The Fc Segment of IgE Influences the Kinetics of Dissociation of a Symmetrical Bivalent Ligand from Cyclic Dimeric Complexes

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Abstract
As part of a systematic effort to determine the features of immunoglobulin E−receptor (IgE−FcεRI) aggregation that are critical for cellular activation, we used fluorescence to examine the dissociation of a soluble bivalent ligand, N,N‘-bis[[ε-[(2,4-dinitrophenyl)amino]caproyl]-l-tyrosyl]-l-cystine ((DCT)2-cys), from soluble bivalent IgE and its bivalent F(ab‘)2 and monovalent Fab‘ fragments. Cross-linking of Fab‘ fragments by (DCT)2-cys is limited to linear dimers, and we find that (DCT)2-cys dissociation from Fab‘ occurs with a single kinetic coefficient [(4.2 ± 0.6) × 10-3 s-1] that corresponds to the lower of the two kinetic coefficients observed with the bivalent IgE [(4.7 ± 0.7) × 10-2 s-1 and (4.4 ± 0.3) × 10-3 s-1]. Similarly, the lower value is obtained for dissociation of (DCT)2-cys that is monovalently bound to IgE after incubation with a large excess of the ligand. (DCT)2-cys can bind to bivalent F(ab‘)2 fragments and form a variety of linear and cyclic aggregates, similarly to IgE, but, unlike IgE, we find that dissociation occurs with a single kinetic coefficient similar to that observed for Fab‘. We find that IgE and its (Fab‘)2 fragments form highly stable cyclic dimer rings with two (DCT)2-cys. We demonstrate that the kinetic coefficients are independent of enhanced fluorescence quenching observed for bound sites in cyclic dimers. Together, the results show that the rate constant for breaking a linear cross-link formed by (DCT)2-cys is the same as that for dissociation of the monovalently bound (DCT)2-cys. Further, they show that opening of a bond in a dimer ring for the F(ab‘)2 fragment occurs with approximately the same dissociation rate constant as opening a bond in a linear cross-link. This rate constant is about three times smaller than that observed with IgE, suggesting that steric strain is caused by apposed Fc segments in cyclic IgE dimers. Such structural interference may affect the functional consequences of IgE−FcεRI aggregation on the cell surface.