenetic analysis, based on the deduced amino acid sequence 2 fish-specific groups, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2) were identified [6]. In addition, chitinase activity has been observed in organs, excluding the stomach, in the class of fish classified as Actinopterygii [11] [12], and a new group fish chitinase-3 (FCase-3), has been identified based on an isoform found in the stomach and kidneys [12]. In con-trast, not even one chitinase gene of the AFCase-1, AFCase-2, and FCase-3 groups has been detected in the stomach of either Prionace glauca, classified as a Chondrichthyes, or the stomach of Latimeria chalmnae, classified as Sarcoptery-gii [12]. These findings suggested the possibility that there may be a new chiti-nase group different from groups of AFCase-1, AFCase-2, and FCase-3, as there were large differences in the amino acid sequences of fish chitinases based on systematic positioning. Therefore, in this paper, two species of fish within the same phylogenetic class, but from different subclasses, that share a similar feeding habitat, feeding on molluscs and crustaceans, the Japanese bullhead shark Hete-rodontus japonicas and Kwangtung skate Dipturus kwangtungensis were selected for analysis. Compared with Actinopterygii, information on Chondrichthyes is

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 138 Open Journal of Marine Science

still limited; however, there are some reports on the occurrence of chitinase within the bodies of several varieties of sharks and stingrays [13], as well as re-ports of cDNA cloning of chitinase genes in the stomach of P. glauca [14]. In addition, there is no literature comparing chitinolytic activity and chitinase mRNA in the body of chondrichthyes. Moreover, it is thought that the examina-tion of chitinase from both animals will assist in the clarification of the speciali-zation of fish chitinase, as there are theories that indicate contrasting evolutio-nary paths for Chondrichthyes and Actinopterygii.

This is the first paper that seeks to clarify the characteristics of Chondrich-thyes chitinase compared with the larger group of fish chitinase. This was per-formed through primary structural analysis, the investigation of chitinase activi-ty, the expression status of mRNA to assess the physiological role in the body, and the use of genetic databases, including phylogenetic analysis.

2. Materials and Methods 2.1. Preparation of Samples

The Japanese bullhead shark H. japonicus and Kwangtung skate D. kwangtun-gensis were purchased from the fishing port in Miura City, Kanagawa Prefec-ture. Each organ was collected, washed with chilled distilled water, and stored at −80˚C until use. Each organ was homogenized with five volumes of 20 mM so-dium phosphate buffer (pH 7.3) and centrifuged at 9000 ×g for 20 min at 4˚C. The supernatant was filtered through filter paper to remove floating fat and used as the crude extract solution. The crude extract solution was stored at −80˚C un-til use.

2.2. Cloning of H. japonicas chitinasec DNA (HjChi) and D. Kwangtungensis chitinase cDNA (DkChi)

The sequences of all primers are presented in Table 1. Total RNA was extracted from the stomach of H. japonicus and D. kwangtungensis using RNA extraction reagent (ISOGEN, Nippon Gene, Tokyo, Japan), in accordance with the manu-facturer’s instructions. The total RNA concentration and purity were measured using ultraviolet and visible spectrophotometry. cDNA was synthesized from 1.0 μg total RNA using a primer for reverse transcription (oligo dT) and cDNA synthesis kit (PrimeScript™ II First Strand cDNA Synthesis Kit, Takara Bio, Shi-ga, Japan), in accordance with the manufacturer’s instructions. The internal se-quences of HjChi and DkChi were amplified using the synthesized cDNA, DNA polymerase (Go Taq® Green Master Mix, Promega, Madison, USA), and dege-nerate primers were designed based on the conserved region of amino acid se-quences of GH family 18 chitinases from several vertebrates, in accordance with the manufacturer’s instructions. The forward and reverse primers were designed from the chitinase gene sequences obtained by the internal sequence amplifica-tion, and the upstream (5') and downstream (3') regions were amplified using the rapid amplification of cDNA ends (RACE) method. The 5' and 3' RACE

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 139 Open Journal of Marine Science

Table 1. Primers used in this study.

Primer name Sequence (5'-3') Length Usage

Oligo dT CTGTGAATGCTGCGACTACGATTTTTTTTTTTTTTTTTTT 40 mer cDNA synthesis

HjChi-a TGYTAYTTYACNAAYTGG 18 mer Conserved region PCR

HjChi-b GAYATHGAYTGGGARTAYCC 20 mer

HjChi-c TTCCARTARTTCATNGCRTARTC 23 mer

DkChi-a GYNGGNGGNTGGAAYTTYGGNAC 23 mer

DkChi-b GAYTGGGARTAYCCNGG 17 mer

DkChi-c AYTTCATNGCRAARTCNAC 19 mer

DkChi-d TARTANGCNARRAANCCNGC 20 mer

3'RACE CTGTGAATGCTGCGACTACGAT 22 mer 3' RACE PCR

HjChi-1 CTGCGTCGTTATGGATTTGATGGT 24 mer

DkChi-1 GACAAGCATCTCTACACTG 19 mer

DkChi-2 TTCCTCAGCTCGGCAAGGTAGTT 23 mer

HjChi-2 AGMSSMRASATGGCAAAGCTT 21 mer 5' RACE PCR

HjChi-3 AGTCCAGGATTTGTCCAAGCTG 22 mer

AAP GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG 36 mer

AUAP GGCCACGCGTCGACTAGTACC 21 mer

DkChi-3 TTGCTCATCCCACCAGCAAC 20 mer

DkChi-4 ATCAGCTCCTGAGCCAG 17 mer

DkChi-5 CGAGATCCAGGATATTCCC 19 mer

DkChi-6 AAAGCCGTACCCTCTCAGG 19 mer

DkChi-7 AACTACCTTGCCGAGCTGAGG 21 mer

HjChi-4 AGAGGCGAGATGGCAAAGCTTCT 23 mer Full length amplification

DkChi-8 CTCCTGCCCAGAACAGTAAC 20 mer

DkChi-9 CCCAAGACTGTGGTCAAC 18 mer

β-actin-a GAAAGACAGTTACGTTGGTG 20 mer Organ expression

β-actin-b AGAATCTAGCACAATGCCAG 20 mer

HjChi-5 TCCCAAATTCCCAAGACACT 20 mer

HjChi-6 TCCAACCCATTCATTTCCAC 20 mer

DkChi-10 CTGCCTAATGACAAGGATTAC 21 mer

DkChi-11 GCCGACCCATATTCCATTCT 20 mer

analyses were performed using custom kits (5' and 3' RACE System for Rapid Amplification of cDNA Ends, Invitrogen, Carlsbad, USA) in accordance with the manufacturer’s instructions. Thefull-length chitinase genes were amplified using DNA polymerase (PrimeSTAR® Max DNA Polymerase, Takara Bio), in accordance with the manufacturer’s instructions. The PCR products were electrophoresed

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 140 Open Journal of Marine Science

on a 2% agarose gel and the resolved DNA was extracted using Quantum Prep® Freeze ’N Squeeze spin columns (Bio Rad, Hercules, USA) and ligated into the pGEM-T Easy Vector (Promega). The base sequences were determined using the Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosys-tems, Waltham, USA) and domain prediction was performed using Inter Pro (EMBL-EBI: The European Bioinformatics Institute, European Molecular Biol-ogy Laboratory, Hinxton, England).

2.3. Organ-Specific Gene Expression

Total RNA was extracted from the organs of H. japonicus and D. kwangtungen-sis. cDNA was synthesized from 1.0 μg of total RNA obtained from each organ and an oligo dT primer; 1.0 μg of the synthesized cDNA was amplified using PCR and primers for HjChi, DkChi, and fish β-actin amplification primers. The PCR parameters were 30 cycles of denaturation at 95˚C for 30 s, annealing at 53˚C for 30 s, and extension at 72˚C for 20 s.

2.4. Chitinolytic Enzyme Activity Assay

Chitinolytic Activity was measured using p-Nitrophenyl-N-Acetyl-β-D-Gluco- saminide (pNP-GlcNAc), p-Nitrophenyl Di-N-Acetyl-β-Chitobioside (pNP- (GlcNAc)2), and p-Nitrophenyl Tri-N-Acetyl-β-Chitobioside (pNP-(GlcNAc)3) (Yaizu Suisan Kagaku Industry Co., Ltd., Shizuoka, Japan) as substrates in ac-cordance with the method described by Ohtakara [15], with slight modifications. Briefly, 2.5 μL of enzyme solution and 2.5 μL of substrate (4 mM solution) were added to 10.0 μL of 0.2 M phosphate-0.1 M citrate buffer (pH 6.0), and the solu-tion was incubated at 37˚C for 20 min. After incubation, 65 μl of 0.2 M sodium carbonate solution was added to the solution, and the absorbance of released p- nitrophenol was measured at 420 nm. A unit of chitinolytic enzyme activity (U) was defined as the amount of enzyme that liberated 1 μmol of p-nitrophenol per minute.

2.5. Determination of Optimal pH

When 4 mMpNP-(GlcNAc)n (n = 1 - 3) was used as a substrate, the optimal pH was determined by assaying enzyme activity. Specifically, the solution was incu-bated for 20 min at 37˚C in 0.2 M phosphate-0.1 M citrate buffer (pH 2.5 - 8.0) using the same technique used for the measurement of chitinolytic activity.

2.6. Phylogenetic Analysis of HjChi and DkChi

Phylogenetic analysis based on the deduced amino acid sequences of HjChi and DkChi was performed using the chitinase genes obtained from many organisms. The analysis was performed using ClustalW2 program (EMBL-EBI: The Euro-pean Bioinformatics Institute, European Molecular Biology Laboratory, Hinx-ton, England) and the tree view program. A bacterial chitinase (GenBank: X03657) was used as an outgroup.

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 141 Open Journal of Marine Science

3. Results and Discussion 3.1. Cloning of H. japonicas chitinase cDNA (HjChi) and

D. kwangtungensis chitinase cDNA (DkChi)

Using degenerate primers designed based on the deduced amino acid sequences of several vertebrate chitinases, the internal sequences of the cDNA of the sto-mach of H. japonicas and D. kwangtungensis were amplified by RT-PCR and fragments of 450 bp and 400 bp were obtained, respectively. From the nucleotide sequence of the obtained fragments, upstream and downstream amplification primers were designed. Using the 5'RACE method, the upstream region of the gene fragments, 300 bp and 500 bp, were amplified and the nucleotide sequence of the start codon was included in the gene fragments. In addition, gene frag-ments of 1300 bp and 1000 bp were obtained by the 3'RACE method, and the analysis of the nucleotide sequence identified a stop codon and polyadenylation signals (AATAAA), each found in the 3'UTR. Based on the untranslated regions of these nucleotide sequences, the full-length primer was designed to sandwich an open reading frame (ORF) and using an enzyme with 3'→5' exonuclease ac-tivity, the full-length gene of chitinase was amplified. Based on these methods, we obtained full length chitinase genes from the stomach of both H. japonicas and D. kwangtungensis. The full-length gene of the stomach chitinase of the H. japonicas (HjChi), consisted of 1592 bp and included a 1380 bp ORF encoding 480 amino acids (Figure 1). For D. kwangtungensis, a 1440 bp ORF encoding 478 amino acids was included in the 1601 bp full length stomach chitinase gene (Figure 2). The nucleotide sequences of the full length chitinase genes were reg-istered in DBBJ and the accession numbers were (HjChi: LC215400, DkChi: LC215399) issued. The nucleotide sequences of HjChi and DkChi were analyzed using NCBI BLAST search. The paper claims that HjChi and DkChi demon-strated approximately 70% homology with other Chondrichtyeschitinase (Rhin-codon typus: XP_020390951, P. glauca: AB872008, Scyliorhinus torazame: BAU51439) and approximately 60% with Actinopterygii (Hexagrammos otakii: AB626093, Scophthalmus maximus: KY421058, Paralichthys olivaceus: AB121732). The computed pI/Mw tool was used to estimate the isoelectric point and mole-cular weight of the deduced amino acid sequence (HjChi and DkChi) of HjChi and DkChi, which were 8.52 and 51.6 kDa and 6.59 and 50.6 kDa, respectively. Using the same calculation method, the isoelectric point and molecular weight of chitinase of R. typus were 8.33 and 51.0 kDa (DDBJ: XM_ 020535362) and P. glauca were 8.42 and 52.0 kDa (DDBJ: AB872008) [14], respectively. For P. oli-vaceus, Actinopterygii, the isoelectric points and molecular weights of the chiti-nase varieties were 6.31, 52.6 kDa (fChi1); 5.39, 53.2 kDa (fChi2); and 6.52, 53.1 kDa (fChi3), respectively. These results identified similar molecular weights of the chitinases of Chondrichthyes and Actinopterygii and the differing chitinase of the shark species, which tended to have a higher isoelectric point.

A comparison of the deduced amino acid sequences of several varieties of

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 142 Open Journal of Marine Science

Figure 1. Deduced amino acid sequences and bases for HjChi from the stomach of H. japonicus. DDBJ accession No. LC215400.

previously reported chitinase from P. olivaceus (DDBJ: AB121732, AB121733, AB121734) [16], Homo sapiens (DDBJ: AF290004, U29615) [8] [17], and Mus musculus (DDBJ: EF094027, AY458654) [18] is shown in Figure 3. Based on the domain prediction, beginning at the N-terminal side, all sequences were com-posed of a signal peptide, a catalytic domain, a linker region, and a chitin-bind- ing domain. The domain structure predicted from the deduced amino acid se-quence was a common structure in vertebrate chitinases [19] [20]. The (DXXDXDXE) array, a conserved sequence within the active site of the GH fam-ily18 chitinases [21], was identified in the active site of the catalytic domain and was thought to possess chitin-degrading activity. The linker region of HjChi and DkChi was rich in glycine, and a distance between the catalytic domain and the chitin-binding domain was observed. The flexible, glycine-rich amino acid linker region allows for the flexibility in determining the appropriate distances between domains, which enabled the full demonstration of domain function [22]. Therefore, based on enzymatic degradation, it was inferred that HjChi and DkChi pos-sessed a rational structure. In contrast, a different scenario was predicted for the linker regions of AMCase [8], fChi1, and fChi2 [16], in which serine-containing repetitive sequences are present in addition to glycine.

A G A G G C G A G

A T G G C A A A G C T T C T A C T T G G A A C A G T T G C G G T C T T G T T G G C A T G T C T G G A A C T G G G C T C T 60

M A K L L L G T V A V L L A C L E L G S

G C C T A C A G A C T G G T T T G T T A C T T T A C A A A C T G G G C T C A G T A C A G A C C A G G A G A G G G G A A G 120

A Y R L V C Y F T N W A Q Y R P G E G K

T A C A T G C C C G A G A A T G T G G A C C C T T G C T T G T G C A C G C A T C T G A T C T A T G C T T T T G C T G G G 180

Y M P E N V D P C L C T H L I Y A F A G

A T G A A G G A C A A T C A A A T T A C C A C C T A T G A A T G G A A C G A C G C A A C T C T C T A C C A C T C A T T C 240

M K D N Q I T T Y E W N D A T L Y H S F

A A T G G C C T A A A A A A C A A A A A T G C A A A C C T T A A G A C A C T C C T G G C C A T C G G A G G C T G G A A C 300

N G L K N K N A N L K T L L A I G G W N

T T T G G A A C T C A G A A G T T C A G C G C T A T G G T T T C C A C A G C A G C A A C T C G C C A A A C T T T C A T A 360

F G T Q K F S A M V S T A A T R Q T F I

A A A T C T G T G A T T A G C T T C C T G C G T C G T T A T G G A T T T G A T G G T T T T G A T C T A G A T T G G G A A 420

K S V I S F L R R Y G F D G F D L D W E

T A T C C T G G G T C A A G A G G A A G T C C C C C A C A A G A C A A A C A T C T C T T C A C T G T G T T G G T G C A G 480

Y P G S R G S P P Q D K H L F T V L V Q

G A A C T G A T G G C A G C C T T T G A A G C T G A G G G G A A A A G C T C T G G G A G A C C A A G A C T G C T G C T C 540

E L M A A F E A E G K S S G R P R L L L

T C G G C T G C A G T T G C T G G T G G G A A G A G T A C C A T T G A T T C T G G C T A T G A G A T T C C T C A G C T T 600

S A A V A G G K S T I D S G Y E I P Q L

G G A C A A A T C C T G G A C T T T T T C A A C G T G A T G A C G T A T G A T T T C T T T G G T T C C T G G A G T G C T 660

G Q I L D F F N V M T Y D F F G S W S A

G T C A C T G G A G A A A A T A G C C C T C T T T A T C C T C T C C C A A A T T C C C A A G A C A C T A C C A A C C T A 720

V T G E N S P L Y P L P N S Q D T T N L

T A C T T T A A T G T G G A T T T T G C A A T G A A A T A C T G G A A A G A T C A G G G A G C T C C T G C A G A G A A A 780

Y F N V D F A M K Y W K D Q G A P A E K

T T G A A T G T T G G A T T C C C T A C A T A T G G C C A T A C C T T C A G A C T G G C C T C A T C C A A C A C T G G G 840

L N V G F P T Y G H T F R L A S S N T G

A T T G G A G C C C C A G C A G C A G G T C C T G G A G C A G C T G G G A A G T A C A C C A G G C A G G C T G G A T T C 900

I G A P A A G P G A A G K Y T R Q A G F

C T G G C C T A T T A T G A G A T C T G C A C A T T T T T G A A A C G T G C C A C C G T G A A A T G G A A T G C T C C A 960

L A Y Y E I C T F L K R A T V K W N A P

C A A A T G G T T C C A T A T G C C T A C A G T G G A A A T G A A T G G G T T G G A T A T G A C A A T G T G A A A A G C 1020

Q M V P Y A Y S G N E W V G Y D N V K S

T T T G G C G A C A A G A C T G A G T G G C T G A A A A A G A A C A A C T A T G G A G G A G C T A T G G T G T G G G A T 1080

F G D K T E W L K K N N Y G G A M V W D

C T T G C T C T G G A T G A T T T C T C T G G C A C T T T C T G T G A C C A A G G A C C T T A T C C T C T C A T T A A T 1140

L A L D D F S G T F C D Q G P Y P L I N

A A A C T G C A C A C T G G G C T T G G A A T T T C T G C A G C T T G T G T T C C A T C C A A A A C A A C A C T G C C A 1200

K L H T G L G I S A A C V P S K T T L P

C C A A C T G T T C C T G G T G G T G G C A G C A G T G G C T G T G G G T T C T G C A C T G A C A A A G C T A A T G G G 1260

P T V P G G G S S G C G F C T D K A N G

T A C T A C C C T T A T C C A A A A G A C A A G A G T A A A T T T T A T C A T T G T G T C G A T G G A G T G A C C T A C 1320

Y Y P Y P K D K S K F Y H C V D G V T Y

C A G A T G G A C T G T T C T C C T G G A T T A G T C T T T G A T C C T T C C T G T G A C T G T T G C A G C T G G C C G 1380

Q M D C S P G L V F D P S C D C C S W P

T A A T G A G A A C G C A G A T G T T T G C C A T T A C T C T T C A T T G G T T A T C A T G A A G G T G C A C C T G C T

*

C T T C A G T T T A A C T A C T G A A A T G G C A C G A C T T G G T G G A T A A A G A A C A T T A A T G C A C C A C T G

T A A T T T G T C T C A A C T G A A G A T A T A A A C T G T A G T T T A A A C A G C A A A T A A A A T C A T T G A T T C

A T A C A G C A A A A A A A A A A A A A A A A

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 143 Open Journal of Marine Science

Figure 2. Deduced amino acid sequences and bases for DkChi from the sto-mach of D. kwangtungensis. DDBJ accession No. LC215399.

3.2. Organ-Specific Gene Expression

Organ-specific expression analysis of HjChi and DkChi chitinase genes from the stomach, intestines, liver, kidney, spleen, and gonads of H. japonicas and D. kwangtungensis was performed (Figure 4). HjChi was expressed only in the sto-mach of H. japonicas. This was similar to the observed expression conditions of digestion-related genes, classified as AFCase-1 and AFCase-2 in the stomach of Actinopterygii [16]. However, their expression in D. kwangtungensis displayed a similar profile to that of P. glauca [14] and Latimeria chalumnae [23], with ex-pression in all organs, including metabolic and detoxification organs such as the liver and kidney, hematopoietic organs such as the spleen, and hormone-secreting organs (endocrine organs) such as the gonads. The expression conditions of DkChi were similar to that of FCase-3 obtained from the kidney and expressed in various parts of the body of Sebastiscus marmoratus. These results suggested that, similar to fish chitinase classified as AFCase-1 and AFCase-2, HjChi is mainly responsible for the digestion of food H. japonicas. In D. kwangtungensis, DkChi was responsible not only for digestion, but is also involved in the meta-bolism of chitin oligosaccharides in the liver and kidney, and the possible role

C G A G A T C C A G G A T A T T C C C C T C C T G C C C A G A A C A G T A A C A A G

A T G G C A A A G C T A C T T C T C G G A A C A G C G G T G G T C T T A C T G G C C T G C C T G C A A C T G G G C T C T 60

M A K L L L G T A V V L L A C L Q L G S

G C C T A C A G G C T G G T G T G T T A C T T C A C C A A C T G G G C G C A G T A C A G A C C A G G A G A G G G T A A A 120

A Y R L V C Y F T N W A Q Y R P G E G K

T A C A T G C C C C A G A G C G T G G A C C C T T G C C T T T G C A C A C A C A T C A T C T A C G C C T T C G C T G G A 180

Y M P Q S V D P C L C T H I I Y A F A G

A T G A G G A G C A A T C A G A T C G C C A C C T A T G A A T G G A A C G A C C C A A A A C T C T A C A A G G A G A T C 240

M R S N Q I A T Y E W N D P K L Y K E I

A A C A G C C T G A A G A A C A A G A A T T C A A A C C T C A A G A C T C T G C T G T C C A T T G G A G G C T G G A A C 300

N S L K N K N S N L K T L L S I G G W N

T T T G G C A C C C A G A A A T T C A C C G C G A T G G T T G C C T C A T C A G G A A C T C G G C A A A T C T T C A T C 360

F G T Q K F T A M V A S S G T R Q I F I

A A G T C A G T C A T C G C C T T C C T G A G A G G G T A C G G C T T T G A C G G T C T G G A C A T C G A T T G G G A A 420

K S V I A F L R G Y G F D G L D I D W E

T A T C C T G G A T C T C G A G G A A G T C C T C C C G T T G A C A A G C A T C T C T A C A C T G T C C T G G C T C A G 480

Y P G S R G S P P V D K H L Y T V L A Q

G A G C T G A T G G C T G C G T T C G A G G C C G A G G G G A A A A G C A C A G G A C A G C C A C G A C T G C T G C T C 540

E L M A A F E A E G K S T G Q P R L L L

T C A G C T G C C G T T G C T G G T G G G A T G A G C A A C A T T G A T A C T G G C T A C G A G G T T C C T C A G C T C 600

S A A V A G G M S N I D T G Y E V P Q L

G G C A A G G T A G T T G A C T T C T T C A A C G T G A T G A C C T A C G A T T T C T A T G G T C C C T G G A G C C A C 660

G K V V D F F N V M T Y D F Y G P W S H

G T T A C T G G A G A G A A C A G C C C C C T C T A T G C C C T G C C T A A T G A C A A G G A T T A C A C C A A C A T C 720

V T G E N S P L Y A L P N D K D Y T N I

T A C T T C A A T G T G G A A T A C G C C A T G A A C T A C T G G A A G A G C A A G G G G G C T C C A G T G G A C A A G 780

Y F N V E Y A M N Y W K S K G A P V D K

C T G A A T G T G G G C T T C C C C A C C T A T G G A C A C A C C T T C A G A C T G A C C T C G T C C A G C A C G G C C 840

L N V G F P T Y G H T F R L T S S S T A

G T G G G T G C C C C A G C A G G A G G C G C T G G A C C A G C C G G C A A A T A C A C C C G C C A A G C C G G C T T C 900

V G A P A G G A G P A G K Y T R Q A G F

C T G G C C T A C T A T G A G A T C T G C G G G T T C C T G A A C G G T G C C A C C G C C G A A T G G A A C G C C C C T 960

L A Y Y E I C G F L N G A T A E W N A P

C A G G A C A C C C C G T A C G C C T T C A A G A A T G G A A T A T G G G T C G G C T A T G A C A A C A T C A A G A G C 1020

Q D T P Y A F K N G I W V G Y D N I K S

T T T G G A G A C A A G G T T G A G T G G C T G A A G A A G A A C A A C T T T G G A G G A G C T A T G G T C T G G G A T 1080

F G D K V E W L K K N N F G G A M V W D

C T G G C T C T G G A T G A C T T C T C T G G T G C C T T C T G T C A C C A G G G A C C C T A C C C T C T C A T C A A C 1140

L A L D D F S G A F C H Q G P Y P L I N

A C T C T G C A C T C T G G C C T T G G C A T C T C T G G A G C T T G C G T C C C A T C C A A G A C A A C A T T G A A G 1200

T L H S G L G I S G A C V P S K T T L K

C C A G C G G T C C C A C C T A C T G A T G C T C C T A G T G G T G G T G G A G G T G G T G G G G G T G G T G G A G G C 1260

P A V P P T D A P S G G G G G G G G G G

A G C A G T G G T G G A G G C A G T G G C A G T G G A G G C A G T G G C T T C T G T A A G G G C A A A G C G A A C G G T 1320

S S G G G S G S G G S G F C K G K A N G

A C C T T C C C C G A T C C C A A G G A C A A G A A C A A G T T C T A C C A G T G C G C C A A T G G T C G G A C C T A C 1380

T F P D P K D K N K F Y Q C A N G R T Y

C T G A A G A G C T G T G C C A C T G G A T T A G T C T T C G A T T C A T C C T G T A A A T G T T G C A A C T G G C C T 1440

L K S C A T G L V F D S S C K C C N W P

T A A T G G C C C A A G A C T G T G G T C A A C A A A C A A C T T G G A A C G T C C A T C C C A A A G A T A A T T G T T

*

G C T G T T A A A A C A A T A A A A A A A A G T C A T A A T T T T A A A C A T A A A A A A A A A A A A A A A A A A A A

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 144 Open Journal of Marine Science

Figure 3. Alignment of amino acid sequences of chitinase. D. kwangtungensis (DkChi: stomach LC215399), H. japonicus (HjChi: stomach LC215400), S. torazame (StChi: stomach LC018996), P. olivaceus (fChi-1: stomach AB121732, fChi-2: stomach AB121733, fChi-3: pancreas AB121734), H. sapiens (AMCase: stomach and lung AF290004, Chitotriosidase: U29615), M. muscu-lus (AMCase: stomach EF094027, Chitotriosidase: Tongue AY458654).

Figure 4. Expression of DkChi, HjChi, and β-actin mRNA in organs of H. japonicus and D. kwangtungensis by RT-PCR; β-actin was used as a control. of biological defense is also suggested in the spleen and gonads for one variety of chitinase.

3.3. Distribution of Chitinolytic Enzyme Activity

The distribution of chitinolytic enzyme activity in H. japonicas and D. kwang-tungensis was investigated using pNP-(GlcNAc)n (n = 2, 3) as a substrate for the measurement of chitinase activity (Figure 5(a), Figure 5(c)). The highest activ-ity for pNP-(GlcNAc)3 was observed in the gonads, followed by the stomach and spleen in H. japonicas. However, with the substrate pNP-(GlcNAc)2, the activity

Glycoside family 18 catalytic domainDipturus kwangtungensis _DkChi M A K L L L A T A V V L L A C L E L G S A Y R L V C Y F S N W A Q Y R P G E G K Y F P H N V D P C L C T H L I Y A F A G M - R G N Q I S T Y E W N D P K L Y H A F N G L K N K N G N L K T L L S V G G W 99

Heterodontus japonicus _HjChi M A K L L L G T A V V L L A C L Q L G S A Y R L V C Y F T N W A Q Y R P G E G K Y M P Q S V D P C L C T H I I Y A F A G M - R S N Q I A T Y E W N D P K L Y K E I N S L K N K N S N L K T L L S I G G W 99

Scyliorhinus torazame _StChi M A K L L L G T V A V L L A C L E L G S A Y R L V C Y F T N W A Q Y R P G E G K Y M P E N V D P C L C T H L I Y A F A G M - K D N Q I T T Y E W N D A T L Y H S F N G L K N K N A N L K T L L A I G G W 99

Homo sapiens _AMcase M T K L I L L T G L V L I L N L Q L G S A Y Q L T C Y F T N W A Q Y R P G L G R F M P D N I D P C L C T H L I Y A F A G R - Q N N E I T T I E W N D V T L Y Q A F N G L K N K N S Q L K T L L A I G G W 99

Mus musculus _AMCase M A K L L L V T G L A L L L N A Q L G S A Y N L I C Y F T N W A Q Y R P G L G S F K P D D I N P C L C T H L I Y A F A G M - Q N N E I T T I E W N D V T L Y K A F N D L K N R N S K L K T L L A I G G W 99

Paralichthys olivaceus _fChi1 M G K L L I C V G L A L L L H V Q L G S S Y I L S C Y F T N W G Q Y R P G A G K Y F P T N I D P C L C D H L I Y A F A G M - D S N M I K T Y E W D D E K L Y G Q F Q A L K N Q N S N L K T L L A I G G W 99

Paralichthys olivaceus _fChi2 M G K V L F V T A L A L L L H A Q L G S S Y I L S C Y F T N W A Q Y R P P P T I Y M P N D I D P C L C T H L L Y A F A T I - K N N K L A T Y E W N D V E L Y S Q F N G L K N Q N G N L K T L L S V G G W 99

Paralichthys olivaceus _fChi3 M N K L I L L A G L C L S L G S - L V S S A R L V C Y F T N W S Q Y R P G N G K F M P S D V D P N L C T H L I Y A F S G I N D A N E L V T M E W N D E E L Y K S F N G L K Q R N P N L K T L L A V G G W 99

Homo sapiens _Chitotriosidase M V R S V A W A G F M V L L M I P W G S A A K L V C Y F T N W A Q Y R Q G E A R F L P K D L D P S L C T H L I Y A F A G M - T N H Q L S T T E W N D E T L Y Q E F N G L K K M N P K L K T L L A I G G W 99

Mus musculus _Chitotriosidase M V Q S L A W A G V M T L L M V Q W G S A A K L V C Y L T N W S Q Y R T E A V R F F P R D V D P N L C T H V I F A F A G M - D N H Q L S T V E H N D E L L Y Q E L N S L K T K N P K L K T L L A V G G W 99

Dipturus kwangtungensis _DkChi N F G T Q K F T A M V A S S G N R Q I F I K S V I S F L R G Y G F D G L D V D W E Y P G S R G S P P Q D K H L Y T V L A Q E L M A A F E A E G K S S G Q P R L L L S L A V A G G K N N I D T G Y E V P Q 199

Heterodontus japonicus _HjChi N F G T Q K F T A M V A S S G T R Q I F I K S V I A F L R G Y G F D G L D I D W E Y P G S R G S P P V D K H L Y T V L A Q E L M A A F E A E G K S T G Q P R L L L S A A V A G G M S N I D T G Y E V P Q 199

Scyliorhinus torazame _StChi N F G T Q K F S A M V S T A A T R Q T F I K S V I S F L R R Y G F D G F D L D W E Y P G S R G S P P Q D K H L F T V L V Q E L M A A F E A E G K S S G R P R L L L S A A V A G G K S T I D S G Y E I P Q 199

Homo sapiens _AMcase N F G T A P F T A M V S T P E N R Q T F I T S V I K F L R Q Y E F D G L D F D W E Y P G S R G S P P Q D K H L F T V L V Q E M R E A F E Q E A K Q I N K P R L M V T A A V A A G I S N I Q S G Y E I P Q 199

Mus musculus _AMCase N F G T A P F T T M V S T S Q N R Q T F I T S V I K F L R Q Y G F D G L D L D W E Y P G S R G S P P Q D K H L F T V L V K E M R E A F E Q E A I E S N R P R L M V T A A V A G G I S N I Q A G Y E I P E 199

Paralichthys olivaceus _fChi1 N F G T Q K F T A M V S S P A N R Q T F I T S V I T F L R Q Y E F D G L D I D W E Y P G S R G S P P Q D K E R Y T V L V Q E L M S A F E A E G K K T N R P R L M L T A A V S A G K G T I D S G Y Q I A Q 199

Paralichthys olivaceus _fChi2 N Y G S T G F S Q M V S S P A T R Q T F I S S V I S F L R Q Y E F D G L D I D W E Y P A N R G G S Y D D K E N Y S V F L E E M R S A F E T E A K Q S N R A R L L M S A A V S A G K D T I D S A Y Q I P K 199

Paralichthys olivaceus _fChi3 T F G T R K F T T M V S T E A N R N T F I Q S S I K L L R K H G F D G L D L D W E Y P A A R G S P L E D R Q R F T V L C K E L L E A Y E A E R T A I G K P K L I I S A A V P A G K G T I D A G Y E I A E 199

Homo sapiens _Chitotriosidase N F G T Q K F T D M V A T A N N R Q T F V N S A I R F L R K Y S F D G L D L D W E Y P G S Q G S P A V D K E R F T T L V Q D L A N A F Q Q E A Q T S G K E R L L L S A A V P A G Q T Y V D A G Y E V D K 199

Mus musculus _Chitotriosidase T F G T Q K F T D M V A T A S N R Q T F V K S A L S F L R T Q G F D G L D L D W E F P G G R G S P T V D K E R F T A L I Q D L A K A F Q E E A Q S S G K E R L L L T A A V P S D R G L V D A G Y E V D K 199

Dipturus kwangtungensis _DkChi L G Q I V D F F N V M T Y D F F G P W S H T T G E N S P L Y P L P N N K N L L N L D F N V D Y A M K Y W K T K G A P A D K L N V G F P T Y G H T F R L T S - S S H G V G A P S S G A G P A G Q Y T R Q A 298

Heterodontus japonicus _HjChi L G K V V D F F N V M T Y D F Y G P W S H V T G E N S P L Y A L P N D K D Y T N I Y F N V E Y A M N Y W K S K G A P V D K L N V G F P T Y G H T F R L T S - S S T A V G A P A G G A G P A G K Y T R Q A 298

Scyliorhinus torazame _StChi L G Q I L D F F N V M T Y D F F G S W S A V T G E N S P L Y P L P N S Q D T T N L Y F N V D F A M K Y W K D Q G A P A E K L N V G F P T Y G H T F R L A S - S N T G I G A P A A G P G A A G K Y T R Q A 298

Homo sapiens _AMcase L S Q Y L D Y I H V M T Y D L H G S W E G Y T G E N S P L Y K Y P T D T - G S N A Y L N V D Y V M N Y W K D N G A P A E K L I V G F P T Y G H N F I L S N P S N T G I G A P T S G A G P A G P Y A K E S 298

Mus musculus _AMCase L S K Y L D F I H V M T Y D L H G S W E G Y T G E N S P L Y K Y P T E T - G S N A Y L N V D Y V M N Y W K N N G A P A E K L I V G F P E Y G H T F I L R N P S D N G I G A P T S G D G P A G P Y T R Q A 298

Paralichthys olivaceus _fChi1 I G A V L D Y F H V M T Y D F H G S W E H N V G E N S P L F K G P A D Q - E A M V Y F N V D Y A M N Y W K S N G A P A E K L L V G F P T Y G H T F R L A S - S N T A V G A P A S G P G P A G T F T R Q A 297

Paralichthys olivaceus _fChi2 L G Q A L D M I N V M T Y D F H G S W D P M T G D C S P L F K G P E D Q - G G F I Y F N V D Y A M N Y W K S Q G A P A E K L I V G F P T Y G N T F T L R N P A D H G V G A P I A G A G T P G K Y T Q E A 298

Paralichthys olivaceus _fChi3 L A K Y L D F I N V M T Y D F H G T W E S V T G H H S P L Y K G S H D T - G E H V Y L N T D F A M K Y W R D K G T P V E K L N M G F A T Y G R T F R L S T - Q S S A V G A P I S G P A A A G A F T R E A 297

Homo sapiens _Chitotriosidase I A Q N L D F V N L M A Y D F H G S W E K V T G H N S P L Y K R Q E E S - G A A A S L N V D A A V Q Q W L Q K G T P A S K L I L G M P T Y G R S F T L A S S S D T R V G A P A T G S G T P G P F T K E G 298

Mus musculus _Chitotriosidase I A Q S L D F I N L M A Y D F H S S L E K T T G H N S P L Y K R Q G E S - G A A A E Q N V D A A V T L W L Q K G T P A S K L I L G M P T Y G R S F T L A S S S D N G V G A P A T G P G A P G P Y T K D K 298

Dipturus kwangtungensis _DkChi G F L A Y Y E I C T L L K S - A T M E W N A A Q V V P Y A Y T A S E W I G Y D N V K S F K A K I E W L K K N N F G G V M V W D L A L D D F S G A F C G E G P Y P L I N T L H S G L G I S T S C A P S K T 397

Heterodontus japonicus _HjChi G F L A Y Y E I C G F L N G - A T A E W N A P Q D T P Y A F K N G I W V G Y D N I K S F G D K V E W L K K N N F G G A M V W D L A L D D F S G A F C H Q G P Y P L I N T L H S G L G I S G A C V P S K T 397

Scyliorhinus torazame _StChi G F L A Y Y E I C T F L K R - A T V K W N A P Q M V P Y A Y S G N E W V G Y D N V K S F G D K T E W L K K N N Y G G A M V W D L A L D D F S G T F C D Q G P Y P L I N K L H T G L G I S A A C V P S K T 397

Homo sapiens _AMcase G I W A Y Y E I C T F L K N G A T Q G W D A P Q E V P Y A Y Q G N V W V G Y D N I K S F D I K A Q W L K H N K F G G A M V W A I D L D D F T G T F C N Q G K F P L I S T L K K A L G L Q S A S C T A P A 398

Mus musculus _AMCase G F W A Y Y E I C T F L R S G A T E V W D A S Q E V P Y A Y K A N E W L G Y D N I K S F S V K A Q W L K Q N N F G G A M I W A I D L D D F T G S F C D Q G K F P L T S T L N K A L G I S T E G C T A P D 398

Paralichthys olivaceus _fChi1 G F W A Y Y E I C S F L K Q G A T Q A W D S T Q D V P Y A Y N Q G T W V G Y D N V K S F Q I K I Q W L K Q S G F G G A M V W S L D L D D F S G T F C G Q G R Y P L I N T I K S G L G T G - A S C A S R S 396

Paralichthys olivaceus _fChi2 G E L A Y F E I C G F L K G G A T E V W N Q A Q D V P Y A Y K G N Q W V G Y D N M K S F Q I K V D W L K K S N F G G A M V W T L D M D D Y M G T F C N Q G K Y P L I N V L K K G L N L E Q A S C A P P A 398

Paralichthys olivaceus _fChi3 G F W S Y Y E I C T F L Q G - A S V Q L I E D Q K V P Y A T K N N E W V G Y D N K E S F E T K V R Y L K D N R F G G A F V W A L D L D D F Q G Q Y C Q Q G N Y A L I N Y L R S L L A L D L P P L P T T E 396

Homo sapiens _Chitotriosidase G M L A Y Y E V C S W K G A - - T K Q R I Q D Q K V P Y I F R D N Q W V G F D D V E S F K T K V S Y L K Q K G L G G A M V W A L D L D D F A G F S C N Q G R Y P L I Q T L R Q E L S L P Y L P S G T P E 396

Mus musculus _Chitotriosidase G V L A Y Y E A C S W K - - - - E R H R I E D Q K V P Y A F Q D N Q W V S F D D V E S F K A K A A Y L K Q K G L G G A M V W V L D L D D F K G S F C N Q G P Y P L I R T L R Q E L N L P S E T P R S P E 394

Linker regionDipturus kwangtungensis _DkChi T L K P A V P P T H A P S G G G G G G G G G G - - G G G G G G G G S - - - - - - G F C A G K A N G T Y P D P K D K S K F Y Q C A N G R T Y L E S C A S G L V F D S S C E C C N W A

Heterodontus japonicus _HjChi T L K P A V P P T D A P S G G G G G G G G G G S S G G G S G S G G S - - - - - - G F C K G K A N G T F P D P K D K N K F Y Q C A N G R T Y L K S C A T G L V F D S S C K C C N W P

Scyliorhinus torazame _StChi T L P P T V P - - - - - - - - - - - - - - - - - - - - G G G S S G C - - - - - - G F C T D K A N G Y Y P Y P K D K S K F Y H C V D G V T Y Q M D C S P G L V F D P S C D C C S W P

Homo sapiens _AMcase Q P I E P I T A A P S G S G N G S G S S S S G G S S G G S - - - - - - - - - - - G F C A V R A N G L Y P V A N N R N A F W H C V N G V T Y Q Q N C Q A G L V F D T S C D C C N W A

Mus musculus _AMCase V P S E P V S T P P - - - G S G S G G G S S G G S S G G S - - - - - - - - - - - G F C A D K A D G L Y P V A D D R N A F W Q C I N G I T Y Q Q H C Q A G L V F D T S C N C C N W P

Paralichthys olivaceus _fChi1 N P L P P V T P T Q H A Q P Q P G G G G S S G G S S G G S S S G G S S S G G S - G F C A G K S N G L Y P D A T N K N H F Y E C S Q G K T Y D Q H C A V G L V F D N S C K C C N W A

Paralichthys olivaceus _fChi2 T R L P P I A G V S T T S G E G S S G G S S G G S S G S S G R D S G T S G M N N G F C A G K A N G M Y P N P T N K N Q F Y N C S G G K T Y F Q N C A P G L V F N S A C S C C N W S

Paralichthys olivaceus _fChi3 T P P T Q V I P T K N P P Y V T S H P T P V I P T S P K T P D S - - - - - - - - N F C A A K A G G I Y A K H G S P S S F Y S C A N G I T W I Q N C P A N L V F R Q S C N C C D W P

Homo sapiens _Chitotriosidase L E V P K P G Q P S E P E H G P S P G Q D - - - - - - - - - - - - - - - - - - - T F C Q G K A D G L Y P N P R E R S S F Y S C A A G R L F Q Q S C P T G L V F S N S C K C C T W N

Mus musculus _Chitotriosidase Q I I P E P R P S S M P E Q G P S P G L D - - - - - - - - - - - - - - - - - - - N F C Q G K A D G V Y P N P G D E S T Y Y N C G G G R L F Q Q S C P P G L V F R A S C K C C T W S

477

466

464

480

460

476

473

484

487

←

→ ← Chitin binding domain type 2 →

478

← Signal peptide → ←

Active site

→

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 145 Open Journal of Marine Science

(a)

(b)

(c)

(d)

Figure 5. The distribution of the chitinolytic enzyme activities in the organs of H. japo-nicus and D. kwangtungensis. (a) Chitinase activity of H. japonicus; (b) β-N-Acetylhex- osaminidase activity of H. japonicus; (c) Chitinase activity of D. kwangtungensis; (d) β-N-Acetylhexosaminidase activity of D. kwangtungensis, ■: pNP-(GlcNAc), ■: pNP- (GlcNAc)2, □: pNP-(GlcNAc)3. Bars represent the standard deviation (n = 3).

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 146 Open Journal of Marine Science

was remarkably low in all organs. Similarly, in D. kwangtungensis, the highest activity for the substrate pNP-(GlcNAc)n (n = 2, 3) was observed in the stomach and minimal activity against pNP-(GlcNAc)2 was also detected in the liver. In H. japonicas, the activity of pNP-(GlcNAc)3 was higher than pNP-(GlcNAc)2 in almost all organs, and the chitinase displayed a similar degradation pattern to the chitinase encoded by the gene AFCase-2 [6]. In addition, chitinase activity observed in the stomach of the D. kwangtungensis was similar to that at the de-gradation site of the chitinase coded by the gene classified as an AFCase-1 [6]. Chitinase activity is present in the lymphomyeloid tissue of the Chimaera mon-strosa, in the esophagus and lymphomyeloidepigonal organs of Etmopterus spi-nax and Raja pulchra [13], and in the stomach, liver, and spleen of Mustelus manazo [24]. These findings indicated that chitinase was found in most rays and sharks, where it participates in physiological roles. Organ expression analysis identified HjChi only in the stomach and chitinase activity in H. japonicas was thought to be a result of the chitinase encoded by HjChi. Further, chitinase ac-tivity in the spleen and gonads is thought to be a result of chitinase encoded by a gene other than HjChi. In contrast, DkChi was expressed in all organs of D. kwangtungensis, but chitinase activity was observed only in the stomach, and based on the feeding status of the three samples used in the current study, the possibility of adjustment for expression confined to the stomach exists. Con-versely, Hex activity, which was measured using pNP-GlcNAc as the substrate, was detected in all organs, similar to previous reports [11] [12], with high values in the intestine, spleen, and gonads, and low values in the stomach, liver, kidney of H. japonicas, whereas in D. kwangtungensis, higher activity was found in the liver, kidney, spleen, and gonads and lower activity in the stomach and intestine (Figure 5(b), Figure 5(d)).

3.4. Determination of Optimal pH

The effect of pH on chitinolytic activity was determind in the stomach of the H. japonicus and D. kwangtungensis using pNP-(GlcNAc)n (n = 2, 3) as substrate (Figure 6(a), Figure 6(c)). Optimal values of pH 5.5 and 5.0 for pNP-(GlcNAc)2 and pNP-(GlcNAc)3, respectively, were observed in H. japonicas; for the same substrates in D. kwangtungensis, pH 4.0 and 3.5 were the optimal values, respec-tively. For both substrates, the relative activity in the stomach of H. japonicas and the D. kwangtungensis decreased to 50% or less of the maximal activity from pH 6.0 - 6.5 to basic pH values. Further, in the investigation of the effect of pH on Hex activity, pNP-GlcNAc was used as the substrate, and the optimal pH for Hex activity was pH 4.0 in H. japonicas and D. kwangtungensis (Figure 6(b), Figure 6(d)). The aforementioned results suggested that the optimal pH of sto-mach chitinase of H. japonicas and D. kwangtungensis was similar to that re-ported for the stomach of Actinopterygii [6], and that it may be involved in feeding and digestion, based on the acidic environment because of the stomach acid.

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 147 Open Journal of Marine Science

(a) (b)

(c) (d)

Figure 6. Effect of pH on the chitinolytic enzyme activities in the stomach of H. japoni-cus and D. kwangtungensis. (a) and (b) H. japonicus; (c) and (d) D. kwangtungensis, ▲: pNP-(GlcNAc), ■: pNP-(GlcNAc)2, ●: pNP-(GlcNAc)3.

3.5. Phylogenetic Analysis of HjChi and DkChi

In order to determine the systematic position of the chitinases of Chondrich-thyes, HjChi and DkChi, discovered in this study, and that of other fish chiti-nases, we performed phylogenetic analysis based on the homology of the de-duced amino acid sequence of family 18 chitinases of other organisms (Figure 7). The accession number and the names of all organisms included in the phylo-genetic tree are shown in Figure 7. The results indicated that, similar to previous reports [6] [12], the chitinases expressed in mammalian stomach were classified as members of the AMCase [8] [25], chitinases produced by mammalian ma-crophages were classified as members of the chitotriosidases [17] [18], stomach chitinases of Actinopterygii were classified as members of the AFCase-1 and AFCase-2 [6] [16], chitinases from the kidney of Actinopterygii were classified as members of the FCase-3 [12]; however, HjChi and DkChi were not classified as members of any of the aforementioned groups. In addition, L. chalumnae (Sarcopterygii) [23], Protopterus aethiopicus (LC350289), and Lethenteron ja-ponicum (Cyclostomata) (EU741679) did not belong to these groups. A new group was created to classify Chondrichthyes in which HjChi and DkChi were detected, including R. typus (XM_020535362), P. glauca (AB872008) [14],

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 148 Open Journal of Marine Science

Figure 7. Phylogenetic tree for chitinase amino acid sequences developed using the neighbor joining me-thod in the ClustalW program. The right table lists the scientific name, information, and accession No. of chitinase used in the phylogenetic tree analysis. The numbers in the phylogenetic tree and table correspond to each other. A chitinase from bacteria was used as an outgroup. The scale bar indicates the substitution rate per residue.

Dasyatis akajei (LC350288), Triakis scyllium (LC350287), M. manazo (LC085613), and S. torazame. The chitinases of Chondrichthyes group were identified to be similar to stomach chitinases and were more similar to the chi-tinases produced mainly within the stomach, such as AMCase, AFCase-1, and AFCase-2, more than the mammalian macrophage-produced chitotriosidase and the Actinopterygii produced FCase-3, and was particularly similar to the AF-Case-2. Further, as in this study, the chitinases of Chondrichthyes were placed in the same group, whether of shark or ray origin; thus, we named the four chiti-nases obtained from sharks and rays as Chondrichthyes chitinase. These results indicated that fish possess chitinases with different functions and structures be-cause of adaptation and that this was dependent on the feeding habitat and en-vironment. Consequently, each chitinase group was formed based on a network classification by phylogenetic analysis.

4. Conclusion

Novel chitinase genes were obtained from the stomach of H. japonicas, HjChi (ORF: 1380 bp) and the stomach of D. kwangtungensis, DkChi (ORF: 1440 bp). Although the amino acid sequence of the linker region was unique, the domain structure predicted from the deduced amino acid sequence was a common structure in vertebrate chitinases. Organ-specific analysis identified the expres-sion of HjChi occurred mainly in the stomach of H. japonicas, which indicated a primary role in the digestion of food, whereas DkChi was expressed in all organs

No. Scientific name Information Accession No.1 Serratia marcescens Bacteria chitinase X036572 Gallus gallus Grandular stomach AB0710383 Latimeria chalumnae Stomach AB7048694 Lethenteron japonicum Liver EU7416795 Equus caballus Chitotriosidase EF6947596 Homo sapiens Stomach, Lung, AMCase AF2900047 Homo sapiens Macrophage, Chitotriosidase U296158 Mus musculus Stomach , AMCase EF0940279 Mus musculus Tongue , Chitotriosidase AY458654

10 Rattus norvegicus Stomach , AMCase AY48607411 Rattus norvegicus Spleen , Chitotriosidase DQ28623212 Sarcophilus harrisii AMCase XM00376976613 Epinephelus coioides Chi1 FJ16989514 Epinephelus coioides Chi2 FJ16989415 Hexagrammos otakii Stomach, HoChi-1 AB62609316 Hexagrammos otakii Stomach, HoChi-2 AB62609417 Hexagrammos otakii Kidney, HoChi-3 LC21872618 Paralichthys olivaceus Stomach , fChi1 AB12173219 Paralichthys olivaseus Stomach , fChi2 AB12173320 Paralichthys olivaceus Pancreas , fChi3 AB12173421 Sardinops melanostictus Stomach, SmeChi-1 AB98561022 Sardinops melanostictus Stomach, SmeChi-2 AB985611 23 Sardinops melanostictus Kidney, SmeChi-3 LC11908724 Sebastiscus marmoratus Stomach, SmChi-1 AB686658 25 Sebastiscus marmoratus Stomach, SmChi-2 AB68665926 Sebastiscus marmoratus Kidney, SmChi-3 LC07773327 Thunnus orientalis Whole body, Chi3 AB67842628 Dasyatis akajei stomach, DaChi LC35028829 Dipturus kwangtungens stomach, DkChi LC21539930 Heterodontus japonicus stomach, HjChi LC21540031 Mustelus manazo stomach, MmChi LC08561332 Prionace glauca stomach, PgChi AB87200833 Rhincodon typus spleen XM_02053536434 Scyliorhinus torazame stomach, StChi LC01899635 Triakis scyllium stomach, TsChi LC35028736 Protopterus aethiopicus esophagus, PaChi LC350289

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 149 Open Journal of Marine Science

of D. kwangtungensis, which was suggestive of a variety of physiological roles. Moreover, chitinase activity against pNP-(GlcNAc)3 was comparatively high in the stomach, spleen, and gonads, whereas chitinase activity against pNP- (GlcNAc)2 was substantially lower in all organs. In contrast, a very high value was obtained for chitinase activity against pNP-(GlcNAc)n (n = 2, 3) in the sto-mach of D. kwangtungensis. Hex activity was widely observed throughout the body of both species of fish. In addition, the optimal pH of the stomach chitinase of both H. japonicas and D. kwangtungensis was similar to that reported for the stomach chitinase of Actinopterygii. Based on our phylogenetic analysis, HjChi and DkChi do not belong to previously reported groups; thus, a new group, Chondrichthyes chitinase, was established owing to the similarities with AM-Case, AFCase-1, and AFCase-2, which are mainly expressed in the stomach, un-like chitotriosidase and FCase-3. These results suggested the existence of class- specific chitinase in the fish kingdom. Hence, it was possible to classify chitinase groups, along with the application of phylogenetic analysis. Our studies are ex-pected to lead to efficient chitin degradation and production of chitin oligosac-charides of specific chain length by obtaining knowledge of chitinase of various properties of fish kingdom. In addition, we are going to try expression of protein in future.

Acknowledgements

This work was supported in part by College of Bioresource Science, Nihon Uni-versity Grant (2017).

References [1] Rovertus, J.D. and Monzingo, F.A. (1999) The Structure and Action of Chitinases.

EXS, 87, 125-135.

[2] Slámová, K., Bojarová, P., Petrásková, L. and Křen, V. (2010) β-N-Acetylhexosami- nidase: What’s in a Name…? Biotechnology Advances, 28, 682-693. https://doi.org/10.1016/j.biotechadv.2010.04.004

[3] Muzzarelli, R.A.A. (1977) Chitin. Pergamon Press, Oxford.

[4] Mei, Y.X., Chen, H.X., Zhang, J., Zhang, X.D. and Liang, Y.X. (2013) Protective Ef-fect of Chitooligosaccharides against Cyclophosphamide-Induced Immunosuppres-sion in Mice. International Journal of Biological Macromolecules, 62, 330-335. https://doi.org/10.1016/j.ijbiomac.2013.09.038

[5] Muzzarelli, R.A.A., Boudrant, J., Meyer, D., Manno, N., Demarchis, M. and Paoletti, M.G. (2012) Current Views on Fungal Chitin/Chitosan, Human Chitinases, Food Preservation, Glucans, Pectins and Inulin: A Tribute to Henribraconnot, Precursor of the Carbohydrate Polymers Science, on the Chitin Bicentennial. Carbohydrate Polymers, 87, 995-1012. https://doi.org/10.1016/j.carbpol.2011.09.063

[6] Ikeda, M., Kakizaki, H. and Matsumiya, M. (2017) Biochemistry of Fish Stomach Chitinase. International Journal of Biological Macromolecules, 104, 1672-1681. https://doi.org/10.1016/j.ijbiomac.2017.03.118

[7] Ahmed, N.U., Park, J.I., Jung, H.J., Kang, K.K., Hur, Y., Lim, Y.P. and Nou, I.S. (2012) Molecular Characterization of Stress Resistance-Related Chitinase Genes of Brassica rapa. Plant Physiology and Biochemistry, 58, 106-115.

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 150 Open Journal of Marine Science

https://doi.org/10.1016/j.plaphy.2012.06.015

[8] Boot, R.G., Blommaart, E.F.C., Swart, E., Vlugt, K.G.V.D., Bijl, N., Moe, C., Place, A. and Aerts, J.M.F.G. (2001) Identification of a Novel Acidic Mammalian Chiti-nase Distinct from Chitotriosidase. The Journal of Biological Chemistry, 276, 6770-6778. https://doi.org/10.1074/jbc.M009886200

[9] Arakane, Y. and Muthukrishnan, S. (2010) Insect Chitinase and Chitinase-Like Proteins. Cellular and Molecular Life Sciences, 67, 201-216. https://doi.org/10.1007/s00018-009-0161-9

[10] Gooday, G.W. (1999) Aggressive and Defensive Roles for Chitinases. EXS, 87, 157-169. https://doi.org/10.1007/978-3-0348-8757-1_11

[11] Kakizaki, H., Ikeda, M., Fukushima, H. and Matsumiya, M. (2015) Distribution of Chitinolytic Enzymes in the Organs and cDNA Cloning of Chitinase Isozymes from the Stomach of Two Species of Fish, Chub Mackerel (Scomber japonicus) and Silver Croaker (Pennahiaargentata). Open Journal of Marine Science, 5, 398-411. https://doi.org/10.4236/ojms.2015.54032

[12] SmChi-3 (Submitted to Advances in Bioscience and Biotechnology).

[13] Fange, R., Lundblad, G. and Lind, J. (1976) Lysozyme and Chitinase in Blood and Lymphomyeloid Tissues of Marine Fish. Marine Biology, 36, 277-282. https://doi.org/10.1007/BF00389289

[14] Suzuki, T., Kakizaki, H., Ikeda, M. and Matsumiya, M. (2014) Molecular Cloning of a Novel Chitinase Gene from Blue Shark (Prionace glauca; Chondrichthyes) Sto-mach. Journal of Chitin and Chitosan Science, 2, 143-148. https://doi.org/10.1166/jcc.2014.1050

[15] Ohtakara, A. (1988) Chitinase and β-N-acetylhexosaminidase from Pycnoporus cinnabarinus. Biomass, Part B: Lignin, Pectin, and Chitin, 161, 462-470. https://doi.org/10.1016/0076-6879(88)61059-7

[16] Kurokawa, T., Tuji, S. and Suzuki, T. (2004) Molecular Cloning of Multiple Chiti-nase Genes in Japanese Flounder, Paralichthys olivaceus. Comparative Biochemistry and Physiology—Part B: Biochemistry & Molecular Biology, 138, 255-268. https://doi.org/10.1016/j.cbpc.2004.03.015

[17] Boot, R.G., Renkema, H., Strijland, A., Zonneveld, A.J.V. and Aerts, J.M.F.G. (1995) Cloning of a cDNA Encoding Chitotriosidase, a Human Chitinase Produced by Macrophages. The Journal of Biological Chemistry, 270, 26252-26256. https://doi.org/10.1074/jbc.270.44.26252

[18] Zheng, T., Rabach, M., Chen, N.Y., Rabach, L., Hu, X., Elias, J.A. and Zhu, Z. (2005) Molecular Cloning and Functional Characterization of Mouse Chitotriosidase. Gene, 29, 37-46. https://doi.org/10.1016/j.gene.2005.05.006

[19] Adrangi, S. and Faramarzi, M.A. (2013) From Bacteria to Human: A Journey into the World of Chitinases. Biotechnology Advances, 31, 1786-1795. https://doi.org/10.1016/j.biotechadv.2013.09.012

[20] Reguera, G. and Leschine, S.B. (2003) Biochemical and Genetic Characterization of ChiA, the Major Enzyme Component for the Solubilization of Chitin by Cellulo-monas uda. Archives of Microbiology, 180, 434-443. https://doi.org/10.1007/s00203-003-0611-y

[21] van Aalten, D.M., Synstad, B., Brurberg, M.B., Hough, E., Riise, B.W., Eijsink, V.G. and Wierenga, R.K. (2000) Structure of a Two-Domain Chitotriosidase from Serra-tia marcescens at 1.9-A Resolution. Proceedings of the National Academy of Sciences, 97, 5842-5847. https://doi.org/10.1073/pnas.97.11.5842

M. Watanabe et al.

DOI: 10.4236/ojms.2018.81007 151 Open Journal of Marine Science

[22] Gilkes, N.R., Henrissat, B., Kilburn, D.G., Miller, R.C. and Warren, R.A. (1991) Domains in Microbial Beta-1,4-glycanases: Sequence Conservation, Function, and Enzyme Families. Archive of Microbiological Reviews, 55, 303-315.

[23] Kakizaki, H., Hamaguchi, K., Ikeda, M. and Matsumiya, M. (2014) Cloning of a Novel Chitinase cDNA from the Stomach of the Coelacanth Latimeria chalumnae (sarcopterygii). Journal of Chitin and Chitosan Science, 2, 123-129. https://doi.org/10.1166/jcc.2014.1051

[24] Okutani, K. and Kiniata, M. (1964) Studies on Chitinolytic Enzyme Present in Aquaic Animals-III. Distribution of Chitinase in Digestive Organs of a Few Kinds of Aquatic Animals. The Japanese Society of Fisheries Science, 30, 574-576.

[25] Chen, X.H., Xie, Z.H., Sun, S.J. and Cai, G. (2006) Cloning of a Rat Lung Fibrogenic Factor. Gene, 384, 9-17. https://doi.org/10.1016/j.gene.2006.06.026