Abstract
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.
Highlights
Growth factors orchestrate growth and development in metazoan organisms
We show that Cg-Clp1 activities on mammalian chondrocytes are identical to its mammalian closest homolog YKL-40
Isolation and Sequence Analysis of Cg-Clp1 Full-length cDNA—PCR with degenerate primers whose design was based on the conserved amino acid sequences of the catalytic domain of members of family 18 glycosyl hydrolase (GH) resulted in the amplification of an expected 147-bp sequence
Summary
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). CDNAs were used as templates for PCR amplifications using two degenerated primers designed to anneal to conserved consensus regions of Drosophila imaginal disc growth factors. PCR was performed in a total volume of 50 l with 10 ng of mantle edge cDNA in 10 mM Tris/HCl, pH 9.0, containing 50 mM KCl, 0.1% Triton X-100, 0.2 mM each dNTP, 1 M each primer, 2.5 mM MgCl2, and 1 unit of TaqDNA polymerase (Eurogentec). Double-stranded cDNA from oyster mantle edges was ligated to adaptors, and 25 ng of this template was used to PCR-amplify 5Ј- and 3Ј-RACE fragments using adaptor-specific primers and gene-specific primers deduced from the initial 147-bp fragment sequence. PCR products were subcloned into pGEM-T easy vector using a TA cloning kit (Promega) and sequenced using ABI cycle sequencing chemistry
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