Cloning of a Novel Chitinase cDNA from the Stomach of the Coelacanth Latimeria chalumnae (Sarcopterygii)

Chitinases are widely found in many types of organisms, including bacteria, fungi, rotifers, some algae, plants, invertebrates, and vertebrates, and play several important physiological roles. Particularly, it has been reported that chitinases from fish stomach efficiently decompose crystalline α-chitin compared with chitinases from other species. Fish chitinase cDNAs have been cloned from several kinds of Teleostei and Agnatha. A group of lobe-finned fish, Coelacanths, considered as ancestors of the superclass tetrapoda, are a part of the clade Sarcopterygii and are also known to possess chitinases. Latimeria chalumnae and L. menadoensis are the only two known living Coelacanth species. In our laboratory, we previously purified a chitinase (46 kDa) from the stomach of Coelacanth L. chalumnae, but had not sequenced its cDNA. Here, we report the cloning of the L. chalumnae stomach chitinase cDNA (LcChi) and its expression profile and phylogenetic analysis. First, we extracted total RNA from the stomach of the L. chalumnae. First strand cDNA was synthesized using reverse transcriptase and Oligo dT primer. The cDNA fragment encoding the chitinase was amplified by PCR using the degenerate primers designed based on the conserved amino acid sequence of several kinds of vertebrates. Next, 3′ and 5′ sites of the chitinase cDNA sequence were determined by using the RACE method. The full length chitinase cDNA of L. chalumnae consists of a 1,581 bp sequence, with an open-reading frame of 1,431 bp. The LcChi sequence corresponds to the N-terminal amino acid sequence of the purified chitinase (46 kDa) from L. chalumnae. The deduced amino acid sequence of this chitinase revealed a typical vertebrate chitinase structure containing a signal peptide, a glycoside hydrolase (GH) family 18 catalytic domain, a linker region, and a C-terminal chitin binding domain, and shared 66% of similarity with the human acidic mammalian chitinase. The phylogenetic tree analysis revealed that LcChi does not belong with the group of acidic fish chitinase-1 and acidic fish chitinase-2. This study is the first report of cDNA cloning of chitinase from Sarcoptrygii.