Crystal structure of chicken riboflavin-binding protein (original) (raw)
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The carbohydrates of the isoforms of three avian riboflavin-binding proteins
European Journal of Biochemistry, 1999
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.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1976
The interaction of riboflavin with a protein isolated from egg white has been studied spectrofluorimetrically at different pH values. In 0.1 M phosphate buffer pH 7.0; 1:1 complex formation occurs with the association constant Ka = 7.7.107 M-1. In the presence of 0.033 7o sodium dodecyl sulphate, the complex dissociated with a rate constant of 4.10-2 sec-1 at 29 °C. The binding was sensitive to pH and to the antibodies produced against the protein. On lowering the pH from 7 to 4 the binding affinity decreased approximately 100-fold and below pH 4, the binding could not be detected at all. These data, together with those obtained by measuring the fluorescence intensities of riboflavin in presence of N-bromosuccinimide oxidizedand disulphide reduced apoprotein, suggest that carboxyl functions, 1-2 tryptophan residues and 2-3 disulphide bridges are essential for binding. The emission spectra of the protein under different conditions upon excitation at 280 and 295 nm were analyzed to calculate the quantum yield (Q) and the efficiency of energy transfer (e) from tyrosine to tryptophan residues. From these data it was concluded that the energy transfer did not occur with equal efficiency under all conditions and that the tryptophan residues responsible for the riboflavin binding are more accessible to N-bromosuccinimide oxidation than others.
Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 ω-hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8°C for apoRBP to 72.8°C for RBP–Rf complex. For RBP–FMN complex, the denaturation temperature of 73.0°C was even higher than for the RBP–Rf complex. The other two flavin analogues showed transition temperatures in between 66.9°C and 68.8°C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions , which contributed ∼70% to the total heat effect, has transition temperature in the broad range of 60.5–73.2°C; the other transition temperature is in the narrower range of 65.4–71.1°C. The observed transitions can be related to RBP domains.
Immunochemistry of the glycopeptide derived from avian riboflavin-binding protein
Molecular Immunology, 1979
Immunization of rabbits with a purified glycopeptide from a pronase digest of avian riboflavinbinding protein by a modified method of Maugh (1977) resulted in the production of carbohydrate-specific antibody. A sensitive radioimmunoassay was developed to detect the low levels of antibodies, and the concentrated IgG fraction was used for immunochemical analyses. Chemical modification of the terminal sugars and hapten inhibition of the precipitin reaction between monospecific antibodies and the glycoprotein demonstrated that blinked galactosyl residues were the immunodominant determinants of the glycopeptide. Radioimmunoassay indicated that the glycopeptide. although a structural component of the riboflavin-binding protein, did not contribute to the major antigenic determinants of the native protein. MATERIALS AND METHODS Riboflavin-binding protein The glycoprotein (mol. wt = 32,000) was isolated and purified from the eggs of Single Comb White Leghorn chickens maintained by the Poultry Science Department of The Pennsylvania State University. The isolation procedure for the egg white protein was that of Farrell et al. (1969). The yolk protein was isolated according to the procedure of Miller (1976).
Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies
Journal of Biosciences
Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87-219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182-204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.
Journal of Biosciences, 1990
The antigenecity of tryptic fragments of reduced and carboxymethylated chicken riboflavin carrier protein were studied. The tryptic sites of the native riboflavin carrier protein bound to riboflavin were inaccessible. The molecular weight and the elution profile on high performance liquid chromatography (TSK 545 DEAE) were unaltered at an enzyme to substrate ratio of 1:31. However, carboxymethylated riboflavin carrier protein could be cleaved into 3 or 4 fragments at an enzyme to substrate ratio of 1:250 or 1:125. Chromatographic separation of the tryptic fragments on high pressure liquid chromatography (TSK 545 DEAE) revealed the presence of two fragments with different elution profiles but similar molecular weight 26 ±2 kDa. Only one fragment (associated with peak 2) had the ability to displace chicken riboflavin carrier protein in an homologous chicken riboflavin carrier protein radioimmunoassay. Thus, carboxymethylated ribotlavin carrier protein which does not compete with chicken riboflavin carrier protein in the radioimmunoassay, on mild trypsinization generates a fragment which interacts with chicken riboflavin carrier protein in radioimmunoassay.
Biochemical and immunological aspects of riboflavin carrier protein
Journal of Biosciences
Riboflavin carrier protein which is obligatorily involved in yolk deposition of the vitamin in the chicken egg, is a unique glycophosphoprotein present in both the yolk and white compartments. The yolk and egg white proteins are products of a single estrogeninducible gene expressed in the liver and the oviduct respectively of egg laying birds. Despite the fact that the carbohydrate composition of the yolk and white riboflavin carrier proteins differ presumably due to differential post-translational modification, the proteins are immunologically similar and have identical amino acid sequence (including a cluster of 8 phosphoser residues towards the C-terminus) except at the carboxy terminus where the yolk riboflavin carrier protein lacks 13 amino acids as a consequence of proteolytic cleavage during uptake by oocytes. The protein is highly conserved throughout evolution all the way to humans in terms of gross molecular characteristics such as molecular weight and isoelectric point, and in immunological properties, preferential affinity for free riboflavin and estrogen inducibility at the biosynthetic locus viz., liver. Obligatory involvement of the mammalian riboflavin carrier protein in transplacental flavin transport to subserve fetal vitamin nutrition during gestation is revealed by experiments using pregnant rodent or subhuman primate models wherein immunoneutralisation of endogenous maternal riboflavin carrier protein results in fetal wastage followed by pregnancy termination due to selective yet drastic curtailment of vitamin efflux into the fetoplacental unit. Using monoclonal antibodies to chicken riboflavin carrier protein, it could be shown that all the major epitopes of the avian riboflavin carrier protein are highly conserved throughout evolution although the relative affinities of some of the epitopes for different monoclonal antibodies have undergone progressive changes during evolution. Using these monoclonal antibodies, an attempt is being made to map the different epitopes on the riboflavin carrier protein molecule with a view to delineate the immunodominant regions of the vitamin carrier to understand its structure-immunogenicity relationship.
Biochemical Journal, 1988
The properties of antibodies generated in rabbits against native riboflavin carrier protein (cRCP), riboflavin carrier protein that had been denatured/renatured by SDS treatment (SDS-RCP) or disulphide-bondreduced then S-carboxymethylated (Carb-RCP) were studied. SDS-RCP could displace native RCP in radioimmunoassay (r.i.a.), whereas Carb-RCP could not. By using antibodies raised in five different rabbits against native cRCP, 'l25-labelled Carb-RCP could bind between 0 and 3000 of the native antibodies. Antibodies raised against native RCP appear to be largely directed towards specific conformational determinants of RCP. Carb-RCP displaced native RCP in an r.i.a. using antibodies raised against SDS-RCP. SDS denaturation presumably unmasks cryptic epitopes in native RCP. Carb-RCP was a weak immunogen and elicited, presumably, antibodies to sequential epitope/epitopes. When injected into pregnant mice the antibodies caused neutralization of RCP, leading to termination of pregnancy, indicating highly conserved sequential epitopes in chicken and rodent RCP. Antibodies raised against Carb-RCP or native RCP reacted with CNBr fragments of native RCP, further confirming the presence of sequencespecific antibodies elicited by Carb-RCP.