In vitro synthesis of proteoglycans and collagen in primary cultures of mantle cells from the nacreous mollusk, Haliotis tuberculata: A new model forstudy of molluscan extracellular matrix

In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Ha liotis tuberculata have been established as useful experimental model to in vestigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/ GAGs) and collagen. First, we tested different enzymatic digestion procedur es to find the method that gives the highest percentage of viable and adher ent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collage nase was routinely used. Six days after the initiation of culture, about 80 % of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [H-3]l eucine incorporation remained constant during this period. For the first ti me, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [H-3]-D-gl ucosamine macromolecules synthesized, [H-3]PGs/GAGs represented 43%, divide d into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyalur onic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De no vo synthesis of collagen represents 4.52% +/- 0.84% (SD) with respect to th e total protein synthesis. Such a model will facilitate studies on the synt hesis of PGs/GAGs and collagen as components of the extracellular matrix an d its regulation in Mollusca. Both PGs/GAGs and collagen participate in mol ecular events that regulate cell adhesion, migration, and proliferation. Fu rther studies with this type of in vitro model should provide knowledge abo ut novel aspects of molluscan cell signaling, in relation to extracellular matrix components.