A New Putative Chitinase from Reticulitermes speratus KMT001

The termites used were R. speratus KMT001 (Cho et al., 2010), which were collected in the Bukhan Mountain, Seoul, Korea (latitude: 37.614009, longitude: 126.990545). The termites were grown at 26°C and 70% humidity without light.

A putative chitinase gene was selected using transcriptomic analysis from previous studies (Park et al., 2014) aimed at elucidating its biological function. The amino acid sequence, including the open reading frame (ORF), was inferred from the selected gene sequence. The chitinase gene identified from R. speratus KMT001 was registered in the NCBI database (accession number: KC477099).

A protein BLAST search (http://blast.ncbi.nlm.nih.gov/) was performed on the NCBI website using the amino acid sequence of the putative chitinase to identify homologous genes. For the multiple sequence alignment and phylogenetic analysis, we selected the gene with the highest homology score from each genus among 100 homologous sequences. The putative chitinase sequence in this study and 19 homologous sequences were aligned using ClustalW in the MEGA4 program (https://www.megasoftware.net/mega4/). For the phylo-genetic analysis of the aligned amino acid sequences, a neighbor-joining method was used to assess the evolutionary distance using bootstrap and crossvalidation methods with 1,000 replications to estimate the reliability of the results (Nei and Saitou, 1987; Tamura et al., 2007).

A previous transcriptomic analysis of R. speratus KMT001 using the GS FLX System (Park et al., 2014) provided sequence information for the expressed mRNA. Among the genes whose biological functions were suggested, two contigs (contig00176 and contig 03679) and 17 singletons, which appear to be chitinases, were selected (Table 1). The biological function analysis of contig00176 (NCBI accession number: KC477099) indicated that the closest gene in the NCBI database was a chitinase of Araneus ventricosus, a spider. The gene was 1,300 bp in length, and the complete ORF of the putative chitinase was 1,199 bp. The most similar gene to contig03679 in the NCBI database was a chitinase from Ixodes scapularis, a mite subspecies. In contig03679, only an ORF with a short base sequence, 178 bp, could be identified. The other 17 singletons did not have an identifiable ORF and were excluded from this study.

We searched for genes homologous to the ORF KC477099 in the NCBI database using a protein BLAST search at the National Center for Biotechnology Information (Table 2). The 100 most similar genes were from GH18, distributed over in 19 orders. Among the search results, none were found in the order Blattodea, to which R. speratus belongs. The highest homology with KC477099 was AAN39100 from A. ventricosus with an e-value of 7E-112. In addition to chitinase, chitotriosidase and acidic mammalian chitinase (AMCase), which belong to the same GH18 group, were found to have high homology scores with KC477099.

The orders Decapoda, Diptera, and Mesostigmata in the phylum Arthropoda have homologous chitinase genes in the GH18 group. Members of the class Enteropneusta in the phylum Hemichordata, order Ostreoida in the phyla Mollusca, Rodentia, Perissodactyla, Carnivora, Primates, Squamata, Dasyuromorphia, and Diptera have genes for chitotriosidase and AMCase in GH18 (Bussink et al., 2007; Reardon and Farber, 1995). Homologous chitinase genes have been identified in various animals. Previous studies suggest that AMCase and chitotriosidase in insects, bacteria, and plants are highly homologous to chitinase (Arakane and Muthukrishnan, 2010; Henrissat, 1999). The role of chitinase in non-insect animals and plants is different from that in insects that have a chitin exoskeleton. Chitinase in the former is used for the digestion of chitin for nutrients or as a defense mechanism against insects, but the latter is mainly used for the maintenance of the chitin exoskeleton (Rathore and Gupta, 2015). The results of this study confirm that AMCase and chitotriosidase have high homology scores with chitinases found in insects, bacteria, and plants (Rathore and Gupta, 2015). Hypothetical proteins from Amphoxiformes, Cladocera, Diptera, and Coleoptera have a conserved region, but the active sites of GH18 and their activities remain to be confirmed.

To compare the amino acid sequences of KC477099, one amino acid sequence with the highest homology score in each of the 19 orders among the 100 homologous sequences was selected, and multiple sequence alignments were performed using ClustalW in the MEGA4 program (Fig. 1). KC477099 shared two conserved regions (motifs I and II in Fig. 1) and an active site (shown in bold in Fig. 1) involved in the enzymatic activity of chitinase belonging to the GH18 group with other sequences (Henrissat and Bairoch, 1993; Korb et al., 2012; Lu et al., 2002; Sharma et al., 2011; Terwisscha van Scheltinga et al., 1996; Thomas et al., 2000). KC477099 have 17 active amino acid sites in the GH18 chitolectin chitotriosidase (https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?hslf=1&uid=119351&#seqhrch), which is the family with the most specific hits to KC477099 (Fig. 1). Among the active sites, 16 sites are located in the β strands (Fig. 1).

Fig. 1. Multiple amino acid sequence alignment of KC477099 from R. speratus KMT001 and chitinase from 19 strains obtained from a protein BLAST search of the National Center for Biotechnology Information. Motifs I and II are indicated on the top of the alignments. The bold characteristics indicate the consensus sequences in the active sites of GH18. The star marks on the bottom of the alignments indicate the active sites of GH18 chitolectin chitotriosidase.

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The amino acid sequence of motif II is FDGLDIDWEYP, which is part of the amino acid sequence of KC477099. The Asp-Trp-Glu site in motif II has a significant role in the enzymatic activity of GH18 (Henrissat and Bairoch, 1993; Huang et al., 2010; Synstad et al., 2004; Zhang et al., 2002). Glu¹⁵⁸ in KC477099 may be involved in the cleavage of glycosidic bonds through protonation as a proton donor in the GH18 enzymatic activity (Sinnott, 1990). The switching of Glu¹⁵⁸ to another amino acid significantly reduces its enzymatic activity (Henrissat and Bairoch, 1993). Asp¹⁵⁶ has been suggested as an electrostatic stabilizer in the transition state and is less involved in the enzymatic activity of GH18 than Glu158 (Huang et al., 2010; Synstad et al., 2004). Glu¹⁵⁴ is involved in the ionization state of Glu¹⁵⁸ and affects the pK values of Glu¹⁵⁸ and Asp¹⁵⁶ (Henrissat and Bairoch, 1993). An additional amino acid in motif II that affects the enzymatic activity of GH18 is Trp¹⁵⁷. Trp¹⁵⁷ maintains the abnormal pK values of other amino acids during enzymatic activity. It has an important role in the extension of chitinase activity within an alkaline pH range (Zhang et al., 2002).

To investigate the evolutionary relationships between genes, we analyzed the phylogeny of the 20 genes shown in Fig. 1 (Fig. 2). KC477099 was grouped in the phylum Arthropoda with the Decapoda, Mesostigmata, Araneae, Hymenoptera, Lepidoptera, and Ixodide. The biological function of chitinase in the phylum Arthropoda is the digestion of food, decomposition of the exoskeleton in ecdysis, and defense of the body against toxicity (Henrissat and Davies, 1997; Merzendorfer and Zimoch, 2003).

Fig. 2. Phylogenetic analysis of KC477099 from R. speratus KMT001 and chitinase from 19 strains from a protein BLAST search at the National Center for Biotechnology Information. A neighbor-joining method with a bootstrap of 1,000 replications was used. The order and phylum of each strain are listed on the right side of the figure. KC477099 is indicated using a black arrow. Saccoglossus kowalevskii class is listed because it has not been assigned to an order.

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The phylum Mollusca, order Ostreoida, phylum Hemichordata class Enteropneusta, and phylum Chordata, including orders Perissodactyla, Carnivora, Primates, Rodentia, Squamata, Didelphimorphia, and Dasyuromorphia, were grouped in one of the main branches. All of them were identified as chitotriosidase and AMCase in GH18. Chitotriosidase was the first chitinase found in humans, and AMCase is chitotriosidase expressed in an acidic condition (Reardon and Farber, 1995). In the group of the phylum Arthropoda, the KC477099 branch is separated at the earliest stage. This phylogenetic analysis confirms that the putative chitinase KC477099 of R. speratus KMT001 is unique from other reported chitinases in the phylum Arthropoda and has low homology in the BLAST search.