Characterization of a novel cathepsin L-like protease from Taenia solium metacestodes for the immunodiagnosis of porcine cysticercosis

Abstract

Porcine cysticercosis is an endemic parasitic disease caused by infection with Taenia solium that is found predominantly in developing countries. In order to aid in the development of simple diagnostic approaches, identification and characterization of potential new antigens for immunodiagnostic purposes is desired. The cysteine protease family has previously been found to have important immunodiagnostic properties. These proteases are expressed as zymogens which contain a signal peptide, pro-peptide, and an active domain. Subsequent catalytic cleavage of the pro-peptide converts these zymogens into enzymes. With the use of bioinformatic tools we identified an active domain of a novel cathepsin L-like cysteine protease (TsolCL) in the T. solium genome. The TsolCL gene includes 705 nucleotides (nt) within a single intron and a 633 nt exonic sequence encoding an active protein of 211 amino acids. Sequence alignment and phylogenetic analysis suggest that the TsolCL gene is closely related to genes found in Echinoccocus granulosus and E. multiloculars. In addition, TsolCL was found to have a 61.9%–99.0% similarity to other cathepsin L proteins found in other helminths and mammals. We cloned, expressed, purified, and characterized the recombinant active TsolCL (27 kDa) using the baculovirus-insect cell expression system. TsolCL showed cysteine protease enzymatic activity with the capacity to hydrolyze the Z-Phe-Arg-AMC substrate as well as bovine serum albumin. However, TsolCL was not able to hydrolyze human immunoglobulin. In addition, TsolCL has cathepsin L conserved amino acid residues in the catalytic site (Gln8, Cys14, His159, Asn179 and Trp181) and the motif GCNGG. Using ELISA, TsolCL was able to distinguish circulating IgG antibodies between healthy animals and naturally infected pigs with cysticercosis, showing a moderate sensitivity of 83.33% (40/48; 95% CI: [69.8%–92.5 %]), and a specificity of 83.78% (31/37; 95% CI: [67.9%–93.8%]). In conclusion, a novel cathepsin L-like cysteine protease from a T. solium metacestode was expressed successfully in Baculovirus system and was evaluated as a candidate antigen to diagnose porcine cysticercosis using the ELISA immunoassay.