A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxyl-terminal domain is active, monomeric, and generates antibodies that inhibit enzymatic activity.
trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is multimeric and heterogeneous in size. We show here that limited proteolysis of tans-sialidase with papain yields a single monomeric polypeptide chain of 70 kDa that conserves full enzymatic activity on soluble and membrane-bound substrates. The papain fragment lacks most of the 12-amino acid repeats of the carboxyl-terminal domain that comprises about 50% of the native trans-sialidase. When injected into rabbits, the papain-generated fragment induces antibodies that inhibit trans-sialidase activity and trypomastigote sialylation. The repeats are also not required for the stability of the enzyme or for the correct folding during the biosynthesis in Escherichia coli, but seem essential for trans-sialidase oligomerization. We conclude that trans-sialidase is composed of two structurally and functionally independent domains.
Schenkman
S
S
Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Sao Paulo, Brazil.
Chaves
L B
LB
Pontes de Carvalho
L C
LC
Eichinger
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology* / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010957:Plasmids; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014349:Trypanosoma cruzi / Q000201:enzymology*
D000906:Antibodies; D017931:DNA Primers; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D009439:Neuraminidase