★ Critical IgY amino acid residues for egg yolk transport in birds are proposed. ★ Substitution of Tyr363 located on the interface of Cυ3/Cυ34 domain seriously damages IgY transport into egg yolks. ★ Lack of N-glycosylated carbohydrate chain at Asn407 lowers IgY transport into egg yolks. ★ A hurdle disturbing the infiltration of IgY mutants lies on the inside of granulosa cell layer of ovarian follicles. ★ We characterize selective IgY transport system in ovarian follicles of birds.
In avian species, maternal IgY is selectively incorporated into the egg yolks of maturing oocytes, but the relevance of receptor-mediated uptake is unclear. Here we investigated the critical amino acid residues of IgY required for egg yolk transport by conducting mutational analyses of selected residues located along the Cυ3 and Cυ4 domains of chicken IgY. Recombinant wild-type IgY-Fc (WT) and its mutants were synthesized, and their uptakes into the egg yolks of quail were determined. Among the 17 amino acid residues located on the Cυ3/Cυ4 interface, the substitution of Y363 at the Cυ3 domain to alanine abolished the IgY-Fc uptake into egg yolks. The comprehensive substitution of Y363 with other amino acids revealed that the residue at 363 needs to be allocated with aromatic amino acids to maintain the high transport ability. The deglycosylation of the N-linked carbohydrate chain by substituting N407 at the Cυ3 domain with alanine also caused a marked reduction of IgY-Fc uptake. The microscopic detection of the injected WT and Y363A mutant in ovarian follicles showed that the WT was concentrically accumulated in yolk granules, whereas the Y363A mutant was hardly accumulated in yolk granules, but it had infiltrated into the granulosa cell layer, suggesting that a major hurdle disturbing the infiltration of the Y363A mutant lies on the inside of the granulosa cell layer. The identification of important amino acid residues required for efficient IgY transport enhances our understanding of the molecular mechanisms underlying IgY transport through a specific IgY receptor in ovarian follicles.