Abstract
An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a non-mammalian cell has been investigated (k(+1) = 1.14 +/- 0.46 x 10(5) mol(-1)sec(-1), k(-1) = 2.30 +/- 0.14 x 10(-3) s(-1), and K(a) = 4.95 x 10(7) m(-1)). This is a lower affinity than that recorded for mammalian IgE-high affinity receptor interactions (Ka approximately 10(10) m(-1)) but is within the range of mammalian IgG-high affinity receptor interactions (human: Ka approximately 10(8)-10(9) m(-1) mouse: Ka approximately 10(7)-10(8) m(-1). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.
Highlights
Domain pair must have existed in the common (IgY-like) ancestor prior to its duplication and subsequent divergence in the mammalian lineage
Through our studies receptor identified by Viertlboeck et al [49]; LRC, leukocyte receptor complex; Mb, megabase; my, million years; FACS, flow cytometry; PBS, phosphate-buffered saline; BSA, bovine serum albumin
Nonspecific binding was measured by incubating cells with an excess (350 nM) of for 10 min in PBS containing 1% bovine serum albumin (BSA) unlabeled IgY for 1 h at room temperature before the addition (Sigma, catalog number A7030) before staining for 1 h at 4 °C of 10 nM labeled IgY, and the ␥ counts obtained were subtracted with chicken IgY-(Fab)2 (Rockland, catalog number 003-0104), (Fab)2 ϩ whole IgY, IgY, or buffer before incubation with a mouse monoclonal antibody (Sigma, catalog number C-7295), which was found to be an anti-C2,5 and detection by antifrom the total bound counts
Summary
Inter-chain disulfide bonds are shown as solid (known) or dashed (putative) lines. The domain structure of IgG shown here is that of human IgG1, with the hinge region represented by a curved line linking C␥1 and C␥2. C2-like domain pairs have persisted since their first appearance in a common ancestor that unites all terrestrial vertebrates To assess their effect on the antibody-Fc receptor interaction, we have expressed two avian IgY-Fc fragments that differ only by the presence (Fc2– 4) or absence (Fc3– 4) of the C2 domain pair and measured their binding to the chicken monocyte cell line, MQ-NCSU, as well as that of whole IgY. This is the first report of the cell binding properties of a nonmammalian antibody and addresses the question of whether the “extra” C2 domain pair in IgY necessarily leads to a high affinity interaction
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