UC San Diego

The sea urchin, Strongylocentrotus purpuratus, has a fluid -filled body cavity, the coelom, containing four types of immunocytes called coelomocytes. Within minutes after coelomic fluid is removed from the body cavity, a massive cell-cell adhesion of coelomocytes occurs, referred to as clotting. One function of coelomocyte clotting could be to seal the body cavity following injury, as urchins are hard -bodied and unable to contract tissue around an injury that penetrates the test. Clotting is also thought to function in the cellular encapsulation of foreign material and microbes. Here I report that the intercellular clotting response is mediated by amassin, an extracellular coelomic plasma glycoprotein. Along its length, amassin contains these structural features: a signal peptide, a short predicted b-region, segments of dimerizing coiled- coils, and an olfactomedin (OLF) domain. Amassin forms disulfide-linked multimers required for its biological clotting activity. The functional activities of amassin's constituent protein regions were investigated using various truncated constructs expressed in the yeast, Pichia pastoris. This research revealed that the multimerization occurs by forming disulfide-linked dimers through Cys203 at the end of the coiled-coils region, then tetramers through the N-terminal b-region and/or the first segment of coiled-coils. The OLF domain itself exists in a monomeric state and contains two intradomain disulfide bonds. The OLF domain confers amassin's cell-binding activity and does so in a calcium-dependent manner. Utilizing data from the S. purpuratus Genome Project, I identified a family of OLF proteins, totaling five, that are all actively transcribed in coelomocytes. Phylogenetically, four of the five belong to a subgroup distinct from all other known OLF domains, and the fifth is similar to the most primitive subgroup known as the colmedins. The five OLF proteins of the sea urchin represent an intermediate diversification between those of the protostomes and the vertebrates. Due to the involvement of OLF domains in important developmental cellular events in other animals, I monitored the expression levels for the genes of all five OLF proteins throughout the larval life cycle. Transcripts were highly regulated, often coinciding with the formation of the rudiment, which eventually becomes the adult