THE SPECIFICITY OF SEA-URCHIN HATCHING ENZYME (ENVELYSIN) PLACES IT IN THE MAMMALIAN MATRIX METALLOPROTEINASE FAMILY

The sea urchin hatching enzyme (HEz) is a protease capable of dissolving the fertilization envelope that surrounds the embryo as a protective coat during early development. We have now purified a 37-kDa active enzyme from the supernatant fluid of hatched blastula medium of the species Hemicentrotus pulcherrimus. The purified enzyme was completely inhibited by alpha-2-macroglobulin and the chelating agents EDTA, EGTA, and 1,10-phenanthroline and was slightly inhibited by chymostatin and pepstatin, but was not inhibited by various other serine and thiol protease inhibitors. These results suggest that HEz is a metalloproteinase. Quantitative analyses of the products of HEz's action on various peptides revealed that the enzyme preferentially cleaved the peptide bonds on the amino side of bulky hydrophobic residues, -Leu, -Ile, and -Phe as well as -Tyr, in a similar but more limited fashion than thermolysin. Furthermore, although substance P was a good substrate of HEz, snake venom alpha-protease, and thermolysin, the analogue [Sar9]substance P was a poor substrate for HEz. This analogue was a good substrate for thermolysin and lpha-protease, but the scissile bonds were altered from those of substance P. The failure of HEz and alpha-protease to cleave the P1-P1' bond that meets the primary specificity is ascribed to the presence of an imino acid residue (proline or sarcosine) or the absence of any amino acid at the P2' or P3' position, in contrast to the simple P2' restriction of thermolysin. Since such a tendency to [Sar9]substance P has been reported to be characteristic of stromelysin, one of the mammalian matrix metalloproteinases (MMPs), HEz and possibly alpha-protease are rationally classified as members of the MMP family (collagenase gene family), rather than of the bacterial metalloproteinase family. In this context we propose the name "envelysin" for the HEz of sea urchin, or of wider sources, if established. The HEz of H. pulcherrimus exhibited a unique species specificity; it could not dissolve the fertilization envelope of Anthocidaris crassispina, a species of a neighboring taxonomical family, but could dissolve those of more distantly related species, even if with less efficiency.