DELINEATION OF THE AMINO-ACID-RESIDUES INVOLVED IN TRANSCYTOSIS AND CATABOLISM OF MOUSE IGG1

The MHC class I-related receptor, FcRn, is involved in both the transc ytosis of serum gamma-globulins (IgGs) and in regulating their serum p ersistence. The interaction site of FcRn on the Fc region of rodent Ig G has been mapped to residues at the CH2-CH3 domain interface using si te-directed mutagenesis and x-ray crystallographic analyses. In the cu rrent study, the role of individual residues (H310, H433, and N434) at this interface in mediating the Fc-FcRn interaction has been investig ated using recombinant, mutated Fc hinge fragments derived from mouse IgG1. In addition, two highly conserved Fc histidines (H435 and H436) have been mutated to alanine, and the resulting mutated Fc hinge fragm ents were analyzed in both transcytosis and pharmacokinetic studies in mice and in competition binding assays using recombinant, soluble FcR n. The analyses indicate that mutation of H310, H435, and, to a lesser extent, H436 to alanine results in reduced activity of the re hinge f ragments in both in vivo and in vitro assays. Thus, in addition to the previously defined role of 1253 in the FcRn-IgG interaction, these hi stidines play a key role in mediating the functions conducted by this Fc receptor. The effects of these mutations on binding of Fc hinge fra gments to staphylococcal protein A have also been analyzed and demonst rate a partial, but not complete, overlap of the FcRn and staphylococc al protein A interaction sites on mouse IgG1.