CLONING AND CHARACTERIZATION OF A CDNA CODING FOR ASTACUS EMBRYONIC ASTACIN, A MEMBER OF THE ASTACIN FAMILY OF METALLOPROTEASES FROM THE CRAYFISH ASTACUS-ASTACUS

The astacin family of zinc endopeptidases was named after the digestiv e enzyme astacin isolated from the crayfish Astacus astacus. Employing a reverse transcription/PCR strategy with degenerate oligonucleotide primers specific for two signature sequences of the astacin family. we have isolated a 1602-bp cDNA fi om embryos of developing A. astacus e ggs, which was designated Astacus embryonic astacin (AEA). This cDNA w as found to code for an astacin-like protease domain which accounts fo r the N-terminal half of the predicted protein. The C-terminal half ma inly consists of two complement subcomponent C1r/C1s/embryonic sea urc hin protein Uegf/bone morphogenetic protein 1 (CUB) domains. The metal loprotease domain displays an amino acid sequence identity of 42% with astacin. A higher sequence similarity was found to astacin family mem bers that act as hatching enzymes in different species, e.g. chorioall antoic membrane protein 1 (CAM-1; from quail) and Xenopus hatching enz yme (formerly UVS.2), both of which show 54% identity. and high and lo w choriolytic enzymes (HCE and LCE) from the teleost Oryzias latipes ( 52% and 48% identity, respectively). A relationship to astacin-like ha tching enzymes is further supported by a phylogenetic analysis of the protease domains. Expression of AEA mRNA in developing embryos was fou nd to be restricted to unhatched juveniles (larvae) during the last 8 days before hatching. AEA transcripts could not be detected in various tissues of adult animals or in eggs and embryos from an earlier devel opmental stage. AEA expression starts about 8 days prior to hatching, followed by a strong (18-fold) induction with a maximum at day 4 befor e hatching. Newly hatched juveniles were found not to express the AEA mRNA.