Studies on the neonatal Fc receptor: FcRn (Placental transfer)

The neonatal Fc receptor FcRn binds immunoglobulin G (IgG). It appears to have two functions. It mediates maternofetal transport of IgG across a number of tissues, such as the placenta in humans, and the intestine of neonatal rats and mice. FcRn is also involved in maintaining serum IgG levels. In numerous tissues of the adult, FcRn binds IgG and returns it to circulation. FcRn binds monomeric IgG at an acidic pH, with nanomolar affinity at pH 6.0. At neutral pH the receptor has a very low affinity for IgG. We tested the binding of the human IgG subclasses to the receptor. I found that FcRn will bind all human IgG subclasses. I calculated the binding affinities for each subclass. Each subclass binds FcRn with high affinity, and we observed a binding range of about 1–100 nanomolar. I investigated the impact of the Cγ2 carbohydrate on FcRn binding, and calculated approximate affinities for the Fc fragment of several IgG4 glycoforms. I observed a correlation with the measured binding affinities and the composition of the carbohydrate, but the effect on binding appears to be minor. This thesis also describes several other experiments unrelated to the investigations on binding of human IgG. I attempted to use a yeast two-hybrid screen to isolate proteins that bind the cytoplasmic domain of the rat receptor. I created a number of plasmid constructs that overexpress FcRn cytoplasmic tail as a fusion protein. I describe the purification of these fusion proteins, and their use in in vitro binding experiments attempting to isolate tail binding proteins. I describe a series of co-immunoprecipitation experiments and affinity purification experiments attempting to isolate and characterize potential tail binding proteins.