Sea urchin hatching enzyme (envelysin): cDNA cloning and deprivation of protein substrate specificity by autolytic degradation.
The hatching enzyme (envelysin) of the sea urchin Hemicentrotus pulcherrimus was purified from the medium of hatched blastulae. By cDNA cloning its deduced amino acid sequence and molecular architecture were revealed. The 591-residue precursor with calculated Mr of 66,123 consists of an 18-residue signal sequence, a 151-residue propeptide, and a 422-residue mature enzyme with N-terminal catalytic and C-terminal hemopexin-like domains. As compared with that of Paracentrotus lividus, its amino acid sequence is 69% identical and 10% similar. They share typical structural features with the mammalian MMP gene family members: cysteine 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, supposedly involved in the proenzyme activation by binding Ca2+ ions in seawater. 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), producing the 38K form retaining catalytic activity and substrate specificity against fertilization envelope. The 38K form and 15K fragment were coeluted from a gel-filtration column, suggesting that these two fragments are disulfide-bridged and that the tertiary structure is not much deviated. The 38K form further autolyzed to 32K form by cleaving Tyr(450)-Tyr(451) bond with the loss of protein-substrate specificity, retaining only nonspecific protease activity. Thus, the autolytic release of 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 substrate specificity. Moreover, autolytic degradation of envelysins from 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 activity depends on the extent of the interaction with the C-terminal domain of the enzyme.
Nomura
K
K
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Japan. nomura@tmig.or.jp
Shimizu
T
T
Kinoh
H
H
Sendai
Y
Y
Inomata
M
M
Suzuki
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D001483:Base Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000201:enzymology*; D006023:Glycoproteins / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011480:Protease Inhibitors / Q000494:pharmacology; D012617:Sea Urchins; D016415:Sequence Alignment; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases
D018076:DNA, Complementary; D006023:Glycoproteins; D011480:Protease Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D008666:Metalloendopeptidases; C024344:envelysin