title: Participation of the N-terminal region of Cepsilon3 in the binding of human IgE to its high-affinity receptor FcepsilonRI.
abstract: The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.
authors.affiliation: The Randall Institute, King's College London, 26-29 Drury Lane, London, WC2B 5RL, United Kingdom.
authors.forename: A J
authors.initials: AJ
authors.lastname: Henry
authors.forename: J P
authors.initials: JP
authors.lastname: Cook
authors.forename: J M
authors.initials: JM
authors.lastname: McDonnell
authors.forename: G A
authors.initials: GA
authors.lastname: Mackay
authors.forename: J
authors.initials: J
authors.lastname: Shi
authors.forename: B J
authors.initials: BJ
authors.lastname: Sutton
authors.forename: H J
authors.initials: HJ
authors.lastname: Gould
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007139:Immunoglobulin epsilon-Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014162:Transfection
chemical_list: D007141:Immunoglobulin Fc Fragments; D007139:Immunoglobulin epsilon-Chains; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E