Enhancement of M2 muscarinic receptor-receptor interaction by IgG antibodies in Chagas’disease: Role of epitope specificity and antibody valency

BELTRAME, SABRINA P.; BILDER, CLAUDIO R.; AUGER, SEGIO R.; WALDNER, CLAUDIA I.; GOIN, JUAN C.

Buenos Aires

Congreso; I French-Argentine Immunology Congress; 2010

Sociedad Argentina de Inmunología, French Society of Immunology

In previous reports we showed that circulating IgG antibodies (Ab) from Chagas’disease (ChD) patients can enhance M2 muscarinic receptor-receptor interaction. The aim of this study is to assess the role of epitope specificity and Ab valency in this modulatory effect. HEK 293 cells expressing fusion proteins derived from the M2 muscarinic receptor (M2R) (M2R-RLuc and M2R-YFP) were treated with the IgG fraction from ChD patients (ChD IgG), affinity purified Ab against the second extracellular loop (II-ECL) of the human M2R (anti-M2II-ECL IgG), or the Fab fragments from ChD IgG (ChD Fab), at a 50 µM concentration, and M2 receptor-receptor interaction was assessed by bioluminescence resonance energy transfer (BRET). The anti-M2II-ECL Ab fraction induced an increase in BRET (milibrets: mB) (16.2 ± 2.1 mB) which was similar to that promoted by ChD IgG (13.1 ± 3.2 mB) and significantly higher than the activity of ChD IgG depleted from anti-M2II-ECL Ab (0.2 ± 0.6 mB) or IgG from control subjects (0.3 ± 0.3 mB) (p<0.001). In BRET assays on membranes at low ionic strength buffer conditions, gallamine (100 µM) (a muscarinic allosteric modulator which binds to the II-ECL of the M2R), but not atropine (10 µM) (a muscarinic antagonist which interacts with the orthosteric binding pocket), impaired the effect of ChD IgG on BRET by 56% (p<0.05). Unlike the native ChD IgG, its Fab fragment did not promote an enhancement of BRET on our M2R-RLuc/M2R-YFP cell system (ChD Fab: 1.8 ± 0.56 mB; ChD IgG: 7.5 ± 1.0 mB) (p<0.05). However, incubation of cells with ChD Fab in the presence of an anti-human Fab Ab promoted a recovery of the original effect (ChD Fab + anti-human Fab: 8.6 ± 0.5 mB), indicating that the effect of anti-M2R Ab on BRET requires the integrity of the IgG molecule. Taken together, our data suggest that the enhancement of M2 receptor-receptor interaction by ChD-IgG occurs as a result of receptor crosslinking by bivalent antibodies directed against the II-ECL of the M2R