PURIFICATION AND CHARACTERIZATION OF HUMAN RECOMBINANT IGE-FC FRAGMENTS THAT BIND TO THE HUMAN HIGH-AFFINITY IGE RECEPTOR

The Fc-region of immunoglobulin E (IgE) comprising C(epsilon)2, C(epsi lon)3, and C(epsilon)4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (FcepsilonRIalpha). In orde r to identify the smallest Fc fragment capable of binding to the Fceps ilonRIalpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bin d FcepsilonRIalpha. The smallest fragment that showed FcepsilonRIalpha binding activity spans amino acids 329-547 and lacks the entire C(eps ilon)2 domain. Two active fragments, viz. Fc(epsilon)(315-547) (contai ning Cys328 which is responsible for interchain S-S bonding) and Fc(ep silon)(329-547), have been overexpressed in CHO cells and purified to homogeneity. The purified proteins bind to the FcepsilonRIalpha with h igh affinity, similar to native IgE. SDS-polyacrylamide gel electropho resis analyses indicate that Fc(epsilon)(315-547) is an S-S-linked dim er of apparent molecular mass of 68 kDa. Fc(epsilon)(329-547) appears on SDS-gel as three distinct bands at approximately 32 kDa, both under reducing and nonreducing conditions. However, size exclusion chromato graphy and analytical ultracentrifugation studies suggest that Fc(epsi lon)(329-547) also remains associated as a dimer. The presence of N-li nked glycosylation was detected in both proteins. The deglycosylated f orm of Fc(epsilon)(315-547) was isolated after Endo F/N-glycosidase F digestion and demonstrated to have binding activity comparable to that of the mock-digested protein. These results suggest that the presence of N-linked sugars is not necessary for FcepsilonRIalpha binding. Bot h proteins blocked the release of histamine from RBL cells expressing human FcepsilonRIalpha in a dose-dependent manner. The availability of these recombinant IgE-Fc proteins will be helpful in elucidating the key epitopes essential for the binding of IgE to its high affinity rec eptor.