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.Growth factors orchestrate growth and development in metazoan organisms. Despite the huge variety of growth factors characterized in vertebrates, only a few have been identified in Protostome lineages and are mainly restricted to the Ecdysozoan model organisms Caenorhabditis elegans and Drosophila melanogaster. Thus, the third branch of bilaterian, named Lophotrochozoa, is an obviously understudied group of animals, since none of the major model organisms presently belong to this clade (1). Recent reports show that Lophotrochozoan animals exhibit biological characteristics that are considered ancestral, or at least less derived from the ancestral state than in other established systems that are known (2). Since bivalve mollusks belong to the Lophotrochozoa, they are good candidates to characterize growth factors and then contribute to our understanding of the evolution of growth and developmental regulations in bilaterian animals. Furthermore, a better knowledge of the molecular control of mollusk physiology may help improve the hatchery production of these economically important animals. Unfortunately, the number of growth factor genes identified so far at the molecular level in bivalve mollusks is extremely limited. In the pacific oyster, Crassostrea gigas, only four members of the transforming growth factor-␤ superfamily and their corresponding receptors have already been described (3-6). This lack of knowledge is above all the consequence of both the paucity of genomic sequence information and the difficu...

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Glycoside hydrolase family 18 (GH18) is a phylogenetically conserved group of proteins present in eukaryotes, prokaryotes and viruses. The GH18 family is characterized by a Glyco_18 domain adopting an (a ⁄ b) 8 triose phosphate isomerase-barrel structure that consists of a specific arrangement of eight parallel b-strands, forming the barrel core, surrounded by eight a-helices [1]. This family classification, based only on similarities in amino acid sequences, groups together chitinases and proteins devoid of catalytic activity due to the substitution of a critical amino acid in the catalytic centre. This latter singular class of proteins, called chitinase-like proteins (CLPs), has been identified only recently in plants [2], mammals [3], insects [4] and molluscs [5]. CLPs have been implicated in many biological processes, such as control of nodulation [2] and growth ⁄ differentiation balance during development in plants [6]. Insect CLPs such as imaginal disc growth factors represent the first proliferating polypeptides reported from invertebrates [7]. These mitogenic growth factors cooperate with insulin to stimulate proliferation, polarization and mobility of imaginal disc cells in vitro. Imaginal disc growth factors may also constitute morphogenetic factors controlling embryonic and larval development, and could stimulate the cell growth required for wound healing [8,9]. In mammals, CLPs such as YM1 ⁄ 2 and YKL-40 (40 kDa mammalian protein with the N-terminus YKL) [also known as human cartilage glycoprotein-39 (HC-gp39) in humans] are considered to be cytokines [10,11] 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 CgClp2-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.Abbreviations Cg-Clp1 ⁄ 2, Crassostrea gigas chitinase-like protein 1 ⁄ 2; CLP, chitinase-like protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GH, glycoside hydrolase; HC-gp39, human cartilage glycoprotein-39 (also called YKL-40); LPS, lipopolysaccharide; YKL-40, 40 kDa mammalian protein with the N-terminus YKL.

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