MOLECULAR-CLONING OF THE FIRST METAZOAN BETA-1,3 GLUCANASE FROM EGGS OF THE SEA-URCHIN STRONGYLOCENTROTUS-PURPURATUS

We report the molecular cloning of the first beta-1,3 glucanase from a nimal tissue, Three peptide sequences were obtained from beta-1,3 gluc anase that had been purified from eggs of the sea urchin Strongylocent rotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences, The full-length cDNA shows a pred icted enzyme structure of 499 aa with a hydrophobic signal sequence, A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue, A polyclonal antibody to the native 68-kDa enzyme r ecognizes a single band during early embryogenesis that reappears in t he adult gut, and recognizes a 57-kDa fusion protein made from a full- length cDNA clone for beta-1,3 glucanase, Tile identity of this molecu le as beta-1,3 glucanase is confirmed by sequence homology, by the pre sence of all three peptide sequences in the deduced amino acid sequenc e, and by tile recognition of the bacterial fusion protein by the anti body directed against the native enzyme, Data base searches shaw signi ficant homology at the amino acid level to beta-1,3 glucanases from tw o species of bacteria and a clotting factor from the horseshoe crab, T he homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four di vergent species, These four species were also found to have two homolo gous regions in common with more distantly related plant, fungal, and bacterial proteins, A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes, In p articular, there is an especially deep split within genes taken from t he bacterial genus Bacillus.