Characterization of an atypical family 18 chitinase from the oyster Crassostrea gigas: Evidence for a role in early development and immunity
Introduction
Chitin, a repeating β(1→4)-linked homopolymer of N-acetyl-β-d-glucosamine (GlcNAc), is the second most abundant bio-polymer on earth next to cellulose [1]. Chitin polymer has been found as a structural component in the integuments of arthropods, nematodes, and molluscs, the gut linings of insects, the cell wall of fungi and some algae. Production of chitin depends on chitin synthase while degradation or remodelling of chitin-containing structures requires chitinases. Both synthesis and degradation processes are essential for the normal development and function of many forms of organisms throughout their life span. In chitin-containing organisms, chitinases are essential for maintaining normal life cycle functions such as morphogenesis of arthropods [2] or cell division and sporulation of yeast and other fungi [3]. Chitinolytic enzymes have also been found in organisms that do not contain chitin polymers themselves such as viruses, bacteria, plants and animals. Indeed, chitinases have also an important role in parasite invasion of chitinous hosts. Chitinases are also believed to play a central role in immunity. Several studies have demonstrated that chitinases are produced constitutively or inducibly as pathogen-resistant proteins in plants [4], [5], [6]. Vertebrates also synthesize chitinases to defend themselves against chitin-containing pathogens such as protozoa, fungi, insects and nematodes [7], [8]. In other respects, since some chitinases are expressed in the digestive tract of mammalians [9], [10] and molluscs [11], [12], they are likely involved in digestion of chitin-containing food.
Chitinases catalyze the hydrolysis of the β-1,4-N-acetyl-d-glucosamine linkages in chitin polymers and oligomers. They belong to the O-glycosyl hydrolases (GH) (EC 3.2.1.14), a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety [13]. A new classification of glycosyl hydrolases based on similarities in amino acid sequences has been put forward [14]. This groups the five known chitinase classes into two GH families, each of which exhibits a strict conservation of the catalytic machinery and enzymatic mechanism. Chitinases of family 18 (formerly class III and V) have been well studied, with information available on the three-dimensional structure and the biochemistry of the enzyme reaction. They operate with retention of the anomeric configuration, whereas family 19 members (class I, II, and IV) invert the configuration at the hydrolysis site [15], [16]. Family 18 of GH (GH18) comprises chitinases from various species, including bacteria, fungi, nematodes, arthropods, mammalians and plants [17], but also several mammalian, insect and plant proteins lacking chitinolytic activity due to substitution of a critical acidic amino acid in the catalytic centre. This new class of proteins has been called chitinase-like proteins (CLPs). Recently, we identified the first lophotrochozoan CLP from the oyster Crassostrea gigas. Interestingly, this protein named C. gigas chitinase-like protein 1 (Cg-Clp1) was found to be involved in the control of growth and remodelling processes in a manner similar to its YKL-40 mammalian counterpart. These findings argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels [18]. Though chitinase activities were measured in the saliva and the digestive tract of several molluscs [11], [12], no GH18 chitinase has been identified yet at the molecular level in Lophotrochozoa. Since chitinases are potentially implicated in the control of growth and immune processes, their identification in bivalve molluscs could be valuable for improving hatchery production of these economically important animals.
In this paper, we report the molecular and biological characterization of a singular chitinase from the oyster C. gigas. Tissue distribution and temporal pattern of expression during oyster development were established by real time PCR and in situ hybridization. Involvement of this chitinase in oyster immune defences was investigated by measuring its transcript expression level after both bacterial and LPS challenges.
Section snippets
Animals
Adult oysters C. gigas were purchased from a local oyster farm (Normandie, France). Embryo and larval stages were produced in the IFREMER shellfish laboratory of Argenton (France).
Reverse transcription, cloning and sequencing
Reverse transcription was carried out using oligo(dT)17 as primer, 1 μg mRNA and 200 U Moloney murine leukaemia virus reverse transcriptase (Promega). cDNAs were used as templates for polymerase chain reaction (PCR) amplifications using two degenerated primers designed to anneal to conserved consensus regions of GH18
Isolation and sequence analysis of C. gigas chitinase full-length cDNA
RT-PCR with degenerate primers whose design was based on the conserved amino acid sequences of the catalytic domain of members of GH18 family resulted in the amplification of an expected 147-bp sequence. Cloning and sequencing of this fragment revealed an open reading frame showing amino acid sequence similarity to members of GH18 family. Subsequently, specific primers deduced from this 147-bp sequence were used to perform 5′ and 3′ RACE-PCR to obtain the full-length cDNA. The complete 2144-bp
Discussion
In the present study, we identified Cg-Chit, the first lophotrochozoan chitinase of the GH18 family. The GH18 family is phylogenetically conserved as members are found in almost every groups of life kingdom including bacteria, fungi, plants, nematodes, insects and mammals. In addition to a vast majority of chitinases, this family contains proteins named CLPs which are closely related to chitinases but are lacking this enzymatic activity [33].
Comparative sequence analyses show that Cg-Chit
Aknowledgements
This study was financially supported by the “Conseil Régional de Basse-Normandie” and the “Agence de l’eau Seine-Normandie” and FEDER Presage no. 4474 grant (programme PROMESSE). The authors are indebted to all staff of the Argenton IFREMER experimental hatchery for the production of oyster embryos and larvae. The authors thank Arnaud Huvet (IFREMER, Brest), Elodie Fleury (IFREMER, Brest), Christophe Fleury (University of Caen) and Emeline Furon (University of Caen) for technical assistance.
References (54)
- et al.
A Bombyx mori gene, BmChi-h, encodes a protein homologous to bacterial and baculovirus chitinases
Insect Biochem Mol Biol
(2003) - et al.
Chitinase is required for cell separation during growth of Saccharomyces cerevisiae
J Biol Chem
(1991) - et al.
Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor
Structure
(1994) - et al.
Purification and characterization of human chitotriosidase, a novel member of the chitinase family of proteins
J Biol Chem
(1995) - et al.
Identification of a novel acidic mammalian chitinase distinct from chitotriosidase
J Biol Chem
(2001) - et al.
The 1.8 Å resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18
J Mol Biol
(1996) - et al.
The phylogenetically conserved molluscan chitinase-like protein 1 (Cg-Clp1), homologue of human HC-gp39, stimulates proliferation and regulates synthesis of extracellular matrix components of mammalian chondrocytes
J Biol Chem
(2006) - et al.
NPS@: network protein sequence analysis
Trends Biochem Sci
(2000) - et al.
Protein structure prediction. Implications for the biologist
Biochimie
(1997) - et al.
Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas
Biochem Biophys Res Commun
(2004)
Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc, Haliotis tuberculata
Biol Cell
Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity
J Biol Chem
Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina. cDNA and deduced amino acid sequences
J Biol Chem
Peritrophic matrix proteins
Insect Biochem Mol Biol
Properties of catalytic, linker and chitin-binding domains of insect chitinase
Insect Biochem Mol Biol
Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta
Insect Biochem Mol Biol
Expression of recombinant microfilarial chitinase and analysis of domain function
Mol Biochem Parasitol
Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae
J Biol Chem
Insect chitin: physical state, synthesis, degradation and metabolic regulation
Insect Biochem
Purification and characterization of Bombyx mori chitinases
Insect Biochem Mol Biol
Structural and functional definition of the human chitinase chitin-binding domain
J Biol Chem
Peptidoglycan recognition proteins (PGRPs)
Mol Immunol
Cg-TIMP, an inducible tissue inhibitor of metalloproteinase from the Pacific oyster Crassostrea gigas with a potential role in wound healing and defense mechanisms (1)
FEBS Lett
Cg-TGF-beta, a TGF-beta/activin homologue in the Pacific Oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection
Dev Comp Immunol
Monomeric and polymeric Gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway
Immunity
A putative double role of a chitinase in a cnidarian: pattern formation and immunity
Dev Comp Immunol
Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases
J Exp Biol
Cited by (52)
The glycoside hydrolase 18 family chitinases are associated with development and virulence in the mosquito pathogen Pythium guiyangense
2020, Fungal Genetics and BiologyCitation Excerpt :The oomycete represents a class of filamentous eukaryotes whose cell walls has none or very small amounts of chitin, however significant animal and plant pathogens encode GH18 family to breakdown chitin in host cell walls (McGowan and Fitzpatrick, 2017). Mounting evidence also demonstrate that chitinases participate in remodeling processes during the molting and the hatching of larvae from the eggshell (Adam et al., 1996) and is involved in early embryonic development (Badariotti et al., 2007). Pythium guiyangense X.Q. Su was discovered from Guizhou, China.
Genomic and transcriptomic landscapes and evolutionary dynamics of molluscan glycoside hydrolase families with implications for algae-feeding biology
2020, Computational and Structural Biotechnology JournalUpregulation of cellulase activity and mRNA levels by bacterial challenge in the earthworm Eisenia andrei, supporting the involvement of cellulases in innate immunity
2020, Biochemical and Biophysical Research CommunicationsVariation in transcriptional responses to copper exposure across Daphnia pulex lineages
2019, Aquatic ToxicologyInsights into the intestine immune of Marsupenaeus japonicus under the white spot syndrome virus challenge using RNA sequencing
2019, Veterinary Immunology and Immunopathology