Chicken Riboflavin-binding Protein Research Articles

Riboflavin interactions with the chicken isolated carrier protein

Riboflavin, a member of the B vitamin family, is a water-soluble vitamin that participates in energy metabolism processes via two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), in oxidized and reduced forms. Low levels of riboflavin have been associated with growth and developmental problems. In an effort to investigate the role of hydrogen bonding in the interactions between riboflavin and chicken riboflavin binding protein, the solid state geometry characteristics of a riboflavin derivative stripped of hydroxyl groups except the primary one, N-(6′-hydroxyhexyl)isoalloxazine, were investigated and found that π-stacking and hydrogen bonding involving the isoalloxazine rings are the primary intermolecular interactions. Subsequent comparative fluorescence studies showed that at neutral pH, in presence of the protein, quenching of N-(6′-hydroxyhexyl)isoalloxazine and riboflavin occurred similarly suggesting that the hydroxyl groups were not a key component of the vitamin protein interactions in the binding pocket.

Characterizing Riboflavin Antagonists for Targeted Drug Delivery Applications

Riboflavin ligands present an alternative pathway for targeted drug delivery as riboflavin receptors are over-expressed in breast and prostate cancer cells. We have examined riboflavin and several riboflavin mimicking molecules (antagonists) for targeting the riboflavin binding protein, which acts as a model protein for the riboflavin receptor. Isothermal titration calorimetry (ITC) was used for determining the binding constant of riboflavin (RF) and RF antagonists to chicken riboflavin binding protein (RfBP). The equilibrium dissociation constants determined for riboflavin (Kd = 1.4 nM) and lumiflavin (Kd = 64.2 nM) in 0.1 M phosphate buffer (pH 7.4) by the microcalorimetric method are in close agreement with the binding data previously determined by fluorescence quenching spectrometry. Several of the RF antagonists screened showed dissociation constants in the micromolar range while some exhibited no binding. The RF antagonists will be used in future studies to target riboflavin receptors for cellular uptake as a potential route for the selective delivery of drug molecules to cancer cells that over-express riboflavin receptors.

Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis

Riboflavin-binding protein (RBP) is a glycophosphoprotein found in hen eggs. We previously identified the extraordinary characteristic of RBP in reducing bitterness. For a more detailed study on the mode of action and industrial application of this characteristic, we investigated the microbial production of recombinant RBP (rRBP). We constructed a chicken RBP gene expression vector by inserting the RBP cDNA in pNCMO2, the Escherichia coli–Brevibacillus choshinensis shuttle vector. B. choshinensis HPD31 transformants produced 0.8g/l of processed and unglycosylated RBP in a soluble form in the culture supernatant. However, the expressed RBP was partially dimerized and monomeric RBP was purified by two step anion-exchange and gel-filtration chromatographies. The purified rRBP elicited bitterness reduction against quinine and caffeine, although it largely lost its riboflavin-binding ability. These results indicated that glycosylation and riboflavin-binding ability are not essential for the bitterness reduction of RBP. In addition, we assessed the usefulness of the Brevibacillus system for the expression and secretion of RBP as a new type of bitterness inhibitor.

The primary structure of a novel riboflavin-binding protein of emu (Dromaius novaehollandiae)

Emu riboflavin-binding protein (RBP) was purified from egg white and yolk, and its N-terminal amino acid sequence was determined. The molecular mass of emu RBP was estimated at approximately 48 and 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., 10 kDa larger than chicken RBP. The molecular mass of deglycosylated RBPs indicated that the content of oligosaccharide chain in emu RBP was approximately 3 times greater than that in chicken RBP. The gene encoding the RBP precursor was cloned from emu oviduct cDNA by PCR and found also in the liver and ovary cDNAs as well as oviduct cDNA. The complete cDNA consisted of an open reading frame of 714 bp encoding a protein of 238 amino acids. The amino acid sequence deduced from the cDNA sequence revealed that many essential structural features were conserved in emu RBP including 18 cysteine residues, 2 N-glycosylation sites, a clustered phosphorylation region, and riboflavin-binding sites. Two additional potential N-glycosylation sites were found in the amino acid sequences of RBPs from the emu and other sources such as the turtle and frog, which might in part account for the greater content of oligosaccharide chain of emu RBP as compared to chicken RBP.

Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins

Analysis of commercial samples of chicken ovalbumin by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) showed the presence of several other co-purifying glycoproteins. These were isolated, subjected to tryptic digestion, and two of them were identified as ovomucoid and chicken riboflavin binding-protein following database matching of the peptide masses obtained by MALDI. The N-linked glycans were released from the glycoproteins and their structures were examined by MALDI-MS in combination with exoglycosidase digestion. Ovalbumin was found to be glycosylated mainly with high-mannose and hybrid structures, consistent with profiles obtained on the intact glycoprotein by electrospray. The other glycoproteins contained mainly larger, complex glycans with up to five antennae, many of which had earlier been associated with ovalbumin.

5'-upstream structure of the gene coding for chicken riboflavin-binding protein and its relation to estrogen induction.

To elucidate the mechanism of the estrogen-dependent induction of chicken riboflavin-binding protein (RfBP), we analyzed the 5'-upstream structure of its gene. A noncoding exon exists there, and around this sequence, 9 widely spaced half-palindromic estrogen-response element (ERE) motifs (5'-GGTCA or 5'-TGACC) were found. Furthermore, an imperfect ERE-like palindromic sequence (5'-ATGTCANNNTGACAT-3') was also found at the 2.25 kb upstream region. No consensus palindromic ERE was observed. By luciferase reporter assay, the regions containing the half ERE motifs and the imperfect ERE showed estrogen-dependent enhancer activities, suggesting that these two characteristic sequences might confer estrogen-inducibility upon the chicken RfBP gene. However the activities were lower than that of a consensus ERE. It remains uncertain whether these sequences act cooperatively.

5'-Upstream Structure of the Gene Coding for Chicken Riboflavin-Binding Protein and Its Relation to Estrogen Induction

5'-Upstream Structure of the Gene Coding for Chicken Riboflavin-Binding Protein and Its Relation to Estrogen Induction

Complete Mapping of Divergent Amino Acids Responsible for Differential Ligand Binding of Folate Receptors α and β

The folate receptor (FR) type alpha may be distinguished from FR-beta by its higher affinity for the circulating folate coenzyme, (6S)-5-methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-beta (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-beta to the functional equivalent of FR-alpha should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-betaL49A/FR-alpha constructs including progressively shorter segments of FR-alpha downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-beta with the corresponding FR-alpha residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-alpha. The results also exclude a role for other residues in FR-alpha in determining its functional divergence. A homology model of FR-alpha based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket.

Regulation of the flavin redox potential by flavin-binding antibodies.

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.

Crystal structure of chicken riboflavin-binding protein.

The crystal structure of chicken egg white riboflavin-binding protein, determined to a resolution of 2.5 A, is the prototype of a family that includes other riboflavin- and folate-binding proteins. An unusual characteristic of these molecules is their high degree of cross-linking by disulfide bridges and, in the case of the avian proteins, the presence of stretches of highly phosphorylated polypeptide chain. The structure of chicken egg white riboflavin-binding protein is characterized by a ligand-binding domain and a phosphorylated motif. The ligand-binding domain has a fold that appears to be strongly conditioned by the presence of the disulfide bridges. The phosphorylated motif, essential for vitamin uptake, is made up of two helices found before and after the flexible phosphorylated region. The riboflavin molecule binds to the protein with the isoalloxazine ring stacked in between the rings of Tyr75 and Trp156. This geometry and the proximity of other tryptophans explain the fluorescent quenching observed when riboflavin binds to the protein.

Sequence of a cDNA encoding turtle riboflavin-binding protein: a comparison with avian riboflavin-binding protein

To search for the existence and distribution of a riboflavin-binding protein (RfBP), total RNA from estrogen-treated oviparous animals were screened by Northern hybridization using chicken RfBP cDNA as a probe. Besides avian livers and oviducts, RfBP mRNA was found in turtle liver, but not in the turtle oviduct. To elucidate the structure of the RfBP from a reptilian source, we constructed a cDNA library from estrogen-injected turtle liver, and a full-length turtle RfBP-encoding cDNA was cloned and sequenced. The open reading frame (ORF) encoded 242 amino acids (aa) including a signal peptide of 18 aa. There is an overall 71.3% aa identity between the deduced aa sequences of turtle and chicken. The aa sequence of turtle and chicken RfBP also show more than 30% similarity to a fragment of folate-binding protein (FBP). Six Trp and nine pairs of Cys residues are conserved between the two RfBPs with only one pair of Cys residues missing in FBP. The two Asn-linked glycosylation sites found in chicken RfBP are conserved in turtle RfBP, but only one of which is conserved in FBP. However, there is an additional potential N-glycosylation site in the turtle sequence and this may provide a better explanation for the greater molecular weight of the turtle protein than chicken RfBP. Turtle RfBP contains a region of nine Ser and five Glu residues which is present in mature chicken RfBP as eight phosphorylserine clusters forming a highly anionic region at the C terminus, but this region is not found in FBP.

Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein.

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.

Phosphorylation heterogeneity of tryptic phosphopeptides of chicken riboflavin-binding protein

The tryptic phosphopeptide of hen egg white riboflavin-binding protein has been found to exist as a mixture of peptides which differ only with respect to the number of covalently bound phosphoryl groups. Anion-exchange chromatography was used to separate homologues of the tryptic phosphopeptide of egg white riboflavin-binding protein. Four peptide peaks were obtained and analyzed using plasma desorption mass spectrometry. Molecular ions obtained agree closely with calculated molecular weight values for phosphopeptides with 8, 7 and 5 phosphoryl groups. Amino acid analyses showed that the octa- and hepta-phosphorylated peptides were pure and had the same amino acid compositions.

Highly phosphorylated region of chicken riboflavin-binding protein: chemical characterization and phosphorus-31 NMR studies

Phosphate residues on chicken riboflavin-binding protein (RBP) have been implicated in the recognition and deposition of the protein-vitamin complex in egg yolk by the ovary. We demonstrate that all of the phosphate of RBP is linked to serine and that most if not all of the phosphoserine residues are contained in a single tryptic peptide having a composition SerP8, Glx6, His2, Leu2, Ala, Met, Lys. Despite differences in the tissue of synthesis, RBPs isolated from egg yolk and egg white yield phosphopeptides virtually identical in their amino acid composition and 101-MHz 31P NMR spectra. This implies that posttranslational phosphorylation of RBP is the same in liver and oviduct. The 31P NMR spectrum of the phosphopeptide is quite different from that of the phosphoprotein. The 31P NMR spectra of egg yolk and egg white RBP are quite similar but not identical and are unaffected by the binding of riboflavin. Optimal resolution of seven phosphorus resonances was obtained near pH 7.0. The titration behavior of all the phosphoserine residues is similar. We propose that this highly anionic peptide, exposed at the surface of RBP, is the principal determinant for the uptake of RBP by the vitelline membrane of the ovarian follicle.

Dephosphorylation of chicken riboflavin-binding protein and phosvitin decreases their uptake by oocytes.

Riboflavin-binding protein (RBP) and phosvitin are phosphoglycoproteins transferred from the plasma of laying hens into the yolk of developing oocytes. We have examined the effect of phosphate removal on this yolk deposition process. Unmodified yolk RBP and phosvitin contain, respectively, 8.3 and 109 residues of phosphate/molecule. Complete dephosphorylation of yolk RBP caused a 20-min decrease in the plasma clearance half-life an 87% decrease in the uptake of the protein into oocytes in vivo. Although partially desialylated, dephospho-yolk RBP was identical with the native protein by several criteria, including riboflavin-binding capacity, mobility on SDS-polyacrylamide gels, and circular dichroism. A series of partially dephosphorylated yolk RBP samples, prepared by limited enzymatic hydrolysis, was indistinguishable from native yolk RBP by all criteria except phosphate content. Removal of the 1st phosphate residue decreased uptake of yolk RBP into oocytes by about 60%. Uptake into oocytes could not be restored to dephospho-yolk RBP by addition of anionic groups by succinylation. However, succinylation of native yolk RBP decreased its deposition into oocytes to the same extent as dephosphorylation. Partial dephosphorylation of phosvitin also had marked effects. The plasma clearance of dephosphophosvitin (70% of phosphate removed) was much faster than native phosvitin. After 4 h, 15% of injected 125I-phosvitin remained in circulation compared with only 3.8% of 125I-dephosphophosvitin. The uptake of dephosphovosvitin into oocytes was 79% less than that of native phosvitin. In vitro, 125I-phosvitin bound specifically to a preparation of oocyte plasma membranes as indicated by competition with unlabeled phosvitin but not with RBP. The specific binding of dephosphophosvitin was 96% less than that of native phosvitin and it could be displaced equally well by phosvitin or yolk RBP.

Carbohydrate compositional effects on tissue distribution of chicken riboflavin-binding protein

Riboflavin-binding proteins (RBP) purified from chicken egg white, yolk and the serum of laying hens differ in their carbohydrate compositions reflecting tissue-specific modifications of a single gene product. All three are complex glycoproteins having more than twice as many N-acetylglucosamine residues (>12) as mannose residues (approx. 6). Egg white RBP is distinctive in having only one sialic acid and two galactose residues. Serum RBP contains approx. five sialic acid and seven galactose residues. In addition there is one residue of fucose. The carbohydrate composition of yolk RBP indicates the hydrolysis, respectively, of one, one, two and 3 residues of sialic acid, fucose, galactose, and N-acetylglucosamine from its precursor, serum RBP. The effect of these differing levels of glycosylation on plasma clearance, ovarian uptake and tissue distribution of 125I-labeled riboflavin-binding proteins in laying hens were compared. 2 h after intravenous injection, 19% of the egg white RBP, 29% of the yolk RBP, and 37% of the serum RBP remained in circulation. The kinetics of plasma clearance was distinctly biphasic for each of the radioiodinated proteins. The initial rapid-turnover component ( t 1 2 =13 min ) ranged from 27% of the serum RBP sample to 48% of the egg white RBP sample. The remaining slow-turnover components were cleared with half-lives fo 81 min (egg white RBP), 101 min (yokl RBP), and 121 min (serum RBP). 16 h after injection, only 4% of the egg white RBP was deposited in the yolk of developing oocytes while about 12% of the serum RBP and yolk RBP was deposited. This higly significant difference is apparently due to preferential, carbohydrate-dependent clearance of egg white RBP by the liver rather than preferential uptake of serum and yolk RBP by the ovarian follicle. We find no evidence for carbohydrate-directed uptake of riboflavin-binding protein by the ovarian follicle.

Purification and properties of riboflavin-binding protein from the egg white of the duck ( Anas platyrhynckos)

Riboflavin-binding protein was purified from the egg white of domestic duck and some of its properties were investigated. The protein was homogeneous by the criteria of gel filtration on Sephadex G-100 and electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, had molecular weight of 36 000 ± 1000 and, unlike the chicken egg white protein ( M r 32 000 ± 2000 ), was devoid of covalently-bound carbohydrate. It was similar to the chicken riboflavin-binding protein in its behavior on ion-exchange celluloses and affinity to interact with the flavin and its coenzymes, but differed significantly in amino acid composition in that it completely lacked proline and contained less of methionine and arginine. The protein partially cross-reacted with the specific antiserum to chicken riboflavin-binding protein with a spur during immunodiffusion analysis.