SEA-URCHIN HATCHING ENZYME (ENVELYSIN) - CDNA CLONING AND DEPRIVATIONOF PROTEIN SUBSTRATE-SPECIFICITY BY AUTOLYTIC DEGRADATION

The hatching enzyme (envelysin) of the sea urchin Hemicentrotus pulche rrimus was purified from the medium of hatched blastulae. By cDNA clon ing its deduced amino acid sequence and molecular architecture were re vealed. The 591-residue precursor with calculated M-r of 66 123 consis ts of an 18-residue signal sequence, a 151-residue propeptide, and a 4 22-residue mature enzyme with N-terminal catalytic and C-terminal hemo pexin-like domains. As compared with that of Paracentrotus lividus, it s amino acid sequence is 69% identical and 10% similar. They share typ ical structural features with the mammalian MMP gene family members: c ysteine switch, zinc-binding signature, methionine-turn, Cys residues near both ends of hemopexin-like domain, etc. However, its propeptide has a 70-residue extra sequence with an Asp- and Glu-rich stretch, sup posedly involved in the proenzyme activation by binding Ca2+ ions in s eawater. The hinge region is also longer than those of most MMPs, with an extra sequence rich in Thr and Arg residues. Mature 50K enzyme is highly susceptible to autolytic cleavage at Gln(503)-Leu(504), produci ng the 38K form retaining catalytic activity and substrate specificity against fertilization envelope. The 38K form and 15K fragment were co eluted from a gel-filtration column, suggesting that these two fragmen ts are disulfide-bridged and that the tertiary structure is not much d eviated. The 38K form further autolyzed to 32K form by cleaving Tyr(45 0)-Tyr(451) bond with the loss of protein-substrate specificity, retai ning only nonspecific protease activity. Thus, the autolytic release o f 2/3 of the C-terminal domain reduced the highly specific enzyme to a common nonspecific protease, implying that the size and structure of almost the entire hemopexin-like domain is essential for the protein s ubstrate specificity. Moreover, autolytic degradation of envelysins fr om the two species follow quite different pathways despite their high homology in structure. The 38K and 32K forms were inhibited by bovine TIMP-1 with different IC50 values, indicating that its inhibitory acti vity depends on the extent of the interaction with the C-terminal doma in of the enzyme.