Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.
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.
Authié
E
E
International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
Muteti
D K
DK
Mbawa
Z R
ZR
Lonsdale-Eccles
J D
JD
Webster
P
P
Wells
C W
CW
D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan / Q000737:chemistry / Q000302:isolation & purification*; D003546:Cysteine Endopeptidases / Q000276:immunology*; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D013045:Species Specificity; D014348:Trypanosoma congolense / Q000201:enzymology* / Q000276:immunology* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D003546:Cysteine Endopeptidases