Aspartyl proteases in Caenorhabditis elegans - Isolation, identification and characterization by a combined use of affinity chromatography, two-dimensional gel electrophoresis, microsequencing and databank analysis

Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal pr oteolytic activity under acidic pH conditions. About 90% of this activity i s 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 sin gle-step affinity chromatography on pepstatin-agarose. Analysis of the puri fied fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membran es, all bands were successfully subjected to N-terminal microsequencing. On 2D gels, the purified protein bands split into 19 spots which, after ren ewed microsequencing, were identified as isoelectric variants of the six pr oteins already described. The N-termini obtained for these proteins could b e correlated to genomic DNA sequences determined in the course of the C. el egans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the s ix coding sequences identified in this study were found to contain the typi cal active-site consensus sequence of aspartyl proteases and displayed an o verall amino acid identity between 25 and 66% as compared to aspartyl prote ases from other organisms. In addition to the five aspartyl proteases detec ted at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomi c DNA of C. elegans which has recently been completely sequenced.