title: Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor.
abstract: The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and C epsilon 4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and lacks the entire C epsilon 2 domain. Two active fragments, viz. Fc epsilon(315-547) (containing Cys328 which is responsible for interchain S-S bonding) and Fc epsilon(329-547), have been overexpressed in CHO cells and purified to homogeneity. The purified proteins bind to the Fc epsilon RI alpha with high affinity, similar to native IgE. SDS-polyacrylamide gel electrophoresis analyses indicate that Fc epsilon(315-547) is an S-S-linked dimer 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 chromatography and analytical ultracentrifugation studies suggest that Fc epsilon(329-547) also remains associated as a dimer. The presence of N-linked glycosylation was detected in both proteins. The deglycosylated form 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 Fc epsilon RI alpha binding. Both proteins blocked the release of histamine from RBL cells expressing human Fc epsilon RI alpha 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 receptor.
authors.affiliation: Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
authors.forename: M
authors.initials: M
authors.lastname: Basu
authors.forename: J
authors.initials: J
authors.lastname: Hakimi
authors.forename: E
authors.initials: E
authors.lastname: Dharm
authors.forename: J A
authors.initials: JA
authors.lastname: Kondas
authors.forename: W H
authors.initials: WH
authors.lastname: Tsien
authors.forename: R S
authors.initials: RS
authors.lastname: Pilson
authors.forename: P
authors.initials: P
authors.lastname: Lin
authors.forename: A
authors.initials: A
authors.lastname: Gilfillan
authors.forename: P
authors.initials: P
authors.lastname: Haring
authors.forename: E H
authors.initials: EH
authors.lastname: Braswell
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E