Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Abstract

Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues. Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges.Keywords : riboflavin carrier protein; refolding; monoclonal antibodies.Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg white, yolk and serum of laying hens and other birds [1,2]. The egg-white protein is synthesized in the oviduct of the laying hen [3]. The liver, under the influence of estrogen, synthesizes serum RCP [3,4] which is then deposited through receptor-mediated uptake into the yolk of the developing egg [4]. RCP delivers riboflavin to the developing embryo in the egg, and birds that harbor a mutant RCP gene fail to lay eggs that hatch, as a result of insufficient deposition of the vitamin in the egg [5,6]. Egg-white RCP differs from the yolk and serum RCP only in the pattern of glycosylation [7] and removal of the C-terminal 11Ϫ13 amino acids in yolk RCP, presumably as a result of proteolytic cleavage during oocyte uptake [8]. A stretch of phosphoserine residues is present towards the C-terminal end of RCP [9]. We and others have shown the critical importance of these residues in mediating oocyte-receptor-binding and uptake of RCP during egg development [10,11].RCP is a complex protein with 18 cysteine residues all involved in disulfide-bridge formation [12]. Early studies had indicated that a single disulfide bridge appears to be critical for riboflavin binding [13], but the specific disulfide bridge involved ha...

Oxidative Protein Folding in Vitro: A Study of the Cooperation between Quiescin-Sulfhydryl Oxidase and Protein Disulfide Isomerase

“…Although glutathione redox buffers (typically with a GSH/GSSG ratio of 5/1) are widely used in refolding studies, they are ineffective with reduced RfBP over 30 min (Figure 7 top) in agreement with earlier studies (37, 38). Conceivably the complexity of a 9-disulfide client protein, with millions of potential disulfide isomers, may limit the effectiveness of simple redox buffers when compared to a 4-disulfide containing protein (with a maximum of 105 isomers).…”

Oxidative Protein Folding and the Quiescin–Sulfhydryl Oxidase Family of Flavoproteins

“…7; regain of activity was assessed by fluorescence quenching as riboflavin rebinds to functional apoprotein). Since flavin binding to apo-RfBP occurs only after substantially all of the native pairings have been secured (76), this convenient continuous fluorescence assay provides useful insights into the factors that modulate the oxidative folding of proteins with complex disulfide connectivities.…”

Downregulation of leaf flavin content induces early flowering and photoperiod gene expression in Arabidopsis

“…The estrogen-dependent and fecundation-induced expression patterns are also found in mammals [21]. Regarding to the RfBP protein, it is mainly produced in the blood plasma of podocyte and localizes to the plasma membrane via the N-terminal ligand-binding domain [23,24]. RfBP also employs the C-terminal phosphorylated domain to tightly bind riboflavin in a 1:1 molar ratio [24–26].…”