ANALYSIS OF THE PH-DEPENDENCE OF THE NEONATAL FC RECEPTOR IMMUNOGLOBULIN-G INTERACTION USING ANTIBODY AND RECEPTOR VARIANT

The neonatal Fc receptor (FcRn) binds maternal immunoglobulin G (IgG) from ingested milk in the gut (pH 6.0-6.5) and delivers it to the bloo dstream of the newborn (pH 7.0-7.5). A soluble version of FcRn reprodu ces the physiological pH-dependent interaction with IgG, showing high- affinity binding at pH 6.0-6.5 but weak or no binding at pH 7.0-7.5. W e have studied the pH dependence of the FcRn/IgG interaction using a s urface plasmon resonance assay to measure kinetic and equilibrium cons tants. We show that the affinity of FcRn for IgG is reduced about 2 or ders of magnitude as the pH is raised from 6.0 to 7.0. A Hill plot ana lysis suggests that several titrating residues participate in the pH-d ependent affinity transition. Histidine side chains are likely candida tes for residues that titrate between pH 6.0 and 7.0, and previous bio chemical and structural work identified several histidines on the Fc p ortion of IgG that are located at the FcRn binding site. Using mutant Ige molecules and IgG subtype variants that differ in the number of hi stidines at the IgG/FcRn interface, we demonstrate that IgG histidines located at the junction between the C(H)2 and C(H)3 domains (residues 310 and 433) contribute to the pH-dependent affinity transition. Expe riments with a mutant FcRn molecule show that two histidines on the Fc Rn heavy chain (residues 250 and 251) also contribute to the pH depend ence of the FcRn/IgG interaction. These results are interpreted using the crystal structures of FcRn and an FcRn/Fc complex.