Characterization and Expression of Conserved Skeletal Proteins in Echinoderms

Abstract

Specification of the cell fate of skeleton‐forming cells in developing sea urchin embryos has been well characterized and the skeletal proteome of both adult and larval skeleton has been determined. Little is known, however, of the mechanism and proteins involved in the mineralization process. A number of spicule matrix proteins have been identified in sea urchins and are present in the organic matrix during spicule formation. However, loss of function studies have indicated they may not be required for mineralization. We have taken a comparative, evolutionary approach to try and elucidate what proteins may be required for the process of mineralization. Here we report the characterization of the skeletal proteome of the sea star Patiria miniata. Our hypothesis is that there are proteins utilized in skeleton formation that are conserved between all echinoderm groups, and possibly among all deuterostomes. First we have isolated skeletal proteins from P. miniata, and then used LC/MS/MS to obtain peptide sequences of those proteins. Characterization of skeletal proteins was accomplished utilizing the NCBI non‐redundant protein database. By comparing the proteins present in all mineralized tissues of the sea urchin with those found in other echinoderm groups we have identified a set of conserved proteins containing similar functional domains. The skeletal proteome from the two echinoderms, P. miniata and Strongylocentrotus purpuratus, groups also shared novel proteins with similar functional domains. We have analyzed the sequences of the proteins we identified as potentially important to determine the evolutionary relationship and to identify the sequences most highly conserved. Additionally, some of these proteins are also found in vertebrate bone. We have also shown that some skeletal proteins prevalent in one group are not found in other echinoderms. This suggests independent co‐option of proteins utilized in mineralization in some groups. The conserved proteins were then analyzed for temporal expression at eight different stages of the developing embryo of S. purpuratus. Some of these stages, such as mesenchyme blastula, were predicted to show higher level of expression for some of the conserved skeletal proteins. Utilizing gel electrophoresis and quantitative polymerase chain reaction (qPCR), we analyzed the temporal expression of conserved skeletal proteins and the relative expression of those proteins during the eight stages of the developing embryo. We found that expression of the conserved skeletal proteins varied, but all were present when skeleton formation occurred in the embryo.Support or Funding InformationNIH/NIGMS T34GM008074, NSF grant 09097979