The carbohydrates of the isoforms of three avian riboflavin‐binding proteins

Abstract

The carbohydrate chains of nine isoforms of chicken egg-white riboflavin-binding protein (RfBP) and six isoforms each of quail egg-white and yolk RfBP have been structurally characterized. The two N-glycosylation sites, Asn36 and Asn147, of the most abundant isoform of each of the three proteins were analyzed in further detail leading to the identification of different glycosylation patterns. In both chicken and quail egg-white RfBP the carbohydrates attached to position 36 had a lower degree of branching and, in the case of the quail protein, this site was only partially glycosylated. A very heterogeneous mixture of complex structures was characteristic of the other glycosylation site. Analysis of the two sites in quail yolk RfBP confirmed this result which agrees with what has been established for hen yolk RfBP. The presence in the three proteins of a highly heterogeneous mixture of differently branched glycans suggests that the differences in isoelectric points, which is a peculiarity of the different isoforms, are probably indeed due to differences in carbohydrate structure.Keywords: riboflavin-binding protein; phosphoglycoprotein; oligosaccharide; structural characterization; MS.The best characterized riboflavin-binding proteins are those of avian origin and in particular those of the hen [1]. Three different proteins have been characterized in this species and they have been shown to be the products of the same gene and to have undergone different post-translational modifications [2]. The three proteins have a single high-affinity site for riboflavin and are found in the plasma of laying hens and in the egg yolk and white. The first two molecules are synthesized by the liver and the third is a product of the oviduct [3,4]. The function of plasma RfBP is that of vitamin transport to the developing oocytes, into which it is incorporated loosing in the process the last 11±13 amino acids which are thus absent from the yolk protein [5]. Egg white RfBP is believed to serve mainly as a storage protein and it is not clear why most of it is found in the apo form. The presence of estrogen-induced pregnancy-specific RfBPs in the plasma of several mammalian species has been demonstrated [6±9] although none of these proteins, which cross-react immunologically with hen RfBP, have been sufficiently characterized. Hen egg-white and plasma RfBP share the same 219 aminoacid sequence; in yolk RfBP the last 11±13 amino acids are missing [10]. The three molecules are phosphoglycoproteins with the carbohydrates attached at the same positions, Asn36 and Asn147. The structure of the oligosaccharides in hen egg yolk and plasma RfBPs has been determined [11,12]. Although from the sugar composition the carbohydrates of hen egg-white RfBP are known to be different from those of plasma and yolk RfBP, their structure has not been reported. A very special characteristic of the three hen proteins is the presence of a highly phosphorylated region that extends between amino acids 186 and 197, where there can be up to eight phosphates attac...

Comparative proteomic analysis of hen egg yolk plasma proteins during embryonic development

“…In the present study, spot 3 representing RFBP showed a slightly higher MW than theoretical value (Table. ), indicating possible glycosylation or a complex with riboflavin (Amoresano et al, ; Geng et al, ). The abundance of this protein was decreased significantly and almost disappeared after day 14 of incubation (Figures and ), suggesting a complete degradation or being absorbed integrally by the embryo during embryogenesis.…”

Comparative proteomic analysis of chicken, duck, and quail egg yolks

“…Their different sources (e.g. chicken egg white, quail egg white, or yolk) showed various glycosylation patterns as well as diverse tertiary structures [34,35] , indicating their distinct biological functions during embryonic development.…”

Identification of an IgE Reactive Peptide in Hen Egg Riboflavin Binding Protein Subjected to Simulated Gastrointestinal Digestion

“…20,21 In RfBP, the N-linked carbohydrates are found on residues Asn 36 and 147. 22 These positions are not included in the immunoreactive peptide 41−84. Therefore, IgE binds the peptide chain, rather than the carbohydrate moieties, suggesting that a potential IgE cross-reactivity of RfBP and ovomucoid would not be likely related to their carbohydrate moieties.…”