Analysis of the pH dependence of the neonatal Fc receptor/immunoglobulin G interaction using antibody and receptor variants.
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 bloodstream of the newborn (pH 7.0-7.5). A soluble version of FcRn reproduces 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. We have studied the pH dependence of the FcRn/IgG interaction using a surface plasmon resonance assay to measure kinetic and equilibrium constants. We show that the affinity of FcRn for IgG is reduced about 2 orders of magnitude as the pH is raised from 6.0 to 7.0. A hill put analysis suggests that several titrating residues participate in the pH-dependent affinity transition. Histidine side chains are likely candidate for residues that titrate between pH 6.0 and 7.0, and previous biochemical and structural work identified several histidines on the Fc portion of IgG that are located at the FcRn binding site. Using mutant IgG molecules and IgG subtype variants that differ in the number of histidines at the IgG/FcRn interface, we demonstrate that IgG histidines located at the junction between the CH2 and CH3 domains (residues 310 and 433) contribute to the pH-dependent affinity transition. Experiments with a mutant FcRn molecule show that two histidines on the FcRn heavy chain (residues 250 and 251) also contribute to the pH dependence of the FcRn/IgG interaction. There results are interpreted using the crystal structures of FcRn and an FcRn/Fc complex.
Raghavan
M
M
Division of Biology 1569-29, California Institute of Technology, Pasadena 911254, USA.
Bonagura
V R
VR
Morrison
S L
SL
Bjorkman
P J
PJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015374:Biosensing Techniques; D003196:Computer Graphics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007231:Infant, Newborn; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011961:Receptors, Fc / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011961:Receptors, Fc; D006639:Histidine