Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae.

ABSTRACT

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.