Isolation and Characterization of Spicule Matrix Protein.

Abstract

Abstract : Ongoing work in our laboratory has shown that suBMP, a bone morphogenetic protein-i family member, is the homolog of the Drosophila embryonic patterning gene Tolloid. Resequencing the 3' end of the suBMP-1 cDNAs has revealed an extended open reading frame with high homology to human and murine tolloid homologs. Recent studies in the laboratories of Darwin Prockop, Daniel Greenspan, and Effrat Kessler have demonstrated that both human BMP-1 and human Tolloid act as procollagen C-terminal proteinases, releasing the C-terminal propeptide from triple helical procollagen. This proteolytic processing event is necessary for the deposition of collagen fibrils. We have been able to demonstrate the presence of a procollagen C-terminal proteinase (PCP) activity in S. purpuratus extracts containing suBMP- 1. This PCP activity is heat labile, and demonstrates both time and concentration dependent cleavage. We are currently working to express suBMP-1 as a recombinant protein in mammalian cell culture and to determine if suBMP-1 has PCP activity. Previous studies in our lab and others have demonstrated that appropriate collagen processing is necessary for gastrulation and spiculogenesis to occur in the developing sea urchin embryo, as well as for calcium carbonate deposition into growing spicules in primary mesenchyme cell culture. Disruption of collagen hydroxylation or crosslinking blocks spicule growth. Removal of the C-terminal propeptide of triple helical procollagen renders the collagen insoluble, allowing it to be deposited into growing collagen fibrils. SuBMP-1 may also play an important role in this process. If suBMP-1 is responsible for the observed PCP activity in S. purpuratus, then its function will be essential for collagen deposition and therefore sea urchin development.