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Glycoside hydrolase family 18 (GH18) includes chitinases and non-enzymatic chitinase-like proteins (CLPs) with representatives among eukaryotes (animals and plants), prokaryotes and viruses. In Lophotrochozoa, one of the three clades of bilaterian animals, only three members (Cg-Clp1, Cg-Clp2 and Cg-Chit) have been reported from the bivalve mollusc Crassostrea gigas. Here, we describe the cloning and the characterization of two additional chitinases (Cg-Chit2 and Cg-Chit3) and a new CLP (Cg-Clp3) from this species. Cg-Chit2 presents an atypical C-terminal hydrophobic region acting probably as a GPI-anchor signal for plasma membrane attachment. On the contrary, Cg-Chit3 displays a C-terminal truncated structure leading to a possible sequestration in lysosomes. Phylogenetic analyses suggest that CLPs have appeared independently in the three main branches of bilaterian animals, as a result of convergent evolution. Gene expression profiles analyzed by quantitative RT-PCR support the involvement of Cg-Clp3 in embryonic development, adult oyster growth and tissue remodelling during metamorphosis and gonadal restructuring., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg-Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg-Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg-Clp1- and Cg-Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity.

Transforming growth factor-beta (TGF-beta) members represent a widespread protein superfamily in the animal kingdom, but few members have been characterised in lophotrochozoans, a major clade of invertebrates. Here, we report the identification of Crassostrea gigas-TGF-beta (Cg-TGF-beta), a homologue of vertebrate TGF-beta and activin, from the bivalve mollusc C. gigas. Phylogenetic analysis suggests an early ancestral origin of this subgroup of TGF-beta superfamily member. Investigation of the spatio-temporal expression of Cg-TGF-beta gene by real-time quantitative RT-PCR showed a ubiquitous pattern in all adult tissues. These findings imply that Cg-TGF-beta has multiple functions as described for its vertebrate counterparts. Moreover, Cg-TGF-beta was upregulated in haemocytes during infection by a Gram-negative bacterium, suggesting that it could act as a cytokine involved in immunity in molluscs.

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.

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

The family of leucine-rich repeat-containing G protein-coupled receptors (LGRs) shows members in both vertebrates and invertebrates including the most ancestral ones. Although this suggests an early evolutionary origin of this family of receptors, little is known about their diversity in molluscs, a major phylum of bilaterian invertebrates. Based on sequences of mammalian and insect LGRs, we have cloned and characterized a new typical LGR in the bivalve mollusc Crassostrea gigas. This receptor named Cg-LGRB exhibits high degree of amino acid sequence identity with both mammalian and Drosophila LGRs. Phylogenetic analysis indicates that Cg-LGRB belongs to the cluster of type B orphan LGRs and suggests that molluscs likely express the three LGR subgroups identified previously in other animals. Quantitative RT-PCR shows that Cg-LGRB is expressed mainly in the digestive gland and only at moderate levels in other organs and developmental stages. A possible involvement in the control of cytological changes occurring in bivalve mollusc digestive gland is discussed.

Calcitonin is a key hormone involved in the regulation of calcium metabolism in vertebrates. Using oligonucleotide primers derived from consensus sequences of vertebrate calcitonin receptors, we have cloned and characterized the first representative of an invertebrate calcitonin receptor from the bivalve mollusc Crassostrea gigas. This receptor named Cg CT-R exhibits 39% amino acid sequence identity with both human calcitonin and calcitonin gene-related precursor receptors. Cg CT-R is expressed mainly in the gills and the mantle edge as well as at lower levels in muscles, digestive gland, heart, and labial palps. Transfer of animals from seawater to brackish water resulted in a significant decrease of Cg CT-R transcript levels in the gills, thus suggesting a role for ionic balance in molluscs.