Aspartyl proteases in Caenorhabditis elegans

Abstract

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.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

“…Several aspartic proteases have also been described in different parasitic nematodes, including hookworms (Williamson et al 2004), S. stercoralis (Gallego et al 1998), Haemonchus contortus (Longbottom et al 1997) and Onchocerca volvulus (Jolodar et al 2004) and in the free-living nematode Caenorhabditis elegans (Geier et al 1999). In the hookworms, these proteases play an important role in haemoglobin digestion in the intestine and in tissue degradation during the larvae migration in the mammalian host (Williamson et al 2003).…”

Improvement of Crystal Solubility and Increasing Toxicity against Caenorhabditis elegans by Asparagine Substitution in Block 3 of Bacillus thuringiensis Crystal Protein Cry5Ba

“…Consistent with this, our pH assessment with different acid-base indicators (unpublished data) suggested that the pH value is ϳ5.0 in the intestine of the nematode C. elegans. The pH optimum for most protease activity in intestinal crude extracts from C. elegans was found to be ϳ5 (17,42), implying the possibility of a mildly acidic pH in the intestinal lumen (31).…”

Affinity-Based Methods for the Separation of Parasite Proteins

“…microplus (Cruz et al, 2010), 60 kDa and 30 kDa from Trichomonas vaginalis (Sommer et al; and 35 to 52 from Caenorhabditis elegans (Geier et al;.…”