No abstract

No abstract

No abstract

The astacin family of zinc endopeptidases was named after the digestive enzyme astacin isolated from the crayfish Astacus astacus. Employing a reverse transcription/PCR strategy with degenerate oligonucleotide primers specific for two signature seqences of the astacin family, we have isolated a 1602-bp cDNA from embryos of developing A. astacus eggs, which was designated Astacus embryonic astacin (AEA). This cDNA was found to code for an astacin-like protease domain which accounts for the N-terminal half of the predicted protein. The C-terminal half mainly consists of two complement subcomponent C1r/ C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein 1 (CUB) domains. The metalloprotease domain displays an amino acid sequence identity of 42% with astacin. A higher sequence similarity was found to astacin family members that act as hatching enzymes in different species, e.g. chorioallantoic membrane protein 1 (CAM-1 ; from quail) and Xenopus hatching enzyme (formerly UVS.2), both of which show 54% identity, and high and low choriolytic enzymes (HCE and LCE) from the teleost Oryzias latipes (52% and 48% identity, respectively). A relationship to astacin-like hatching enzymes is further supported by a phylogenetic analysis of the protease domains. Expression of AEA mRNA in developing embryos was found 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 developmental stage. AEA expression starts about 8 days prior to hatching, followed by a strong (18-fold) induction with a maximum at day 4 before hatching. Newly hatched juveniles were found not to express the AEA mRNA.Keywords : Astacus astacus; astacin; metalloprotease; gene expression ; hatching enzyme.The astacin family of zinc endopeptidases [1Ϫ3] was named genetic organization [7], represents the only astacin family member with a purely digestive function, contributing to the deafter the digestive enzyme astacin, isolated from the stomachlike cardia of the freshwater crayfish Astacus astacus [4]. As-gradation of protein diet in the crayfish stomach. All other members of this protease family may be classified by their function tacin family members are characterized by the consensus sequences HEXXHXXGFXHEXXRXDRD, containing the zinc-into one of three groups. The membrane-bound meprins [8,9] found in the small intestine and kidney of mammals are believed binding motif, and SXMHY, the so-called Met turn. These highly conserved regions are part of a 200-amino-acid sequence, to be involved in the processing of biologically active peptides.A second class of astacins comprises morphogenetically active which constitutes the entire mature crayfish astacin. All other astacin family members [with the exception of the fish hatching proteins found in many vertebrate and invertebrate species, e.g. BMP-1 [10, 11] or tolloid [12, 13]. Astacin-like enzymes have enzymes high and low choriolytic enzymes (HCE and LCE)] [...

Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin.We have purified enzymes responsible for this proteolytic activity by a single-step affinity chromatography on pepstatin±agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N-terminal microsequencing.On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N-termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active-site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.