Structural basis of the beta1-adrenoreceptor agonist-like properties of antibodies against the C-terminal end of the Trypanosoma cruzi Ribosomal P proteins.

C. SMULSKI; V. LABOVSKY; G. LEVY; L. SIMONETTI; V. GRIPPO; K. GÓMEZ; M.J. LEVIN

Ashburn, Virginia, USA.

Simposio; HHMI Meeting of International Research Scholars.; 2006

Howard Hughes Medical Institute (HHMI)

Human antibodies from patients with Chagas heart disease and monoclonal antibodies against the carboxy-terminal end (B cell epitope R13) of the ribosomal P2b protein of Trypanosoma cruzi (TcP2b) have been found to cross-react and stimulate the b1 adrenergic receptor (b1-AR). To study the structural basis of this cross-reactivity, the variable regions of the anti-R13 monoclonal antibody mAb17.2 were cloned, sequenced, and used to express four different scFv constructs in Escherichia coli. Two of them were characterized for their physico-chemical and immunochemical properties. We showed that scFvC5 is a dimer and binds to TcP2b with an affinity of Kd=8 nM, whereas scFvB7 is monomeric and exhibits decreased affinity to TcP2b (Kd=46 Nm). Interestingly, while C5 induces an increase in cAMP levels in CHO cells transfected with the human b1 adrenoceptor, B7 has no intrinsic effect on these cells but blocks isoproterenol-induced stimulation. The agonist-like activity of scFvC5 and the antagonist activity of scFvB7 were both confirmed in vitro in neonatal rat cardiomyocytes and in vivo in the beating frequency of heart after passive transfer in mice. We demonstrated also that scFvC5 and scFvB7 cross-react with rhodopsin. Interestingly, passive   transfer allowed us to follow by electro-retinography the effect of the clones on animal vision. Our results suggest that scFvC5 blocks rhodopsin function by inducing a selective dysfunction of the rods. Molecular modeling of the variable region of mAb17.2 pinpointed the amino acids that are critical for recognition of the parasite peptide EDDDMGFGLF of TcP2b, the peptide ESDEARRCYN of the second extracellular loop of the b1-AR, and a similar region of the second extracellular loop of rhodopsin. These results substantiate the structural basis for the cross-reaction of anti-TcP2b antibodies with G protein–coupled receptors and stress the importance of mono- or bivalent recognition in the antibodies’ physiological effects.