Production of Recombinant Chicken IgY-Fc and Evaluation of Its Transport Ability into Avian Egg Yolks

Abstract

Maternal immunoglobulin (Ig) Y, the avian counterpart of mammalian IgG, is e ciently transported into the yolks of maturing oocytes. We have previously shown that the Fc region plays a critical role in the IgY transport into avian egg yolks. The aim of this study was to produce recombinant IgY-Fc and to evaluate its transport ability into avian egg yolks. Two basic expression vectors were constructed: one mainly expressed three constant regions of chicken IgY heavy chain (Fc) that contained three cysteine residues (C , C , C) involved in inter-heavy chain disulfide bonds; and the other mainly expressed two constant regions (Fc) that contained two cysteine residues (C , C). SDS-PAGE and Western blotting analyses indicated that both Fc and Fc were separated into multiple protein bands under non-reducing conditions, suggesting incomplete formation of inter-chain disulfide bonds. To prevent the incomplete disulfide bond formation, C and C were mutated individually to serine by site-directed mutagenesis. As expected, each of the mutated recombinant IgY-Fc produced a single protein band by SDS-PAGE analysis. When intravenously-injected into laying quail, the two mutants, Fc C S and Fc C S, were transported into the egg yolks at high levels, whereas the other two mutants, Fc C S and Fc C S, were transported into the egg yolks at lower levels. In conclusion, we have succeeded in producing a recombinant IgY-Fc retaining a high transport ability into the avian egg yolks; this protein will be provided as a useful tool for studying the mechanism of IgY transport into egg yolks. : egg yolk, IgY transport, inter-chain disulfide bond, quail, recombinant IgY-Fc

Maternal Transfer of Immunoglobulins into Egg Yolks of Birds

“…They mutated "C347" to the serine of IgY-Fcυ2-4, and this recombinant also retained high affinity binding to the chicken leukocyte IgY-Fc receptor, CHIIR-AB1. By introducing this methodology, we have succeeded in producing two recombinant chicken IgY-Fc(s) with different sizes, IgYFcυ2-4 (C347S) and IgY-Fcυ3-4 (C340S), retaining a high transport ability into the egg yolks of quail (Bae et al, 2010a). Interestingly, the transport ability of the IgY-Fcυ3-4 into the egg yolks corresponded closely with that of the IgY-Fcυ2-4, suggesting that the presence of Cυ3 and Cυ4 domains but not Cυ2 domain is important to maintain the transport ability of IgY.…”

“…A recent study showed that ggFcR mainly interacts with the Cυ2 domain of IgY (Schreiner et al, 2012). However, the Cυ2 domain is not essential to maintain IgY-Fc transport capability into egg yolks (Bae et al, 2010a), suggesting that the likelihood of ggFcR involvement in IgY transport is low. A third receptor, CHIR-AB1, is a member of the leukocyte receptor family (Viertlboeck et al, 2005(Viertlboeck et al, , 2007.…”

A review on the application of chicken immunoglobulin Y in aquaculture

“…The heavy chains of IgY feature a variable region (VH) and four constant regions (CH1–CH4); in comparison, IgG features a variable region (VH) and three constant regions (CH1–CH3) (Figure 1). 17,19 Unlike IgG, IgY lacks a hinge region, which reduces swing and flexibility, thus resulting in increased stability 11 . The isoelectric point of IgY (5.7–7.6) is lower than that of IgG (6.1–8.5) 20 .…”

“…The heavy chains of constant regions (CH1-CH3) (Figure 1). 17,19 Unlike IgG, IgY lacks a hinge region, which reduces swing and flexibility, thus resulting in increased stability. 11 The isoelectric point of IgY (5.7-7.6) is lower than that of IgG (6.1-8.5).…”

Amino acid substitution in the Cυ3 domain causes either elevation or reduction of IgY uptake into egg yolks of quail

“…First, Fc 3-4 was utilized for an uptake study, since the C 2 domain of Fc 2-4 is not essential for Fc uptake in chicken IgY (Bae et al, 2010a). Seven Fc 3-4 mutants were produced and were analyzed by SDS-PAGE (Fig.…”