M. Basu et al., PURIFICATION AND CHARACTERIZATION OF HUMAN RECOMBINANT IGE-FC FRAGMENTS THAT BIND TO THE HUMAN HIGH-AFFINITY IGE RECEPTOR, The Journal of biological chemistry, 268(18), 1993, pp. 3118-3127
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.