Mapping of a B-cell epitope present in the neuraminidase of Trypanosoma cruzi.
We have previously shown that a polyclonal (rabbit anti-TCNA) and a mouse monoclonal antibody (TCN-2) against the neuraminidase of Trypanosoma cruzi (TCNA) inhibit enzyme activity, immunoprecipitate active enzyme, enhance in vitro infection, and identify a subpopulation of extracellular trypomastigotes. We now report on the identification of a synthetic peptide that contains the epitope recognized by these antibodies. The synthetic peptide (TR) is a dodecamer (D-S-S-A-H-G-T-P-S-T-P-A) deduced from the DNA sequence of the long tandem repeat (LTR) domain present in the TCNA carboxyterminus. By ELISA, rabbit anti-TCNA bound to TR coupled to ovalbumin, and the binding was inhibited by soluble TR but not by BR (Y-S-V-D-D-G-E-T-W-E), a peptide derived from the N-terminal domain of the enzyme. TCN-2 recognized TR, and this reaction as well as TCN-2 binding to endogenous TCNA could be inhibited by soluble TR but not by BR. These results indicate that the rabbit anti-TCNA and TCN-2 react with the LTR region of TCNA. Antibodies to TR reacted by immunoblot with the TCNA of the Silvio X-10/4, MV-13 and Y-H6 strains, identifying the same molecular polymorphism previously observed with the rabbit anti-TCNA and TCN-2. Furthermore, anti-TR antibodies immunoprecipitated active enzyme and immunofluorescence analysis revealed that anti-TR and TCN-2 antibodies detected equally well the differential expression of their epitopes in intra- and extracellular trypomastigotes. Moreover, expression of TR and TCN-2 epitopes on the different stages of T. cruzi paralleled the stage-specificity of TCNA activity. TCN-2 prevented desialylation by TCNA of intact cells but not of soluble glycoconjugates, indicating that TCN-2 epitope is probably not associated with the enzyme catalytic site, in agreement with the predicted sequence of the TCNA gene. Finally, analysis of the humoral response of a Chagasic patient to different areas of the TCNA molecule indicated that the antibody response is predominantly against TR suggesting that the tandem repeat is the immunodominant domain of TCNA.
Prioli
R P
RP
New England Medical Center Hospitals, Department of Geographic Medicine and Infectious Diseases, Boston, MA 02111.
Ortega-Barria
E
E
Mejia
J S
JS
Pereira
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000276:immunology* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011233:Precipitin Tests; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000276:immunology; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D009439:Neuraminidase