Structural basis of the b1 adrenoreceptor agonist-like properties of antibodies against the C-terminal end of the Trypanosoma cruzi ribosomal P protein

SMULSKI CR; VIVIAN LABOVSKY; G. V. LEVY; SIMONETTI L; GRIPPO VANINA; GÓMEZ KA; LEVIN MJ

Congreso; HHMI Meeting of International Research scholars; 2006

Human antibodies from patients with Chagas heart disease and monoclonal antibodies (or mAb) to the carboxy-terminal end (B cell epitope R13) of the ribosomal P2b protein of Trypanosoma cruzi (TcP2b) crossreact with the b1 adrenergic receptor (b 1-AR). Two single chain Fv fragments (scFv) C5 and B7 derived from the variable regions of the anti-R13 mAb 17.2 were expressed. scFv C5 was a dimer and bound to TcP2b with an affinity  of Kd 8 nM, whereas scFv B7 was monomeric and had less affinity than scFv C5 for TcP2b, Kd 46 nM. The affinity constant of scFv C5 to the second extracellular loop of the human b1-AR was of 10 uM. Moreover, scFv C5 induced an increase in cAMP levels of CHO-K cells transfected with the human b1-AR; scFv B7 had no effect but blocked isoproterenol stimulation. The agonist-like activity of scFv C5 and the antagonist activity of scFv B7 were both confirmed in vivo on heart beating frequency after their passive transfer to mice. Molecular modeling of the variable region of mAb 17.2 indicated which amino acids were likely to be involved in recognizing both peptide EDDDMGFGLF, derived from the R13 epitope of TcP2b, and peptide ESDEARRCYN from the second extracellular loop of the human b1-AR. It is plausible that the recently described cross-reaction of mAb 17.2 with rhodopsin can also be explained by this model. The physiological effects of this type of anti-T. cruzi antibodies may increase the liability of patients with Chagas disease protein of Trypanosoma cruzi (TcP2b) crossreact with the b1 adrenergic receptor (b 1-AR). Two single chain Fv fragments (scFv) C5 and B7 derived from the variable regions of the anti-R13 mAb 17.2 were expressed. scFv C5 was a dimer and bound to TcP2b with an affinity  of Kd 8 nM, whereas scFv B7 was monomeric and had less affinity than scFv C5 for TcP2b, Kd 46 nM. The affinity constant of scFv C5 to the second extracellular loop of the human b1-AR was of 10 uM. Moreover, scFv C5 induced an increase in cAMP levels of CHO-K cells transfected with the human b1-AR; scFv B7 had no effect but blocked isoproterenol stimulation. The agonist-like activity of scFv C5 and the antagonist activity of scFv B7 were both confirmed in vivo on heart beating frequency after their passive transfer to mice. Molecular modeling of the variable region of mAb 17.2 indicated which amino acids were likely to be involved in recognizing both peptide EDDDMGFGLF, derived from the R13 epitope of TcP2b, and peptide ESDEARRCYN from the second extracellular loop of the human b1-AR. It is plausible that the recently described cross-reaction of mAb 17.2 with rhodopsin can also be explained by this model. The physiological effects of this type of anti-T. cruzi antibodies may increase the liability of patients with Chagas disease