Amino acid residues that influence Fc epsilon RI-mediated effector functions of human immunoglobulin E.
Immunoglobulin E (IgE) mediates its effector functions via the Fc region of the molecule. IgE binding to and subsequent aggregation of the high-affinity receptor (Fc epsilon RI) by allergen plays a pivotal role in type I hypersensitivity responses. Earlier studies implicated the C epsilon 2 and 3 interface and the A-B loop in C epsilon 3 in the IgE-Fc epsilon RI interaction. These regions and glycosylation sites in C epsilon 3 were now targeted by site-specific mutagenesis. IgE binding to Fc epsilon RI was compared with surface plasmon resonance (SPR) measurements, which assessed the binding of the soluble extracellular domain of Fc epsilon RI to IgE. Kinetic analysis based on a pseudo-first-order model agrees with previous determinations. A more refined SPR-based kinetic analysis suggests a biphasic interaction. A model-free empirical analysis, comparing the binding strength and kinetics of native and mutant forms of IgE, identified changes in the kinetics of IgE-Fc epsilon RI interaction. Conservative substitutions introduced into the A-B loop have a small effect on binding, suggesting that the overall conformation of the loop is important for the complementary interaction, but multiple sites across the C epsilon 3 domain may influence IgE-Fc epsilon RI interactions. Asn394 is essential for the generation of a functional IgE molecule in mammalian cells. A role of Pro333 in the maintenance of a constrained conformation at the interface between C epsilon 2-3 emerged by studying the functional consequences of replacing this residue by Ala and Gly. These substitutions cause a dramatic decrease in the ability of the ligand to mediate stimulus secretion coupling, although only small changes in the association and dissociation rates are observed. Understanding the molecular basis of this phenomenon may provide important information for the design of inhibitors of mast cell degranulation.
Sayers
I
I
Krebs Institute for Biomolecular Research, MBB, University of Sheffield, UK.
Cain
S A
SA
Swan
J R
JR
Pickett
M A
MA
Watt
P J
PJ
Holgate
S T
ST
Padlan
E A
EA
Schuck
P
P
Helm
B A
BA
D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D005822:Genetic Vectors; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D015202:Protein Engineering; D051381:Rats; D011940:Receptor Aggregation; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D008024:Ligands; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E