Purification, molecular characterization and gene expression analysis of an aspartic protease (Sc-ASP113) from the nematode Steinernema carpocapsae during the parasitic stage

Abstract

Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. During invasion, this nematode is able to express many proteases, including aspartic proteases. Genes encoding these aspartic proteases have been identified in the EST, and aspartic protease has been found in excretory–secretory products. The total protease was shown to digest blood hemoglobin in a zymogram gel. When the protein was partially purified by pepstatin affinity chromatography, it was observed to have high activity against both hemoglobin and the synthetic substrate Phe-Ala-Ala-Phe-(4NO2)-Phe-Val-Leu (4-pyridylmethyl) ester. The protein was confirmed by mass spectrometry and was found to be encoded by the gene sc-asp113. A cDNA encoding aspartic protease was cloned based on the EST fragment, which was constructed in our lab. The full-length cDNA of Sc-ASP113 consists of 1257 nucleotides encoding a protein with multiple domains, including a signal peptide (aa 1–15), a propeptide region (aa 16–45), and a typical catalytic aspartic domain (aa 68–416). The cleavage site of the signal peptide is predicted to be between Ala15 and Ala16. The putative 418 amino acid residues have a calculated molecular mass of 44,742Da and a theoretical pI of 5.14. BLAST analysis showed 33–56% amino acid sequence identity to aspartic proteases from parasitic and free living nematodes. Expression analysis showed that the sc-asp113 gene was up-regulated during the initial parasitic stage, especially during L3 inside the gut. In vitro, we showed that treatment with insect homogenate for 6h is sufficient to induce the expression of this protease in treated infective juveniles. Sequence comparison and evolutionary analysis revealed that Sc-ASP113 is a member of the aspartic protease family with the potential for tissue degradation. Phylogenetic analysis indicates that Sc-ASP113 branched between Haemonchus contortus and Steinernema feltiae proteases. Homology modeling showed that Sc-ASP113 adopts a typical aspartic protease structure. The up-regulation of Sc-ASP113 expression indicates that this protease could play a role in the parasitic process. To facilitate the exploration of this protease as a virulence factor, here we describe the purification of the protease and its molecular characterization in S. carpocapsae.