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.