Identification of a 33-kilodalton immunodominant antigen of Trypanosoma cangolense as acysteine protease

• Published in: Molecular and biochemical parasitology Vol. 56; no. 1; pp. 103 - 116
• Main Authors: Authié, Edith, Muteti, David K., Mbawa, Zeres R., Lonsdale-Eccles, John D., Webster, Paul, Wells, Clive W.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1992
• Subjects: Antigen; Cysteine protease; Trypanosoma congolense; PBS; SDS; IFA; PSG; E-64; HRP; kDa; Fbg; PAGE; 2D IEF/SDS-PAGE; mAb; Antigen; pAb; BSA; Cysteine protease; Trypanosoma congolense; Z-Phe-Arg-NHMec; Mes; Ig; CFA; ELISA
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/0166-6851(92)90158-G

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl- l-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.