On the basis of our preliminary observation that a crude extract of the stomach of the toad Bufo japonicus exhibited a chitinase activity with its optimum pH around 3.0, we undertook molecular cloning of a cDNA encoding this putative gastric chitinase. By use of 2 degenerate oligonucleotide primers derived from the 2 conserved regions of the vertebrate chitinases, a reverse transcription-PCR product was obtained. This product was used as a probe to screen a cDNA library constructed from the toad stomach. The longest positive clone was revealed to contain an open reading frame for a putative chitinase protein of 484 amino acids, which protein exhibited sequence similarity to the known vertebrate chitinases. Our data also revealed this putative gastric chitinase to be distinct from the chitinase that we had previously isolated from the pancreas of the same species. In this putative gastric chitinase, both the N-terminal catalytic domain and the C-terminal chitin-binding domain were perfectly conserved, suggesting this protein to function as chitinase in the toad stomach.
INTRODUCTION
Chitin is a β-1,4-linked homo polymer consisting of N-acetylglucosamine residues that constitutes the cell walls of fungi, the exoskeletons of arthropods including crustaceans and insects and the cuticles of annelids and mollusks (Gooday, 1996; Robyt, 1998). In vivo hydrolysis of chitin to acetylglucosamine is performed by the sequential action of a polysaccharidase (chitinase) and an oligosaccharidase (chitobiase; Jeuniaux, 1993). The presence of chitinolytic enzymes has been reported in various invertebrate species. Information about such enzymes in vertebrates, however, is limited. Recently, mammalian chitinases such as chitotriosidase from human macrophages (Renkema et al., 1995), acidic mammalian chitinase (AMCase) from the mouse stomach (Boot et al., 2001) and chitin-binding protein b04 (CBPb04) from bovine serum (Suzuki et al., 2001) have been identified. In lower vertebrates, there are several reports on the presence of chitinase activity mostly in digestive organs (Micha et al., 1973; Jeuniaux, 1993; Marsh et al., 2001). However, isolation and molecular characterization of chitinases in lower vertebrates have scarcely been attempted. Very recently, we purified a protein possessing a chitinolytic activity from the toad pancreas (Oshima et al., 2001). In fact, this chitinolytic protein was the first amphibian chitinase to be identified, as well as the first pancreatic chitinase to be found in vertebrates. During the course of our study on this toad pancreatic chitinase, we noticed the existence of chitinase activity in the stomach of the toad as well. This finding prompted us to perform molecular cloning of this putative gastric chitinase of the toad. Our results are herein reported.
MATERIALS AND METHODS
Isolation of total RNA and RT-PCR
Total RNA was isolated from the toad stomach by use of Isogen (Nippon Gene, Toyama, Japan). First-strand cDNA synthesis was performed on 3 μg of total RNA by using SuperScript II reverse transcriptase (GIBCO BRL, Rockville, MD) with oligo(dT)12–18. After thermal denaturation of the enzyme, the cDNA was precipitated with ethanol and used as template for PCR amplification with degenerate primers. The primers were constructed on the basis of highly conserved regions of known vertebrate chitinases: 5′-CA (AG) TA (CT) (AC) G (ACGT) CC (ACGT) GA (CT) CA (AG) GG-3′ as a sense primer and 5′-C (ACGT) A (AG) (AG) TC (AGT) AT (ACGT) GCCCA (ACGT) ACCAT-3′ as an antisense primer. The conditions of the PCR reaction were denaturation at 94°C for 5 min followed by denaturation (94°C, 30 sec), annealing (55°C, 1 min) and extension (72°C, 1 min) reactions for 30 cycles. The amplified cDNA fragment was subcloned into plasmid pT7-blue (Novagen, Darmstadt, Germany). The plasmid containing the cDNA encoding toad gastric chitinase was used to transform JM109 competent cells (TaKaRa, Shiga, Japan) and was subjected to sequence analysis.
Construction and screening of a cDNA library from the toad stomach
A cDNA library of the toad stomach was constructed by using EcoRI-digested Lambda ZAP II (Stratagene, La Jolla, CA). The PCR-amplified chitinase cDNA fragment was labeled with [α-32P]dCTP by the random-priming method using a BcaBEST Labeling Kit (TaKaRa) and was used to screen the cDNA. Hybridizations were performed at 60°C for 16 hr with the labeled cDNA encoding a partial sequence of toad chitinase. Clones giving positive signals were obtained by In vivo excision. The cDNA sequences were analyzed by a cycle sequencing method on a DNA sequencer Model 4000L (LI-COR, Lincoln, NE). By use of a computer program (GENETYX-MAC), the molecular weight and isoelectric point of the putative chitinase predicted from a cDNA encoding the mature protein were calculated.
Northern blot analysis
Total RNAs extracted from various organs were electrophoresed in 1% formaldehyde-agarose gel and transferred to a nylon membrane. The RNAs were fixed on the membrane by UV-cross-linking. Hybridizations with the radiolabeled cDNAs were performed for 16 hr at 60°C following addition of the probe to the pre-hybridization solution (Sambrook et al., 1989). The filters were washed for 30 min at 60°C with 0.1-fold standard saline citrate containing 0.1% SDS and placed in contact with X-ray film (Eastman Kodak, Rochester, NY) for 16 h at −80°C.
RESULTS
One PCR product (997 bp) was obtained by using as primers synthetic nucleotides coding for a conserved region of vertebrate chitinase sequences. By employing this product as a probe, we isolated chitinase cDNA from a toad stomach cDNA library. The nucleotide sequence of the longest clone obtained was analyzed. This clone was estimated to be 1541 bp long, and it had an open reading frame of 1452 bp encoding the toad putative gastric chitinase protein consisting of 463 amino acid residues and a putative signal peptide of 21 amino acid residues (Fig. 1). The molecular mass and isoelectric point of the predicted mature protein were calculated to be 50 kDa and 4.89, respectively.
Fig. 1
Nucleotide and deduced amino acid sequences of putative tGCase cDNA. Annealing positions of primers for RT-PCR are boxed. The putative signal peptide region is underlined. An asterisk indicates the termination codon. A polyadenylation signal (AATAAA) is indicated by boldface type.
Comparison of the amino acid sequence between the putative toad gastric chitinase (tGCase) and known vertebrate chitinase family proteins revealed homologies of 75.9, 70.3, 52.1 and 50.2% with mouse AMCase, bovine CBPb04, toad pancreatic chitinase and human chitotriosidase, respectively. Like these vertebrate chitinases, this putative tGCase was predicted to contain an N-terminal catalytic domain and a C-terminal chitin-binding domain (Fig. 2).
Fig. 2
Amino acid sequence comparison of putative tGCase, mouse (m) AMCase (Boot et al., 2001; accession number AF290003), bovine (b) CBPb04 (Suzuki et al., 2001; accession number AB051629), tPCase (Oshima et al., 2001; accession number AJ345054) and human (h) chitotriosidase (Boot et al., 1995; accession number U29615). Identical amino acid residues among the 5 chitinases are shown in white letters. Identical residues among any 3–4 chitinases are shadowed. The catalytic center for chitinase activity is underlined and the chitin-binding site, doubly underlined.
Northern blot analysis revealed the toad putative gastric chitinase mRNA to be 1.5 kb long and to be expressed in the stomach but not in other organs so far tested (Fig. 3).
Fig. 3
Northern blot analysis of RNA from various organs of Bufo japonicus for detection of mRNA for tGCase. Total RNAs of the stomach (lane 1), brain (lane 2), kidney (lane 3), large intestine (lane 4), liver (lane 5), lung (lane 6), olfactory epithelium (lane 7), pancreas (lane 8) and small intestine (lane 9) were hybridized with the radiolabeled tGCase cDNA. The amount of the applied RNA was 15 μg in each case except for the stomach, where 5 μg RNA was applied.
DISCUSSION
Considering that amphibians eat chitin-covered preys, it is highly probable that chitinolytic enzymes would be required for the digestion of the ingested animals. In fact, Micha et al. (1973) demonstrated chitinase activity in the gastric mucosa and pancreas of 4 species of amphibians, i. e., Rana temporaria, Bufo marinus, Salamandra salamandra taeniata and Triturus alpestris alpestinus. However, it was only very recently that the isolation and molecular characterization of an amphibian chitinase was done. We isolated from the pancreas of the toad Bufo japonicus a 60-kDa protein possessing a potent chitinase activity with a considerable amino acid sequence homology (about 50%) with known mammalian chitinases (Oshima et al., 2002). Using a cDNA encoding this chitinase as a probe, we found the mRNA for this enzyme to be expressed exclusively in the pancreas. We designated this chitinase as toad pancreatic chitinase (tPCase). The optimum pH of tPCase was 6.0.
On the other hand, we noticed that a crude extract of the stomach from the same species exhibited chitinase activity (unpublished data), indicating that another chitinase, perhaps different from tPCase, exists in the stomach of the toad. In the present experiment, we obtained a cDNA clone encoding a putative tGCase from a cDNA library of the toad stomach, and found the predicted amino acid sequence to be distinct from that of tPCase. This putative tGCase was expressed in the stomach but not in other organs so far studied. It is of interest to note that its amino acid sequence showed higher homology with AMCase from the mouse stomach than with other known vertebrate chitinases of extra-stomach origin.
Chitinases are classified into 2 different families, namely, families 18 and 19, on the basis of the amino acid sequence similarity of their catalytic domain (Davies and Henrissat, 1995). Judging from the predicted amino acid sequence of the putative tGCase, this enzyme seems to belong to the family-18 chitinases. In these chitinases, the catalytic center of the chitinase activity was identified in a study using mutant recombinant chitinase (Renkema et al., 1998). In the case of family-18 chitinases, the second Asp (D) and Glu (E) in the DG-D-D-E motif of the N-terminal catalytic domain are considered to be essential for chitinase activity (Bleau et al., 1999). In addition, 6 cysteine residues forming 3 sets of disulfide bonds in the chitin-binding domain are reported to be essential for exerting chitinolysis (Tjoelker et al., 2000). In our putative tGCase, both of these structures were perfectly conserved (Fig. 3), suggesting that tGCase functions as a chitinolytic enzyme in the toad stomach.
Acknowledgments
This study was supported by a grant-in-aid from the Ministry of Education, Science and Culture, of Japan and by a research grant from Waseda University to SK.
