Amino acid substitution in the Cv3 domain causes either elevation or reduction of IgY uptake into egg yolks of quail

摘要

Egg yolks of avian species contain large quantities of immunoglobulin (Ig) Ys transferred from maternal blood circulation. However, it is unclear how maternal IgYs are incorporated into the egg yolks of maturing oocytes. The aim of this study was to identify the amino acid residues required for efficient IgY transport into the egg yolks of quail by utilizing recombinant quail IgY-Fc (qlgY-Fc). Five amino acid residues (361-365 at the Cv3 domain) located on the Cv3/Cu4 interface were individually substituted for alanine residues. The mutants were then intravenously injected into laying quail, and their uptakes into egg yolks were measured by ELISA. Substitution of 1362, Y363 and 1364 for alanine markedly reduced qlgY-Fc uptake into the egg yolks to almost undetectable levels. With respect to the Y363 residue, neither substitution for phenylalanine nor substitution of tryptophan reduced qlgY-Fc uptake, suggesting the necessity of an aromatic side-chains at the Y363 residue. Interestingly, substitution of G365 for alanine or for other polar- or non-polar amino acids elevated qlgY-Fc uptake by 2.5-fold compared to that of the wild-type qlgY-Fc. Analyses of the blood concentrations of the two alanine mutants with a low uptake (Y363A) and a high uptake(G365A) showed that their modified uptakes were not explained by changes in blood clearance. Removal of the N-glycosylated carbohydrate chain at the Cv3 domain by substituting an N408 residue for alanine also resulted in lowered qlgY-Fc uptake. These results emphasize the existence of a selective IgY transport system recognizing the Cv3 domain of IgY, which raises the possibility that an IgY with high transport ability might be engineered by genetic manipulation.