Kinetics of multivalent antigen DNP-BSA binding to IgE-Fc epsilon RI in relationship to the stimulated tyrosine phosphorylation of Fc epsilon RI.
Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to Fc epsilon RI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15 degrees C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of Fc epsilon RI. At 35 degrees C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the time-course for cross-linking by multivalent Ag and Fc epsilon RI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.
Xu
K
K
Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853, USA.
Goldstein
B
B
Holowka
D
D
Baird
B
B
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004140:Dinitrophenols / Q000276:immunology* / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006241:Haptens / Q000378:metabolism*; D007073:Immunoglobulin E / Q000378:metabolism*; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D008024:Ligands; D018448:Models, Immunological; D010766:Phosphorylation; D011485:Protein Binding / Q000276:immunology; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000276:immunology* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004140:Dinitrophenols; D006241:Haptens; D008024:Ligands; D017455:Receptors, IgE; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D007073:Immunoglobulin E; D014443:Tyrosine; D016650:Fluorescein-5-isothiocyanate