Characterization of an atypical family 18 chitinase from the oyster Crassostrea gigas: Evidence for a role in early development and immunity

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Abstract

Despite their physiological significance in immune and growth-controlling processes in plants and animals, no chitinolytic enzyme has been identified yet at the molecular level in Lophotrochozoa, one of the major clades of bilaterian animals. Here, we report the cloning and the characterization of a singular chitinase homologue from the bivalve mollusc Crassostrea gigas (Cg-Chit). This protein displays a modular structure including a conserved catalytic domain attached to a peritrophin-A type chitin-binding domain and an unconventional C-terminal hydrophobic sequence acting as a potential membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues and during development support for the first time the involvement of such a protein in early embryonic development. Furthermore, Cg-Chit encoding gene was transcriptionally stimulated in haemocytes in response to either bacterial or LPS challenge. This suggests that Cg-Chit plays an important role as an immunity effector in molluscs.

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.

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