Development from the inside out: A molecular study of beta-1,3-glucanase and moesin in the sea urchin

Classical observations made by embryologists have recently been analyzed in detail by studying specific molecules during development. The cell cortex is one cellular compartment that influences morphogenesis and cell signalling during development. The dynamics of two molecules that undergo rapid changes in distribution following fertilization, away from and toward the cell cortex, were investigated. I have cloned and characterized {dollar}beta{dollar}-1,3 glucanase and moesin in the sea urchin, which are two maternally derived proteins that play roles during development at the cell cortex-plasma membrane interface.; The enzyme {dollar}beta{dollar}-1,3 glucanase is exocytosed from sea urchin eggs during fertilization, and appears later as a gut digestive enzyme. The structure and function of this egg enzyme is unknown. In order to gain insight into the function and evolution of sea urchin {dollar}beta{dollar}-1,3 glucanase, I have analyzed the comparative biochemistry and molecular structure of this enzyme. Sea urchin {dollar}beta{dollar}-1,3 glucanase has properties of bacterial {dollar}beta{dollar}-1,3 glucanases in vivo and in vitro. I cloned {dollar}beta{dollar}-1,3 glucanase in the sea urchin Strongylocentrotus purpuratus using degenerate oligonucleotides based on peptide sequence in the PCR. This enzyme has strong homology to {dollar}beta{dollar}-1,3 glucanases from Bacillus circulans, a eubacterium, and a horseshoe crab clotting factor, and represents the first animal {dollar}beta{dollar}-1,3 glucanase nucleotide sequence to be derived. A 3.2 kb message is present during early embryogenesis and in the adult gut. Phylogenetic analysis suggests that the gene for {dollar}beta{dollar}-1,3 glucanase has either duplicated prior to the origin of metazoa, or has arisen twice in the metazoa by horizontal transfer. Secondly, I have characterized a homologue of the gene moesin in the sea urchin Lytechinus variegatus (SUmoesin). Affinity purified antibodies made to recombinant fusion proteins were used for immunofluorescence and biochemical analysis. SUmoesin is recruited to the cell cortex immediately after fertilization, and remains apposed to the plasma membrane in an apical location in the regions of cell junctions during development. This localization is coincident with and requires the ongoing polymerization of actin in order to establish and maintain its polarity.