Structural and functional complexity of the humoral response against the Trypanosoma cruzi ribosomal P2 beta protein in patients with chronic Chagas' heart disease

Abstract

High levels of antibodies against the C-terminus of the Trypanosoma cruzi TcP2 beta ribosomal protein, defined by the peptide EEEDDDMGFGLFD, named R13, have been measured in sera from patients with chronic Chagas' Heart Disease (cChHD). These antibodies also recognize an epitope on the second extracellular loop of the beta 1-adrenergic receptor, inducing a functional response on cardiomyocytes. The aim of this study was to gain novel insights into the structural basis of this cross-reactivity as well as to evaluate the origin of anti-M2- cholinergic receptor antibodies, which are also commonly found in cChHD patients. To address these questions we immunopurified anti-R13 antibodies and studied the structural requirements of epitope recognition. Results showed that the immunopurified antibodies recognized a conformation of R13 in which the third Glu residue was essential for binding, explaining their low affinity for the mammalian homologue (peptide H13: EESDDDMGFGLFD). Alanine mutation scanning showed individual variations in epitope recognition in each of the studied patients. The importance of a negatively charged residue at position 3 for the recognition of anti-R13 antibodies was further confirmed by competition experiments using a Ser3-phosphorylated H13 analogue, which had 10 times more affinity for the anti-R13 antibody than the native H13 peptide. Moreover, anti-R13 antibodies stimulated either the beta 1-adrenergic or the M2-cholinergic receptor, in strict agreement with the functional properties of the IgG fractions from which they derived, demonstrating that the same parasite antigen may generate antibody specificities with different functional properties. This may be a clue to explain the high variability of electrophysiological disturbances found in cChHD.

Fig. 1

(a) Functional effect on spontaneously beating rat cardiomyocytes of IgG fractions from cChHD (▪), patients with other non-Chagas cardiomyopathies () and healthy individuals (□). Bars represent change in beating rate caused by addition of IgG fraction from each individual. All results represent one from at least three independent experiments. (b) Chronotropic effect of IgG fractions (▪) among the cChHD population. The specificity of the effect was studied by subsequent addition of the M2-ChR antagonist atropine (□) and the β1-AR antagonist bisoprolol ().VA, Ventricular arrhythmias; SND, Sinus node dysfunction; HC, Healthy Controls.

Fig. 2

(a) Prevalence of anti-R13 antibodies as determined by ELISA in a cChHD population (n = 123), compared to circulating antibody levels in sera from patients with other non-Chagas cardiomyopathies (n = 103) and healthy individuals (n = 17). Insert: Ratio values distribution of antibody levels among the studied groups (for details see Materials and methods). (b) Correlation between severity of heart disease and circulating antibody levels against R13 and T. cruzi epimastigote lysate. Rhomboids represent mean ratio values for each studied group: CH group I (n = 15): severe chronic Chagas cardiomyopathy (♦), CH group III (n = 6): mild chronic Chagas cardiomyopathy (), and CH group IV (n = 3): preclinical chronic Chagas cardiomyopathy with no ECG disturbances (e). Error bars represent standard deviation. Significance of the differences in mean antibody levels between CH group I and CH groups III + IV were determined applying the Wilcoxon test (P < 0·05).

Fig. 3

(a) Immunoblotting of T. cruzi epimastigotes lysate with the IgG fractions of the eight patients that were selected to purify anti-R13 antibodies. (b) Chronotropic effect of total IgG fractions of eight patients with high anti-R13 antibodies levels. Effect of the IgG fraction (), as well as antagonizing effects of 10 µm atropine (□) and 1 µm bisoprolol () were evaluated. (c) Immunoblotting of T. cruzi epimastigote lysate with the eight purified anti-R13 antibodies. The monoclonal antibody mAb17·2 was used as positive control. Arrows indicate the position of the T. cruzi ribosomal P0 protein and the low molecular weight ribosomal P proteins (TcP1, TcP2α and TcP2β) (d) Chronotropic effect of the corresponding immunopurified purified anti-R13 antibodies (). Bars represent mean ± SD of the change in beats/min; *significant differences (P < 0·001) with respect to the preceding bar.

Fig. 4

(a) Comparison of the R13 epitope recognized by the different immunopurified anti-R13 antibodies. Each amino acid of R13 was replaced in turn to alanine with the Spots® method (Materials and Methods) and the anti-R13 reactivity was evaluated. In the table, amino acids essential for recognition are represented with the corresponding letters whereas residues nonessential for recognition are symbolized with –. †The pharmacological effect of each purified antibody as evaluated in Fig. 3 is detailed for comparison; β1, β1-AR stimulating effect; M2, M2-ChR stimulating effect. (b,c) Number of antibodies sensitive to alanine mutation evaluated as the number of anti-R13- antibodies with β1-AR (b), or M2-ChR receptor (c) stimulating properties, that fail to recognize R13 when substituted by alanine at the corresponding position. Abs, antibodies.