The beta1 adrenergic effects of antibodies against the C-terminal end of the ribosomal P2beta protein of Trypanosoma cruzi associate with a specific pattern of epitope recognition

Abstract

BALB/c mice immunized with recombinant Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) develop a strong and specific antibody response against its 13 residue-long C-terminal epitope (peptide R13: EEEDDDMGFGLFD) that has a concomitant beta1-adrenergic stimulating activity. However, other animals that undergo similar immunizations seem tolerant to this epitope. To evaluate further the antibody response against the ribosomal P proteins, 25 BALB/c and 25 Swiss mice were immunized with TcP2beta. From the 50 animals, 31 developed a positive anti-R13 response, whereas 19 were non-responsive. From the 31 anti-R13 positive mice, 25 had anti-R13 antibodies that recognized the discontinuous motif ExDDxGF, and their presence correlated with the recording of supraventricular tachycardia. The other six had anti-R13 antibodies but with a normal electrocardiographic recording. These anti-R13 antibodies recognized the motif DDxGF shared by mammals and T. cruzi and proved to be a true anti-P autoantibody because they were similar to those elicited in Swiss, but not in BALB/c mice, by immunization with the C-terminal portion of the mouse ribosomal P protein. Our results show that the recognition of the glutamic acid in position 3 of peptide R13 defines the ability of anti-R13 antibodies to react with the motif AESDE of the second extracellular loop of the beta1-adrenergic receptor, setting the molecular basis for their pathogenic beta1 adrenoceptor stimulating activity.

Fig. 2

Functional effect of anti-P antibodies from BALB/c mice immunized with TcP2β. Chronotropic effect on neonatal rat cardiomyocytes of IgGs from mice displaying R13⁺/C10^– (a) or R13⁺/C10⁺ (c) profile. The effect of the antibodies was also assessed in the presence of the muscarinic acethylcholine antagonist atropine, β-adrenergic antagonist bisoprolol or after preincubation with H26R or R13 peptides. Mean and s.e. from 10 observations are given. Results show the increase in beats per minute with respect to the baseline beating rate from two representative serum samples from each group. Representative electrocardiograms from mice displaying R13⁺/C10^– (b) or R13⁺/C10⁺ profile (d).

Fig. 1

Reactivity to TcP2β and C-terminal peptides in sera from mice immunized with MBP-TcP2β. Sera (1 : 200 dilution) from BALB/c (B sera) and Swiss (S sera) mice immunized with MBP-TcP2β were assayed against the recombinant protein GST-TcP2β (white bars) and against peptides R13 (black bars), H13 (hatched bars) and C10 (grey bars). The optical density reading at 405 nm is shown. (a) Mice positive for TcP2β, R13 and H13 and negative for C10 (Chagas’-like phenotype); (b) mice positive for TcP2β and negative for R13, H13 and C10 (non-responsive phenotype) and (c) mice positive for TcP2β, R13, H13 and C10 (autoimmune phenotype).

Fig. 4

Reactivity to the MmP0 and C-terminal peptides in sera from mice immunized with MBP-MmP0. Sera (1 : 200 dilution) from BALB/c (a) and Swiss (b) mice immunized with MBP-MmP0 were assayed against the recombinant protein GST-MmP0 and against peptides R13, H13 and C10. The optical density reading at 405 nm is shown. Results from Swiss mice were grouped on profiles C10⁺ and R13^–. (c) Representative electrocardiogram from mice immunized with MmP0 displaying C10⁺ profile. (d) Electrocardiogram from normal BALB/c mouse.

Fig. 3

Evolution of the reactive profile along the immunization protocol. Serial serum samples (1 : 200 dilution) from BALB/c mice immunized with TcP2β were assayed by enzyme-linked immunosorbent assay (ELISA) for reactivity against peptides R13 (▪), H13 (□) and C10 (*). Results from a representative mouse from both the R13⁺/C10^– (a) and R13⁺/C10⁺ (b) profiles are plotted. The ratio R13/H13 at each bleed is also shown.

Fig. 5

Alanine scanning mutagenesis patterns of anti-P antibodies.The indicated amino acids of R13 were replaced by Ala and the reactivity of the mutated peptides was assayed using polyclonal C10⁺ or R13⁺ sera from mice immunized with TcP2β or MmP0. Amino acids involved in antibody binding are in capital letters.