A highly conserved surface loop in the C-terminal domain of ovotransferrin (residues 570-584) is remote from receptor-binding site (original) (raw)
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The Biochemical journal, 1990
A peptide corresponding to a surface loop in the C-terminal domain of chicken ovotransferrin (residues 570-584) was made by solid-phase synthesis and used to immunize rabbits. A 15-amino acid-residue disulphide-linked loop occurs in both domains of all five transferrins for which the sequence is available and lies on the opposite side of the iron-binding site from the interdomain cleft. Polyclonal antibodies to the peptide were specific for non-reduced holo-ovotransferrin and the C-terminal domain, as shown by e.l.i.s.a. and immunoblotting. The antibody did not inhibit binding of ovotransferrin to receptors on chick-embryo reticulocytes but was able to bind ovotransferrin bound to the cellular receptors at 0 degree C. The loop composed of residues 570-584 appears to be remote from the transferrin receptor-binding site.
Monoclonal antibodies to chicken ovotransferrin: epitopic and phylogenetic analysis
Comparative Biochemistry and Physiology Part A: Physiology, 1995
1. The ability of four domain-specific anti-chicken ovotransferrin antibodies to bind to turkey, quail, duck, pheasant and goose ovotransferrins was examined directly by an enzyme-linked immunoassay and indirectly in a competitive radioimmunoassay. 2. The ability of these same ovotransferrins to compete with radioiodinated chicken ovotransferrin for binding to transferrin receptors on chick embryo red blood cells was also tested. 3. The hypothesis that evolutionarily conserved determinants involved in receptor recognition can be predicted by monoclonal antibodies that block binding of ovotransferrin to receptor was found to be incorrect.
2007
Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile ...
X.a.f.s. studies of chicken dicupric ovotransferrin
Biochemical Journal, 1991
A comparison of Cu K-edge x.a.f.s. spectra with that of the equivalent Fe K-edge for chicken ovotransferrin (COT) indicates that the metal ions occupy essentially the same binding sites in the protein. However, in the case of the Cu2+ complex the metal appears to have reduced co-ordination. Changes are observed in the x.a.f.s. of 90%-saturated COT (Cu1.8COT) on freeze-drying. The three-dimensional X-ray structures of rabbit serum transferrin and human lactoferrin have shown that the ferric cations are co-ordinated by four protein ligands and a bidentate carbonate anion in a distorted octahedral arrangement [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1768-1774; Anderson, Baker, Norris, Rice and Baker (1989) J. Mol. Biol. 209, 711-734; Bailey, Evans, Garratt, Gorinsky, Hasnain, Horsburgh, Jhoti, Lindley, Mydin, Sarra & Watson (1988) Biochemistry 27, 5804-5812]. This structural information, together with the differences in e.x.a.f....
The Biochemical journal, 1996
Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/1 oTF/2N were obtained for constructs defining residues 1-319, 1-332 and 1-337-(Ala)3 respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/1 and a foreshortened oTF consisting of residues 1-682 was expressed at a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342-686 and 342-682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the a...
Structural studies on rabbit transferrin: isolation and characterization of the glycopeptides
Biochemistry, 1978
The structure of rabbit transferrin was investigated with regard to number, size, and composition of the heteropolysaccharide units and their relative location on the polypeptide chain. The composition and molecular weight of the Pronase glycopeptides revealed that rabbit transferrin contains two heteropolysaccharide units, each composed of 2 sialic acid residues, 2 galactose residues, 3 mannose residues, and 4 N-acetylglucosamine residues. The composition and molecular weight of the tryptic glycopeptides further sub-
Monoclonal Antibodies to a Purified Human Transferrin Receptor
Scandinavian Journal of Immunology, 1984
BALB/c mice were injecied wiih an affinily-chromalography-purified placcnlal ininsferrin receptor preparation. Spleen cells were fused wiih NS-1 myeloma cells. Sixteen hybrids producing monoclonal antibodies speeific for the transferrin receptor and two hybrids specific for transferrin were identilled by radioimmunoassay (RIA). Five hybrids were seiecied for cloning on the basis of antibody spcdfieity and affmity. None of the antibodies inhibited the binding of transferrin to K562 celts. The binding of aniihody 1D9 to K.S62 cells was partially inhibited by transferrin or a polyclonal goat anli-lransferrin receptor antiserum. Of the five antibodies, two (IIB(ยป and nB2) reacted only with the purified receptor and soluhilized cells and not with whole cells. The other three aniihodies, when tested with normal human cells and leukaemia and luniour cell lines, showed identical reaction patterns. The antibodies precipitated a glycoprt)tein from K.S(i2 eelis with an apparent molecular weight of 94.(K)0, estimated from sodium dodecyl sulphatepolyacrylamide gel eleetrophoretograms run under reducing conditions, and a molecular weight of IHK. IXX) when run under unreduced conditions. All antibodies have a high affinity with K^ values ranging from 1.44 x 10' * to .1.56 x IO'"(i/mol). The antigen precipitated by all five antibodies showed identical peptide maps after partial proteolytic digestion.
The complete amino acid sequence of human serum transferrin
Proceedings of the National Academy of Sciences, 1982
The complete amino acid sequence of human serum transferrin has been determined by aligning the structures of the 10 CNBr fragments. The order of these fragments in the polypeptide chain is deduced from the structures of peptides overlapping methionine residues and other evidence. Human transferrin contains 678 amino acid residues and--including the two asparagine-linked glycans--has an overall molecular weight of 79,550. The polypeptide chain contains two homologous domains consisting of residues 1-336 and 337-678, in which 40% of the residues are identical when aligned by inserting gaps at appropriate positions. Disulfide bond arrangements indicate that there are seven residues between the last half-cystine in the first domain and the first half-cystine in the second domain and therefore, a maximum of seven residues in the region of polypeptide between the two domains. Transferrin--which contains two Fe-binding sites--has clearly evolved by the contiguous duplication of the structural gene for an ancestral protein that had a single Fe-binding site and contained approximately 340 amino acid residues. The two domains show some interesting differences including the presence of both N-linked glycan moieties in the COOH-terminal domain at positions 413 and 610 and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). The locations of residues that may function in Fe-binding are discussed.