Domain-specific monoclonal antibodies to ovotransferrin indicate conservation of determinants involved in avian transferrin receptor recognition (original) (raw)
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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.
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.
Physiological roles of ovotransferrin
Biochimica et Biophysica Acta (BBA) - General Subjects, 2012
Ovotransferrin is an iron-binding glycoprotein, found in avian egg white and in avian serum, belonging to the family of transferrin iron-binding glycoproteins. All transferrins show high sequence homology. In mammals are presents two different soluble glycoproteins with different functions: i) serum transferrin that is present in plasma and committed to iron transport and iron delivery to cells and ii) lactoferrin that is present in extracellular fluids and in specific granules of polymorphonuclear lymphocytes and committed to the so-called natural immunity. To the contrary, in birds, ovotransferrin remained the only soluble glycoprotein of the transferrin family present both in plasma and egg white. Substantial experimental evidences are summarized, illustrating the multiple physiological roles of ovotransferrin in an attempt to overcome the common belief that ovotransferrin is a protein dedicated only to iron transport and to iron withholding antibacterial activity. Similarly to the better known family member protein lactoferrin, ovotransferrin appears to be a multi-functional protein with a major role in avian natural immunity. Biotechnological applications of ovotransferrin and ovotransferrin-related peptides could be considered in the near future, stimulating further research on this remarkable protein. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.
The cDNA sequence and primary structure of the chicken transferrin receptor
Gene, 1991
Recombinant cDNA clones encoding the chicken transferrin receptor (cTR) have been isolated and sequenced. Comparison of the deduced primary structure of cTR with those of the human transferrin receptor (hTR) and mouse transferrin receptor (mTR) shows that their size, hydropathy profile, location of sites for posttranslational modifications, and domain organization are highly similar. The cytoplasmic domain of cTR contains the motif Tyr-Xaa-Arg-Phe (YXRF) that is the recognition signal for high-efficiency endocytosis of hTR. The cTR has several highly conserved regions within its extracellular domain, including those flanking the putative N-glycosylation sites. Overall, however, the extracellular domain of cTR is only 53 % identical to the extracellular domains of hTR and mTR. The cTR also lacks three of the six Cys residues found in the extracellular domains of the mammalian TRs. These differences can account for functional and structural properties that distinguish cTR and m~m~ian TRs. cTR indicate that they have a similar structure also
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...
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 'C. The loop composed of residues 570-584 appears to be remote from the transferrin receptor-binding site. EXPERIMENTAL Materials Bio-Gel P-6DG desalting resin and Affigel-15 were obtained from Bio-Rad Laboratories. Nai2iI was from Amersham (carrier-free; 100 mCi/ml in NaOH solution). lodogen and m-maleimidobenzoic acid N-hydroxysuccinimide ester were from Pierce Chemical Co. Goat anti-(rabbit IgG) antibody and its horseradish peroxidase conjugate came from Organon Teknika-Cappel. Microtitre plates were from Dynatech. Keyhole-limpet haemocyanin was obtained from Calbiochem. Nitrocel-Vol. 266
Receptor recognition sites reside in both lobes of human serum transferrin
The Biochemical journal, 1997
The binding of iron by transferrin leads to a significant conformational change in each lobe of the protein. Numerous studies have shown that the transferrin receptor discriminates between iron-saturated and iron-free transferrin and that it modulates the release of iron. Given these observations, it seems likely that there is contact between each lobe of transferrin and the receptor. This is the case with chicken transferrin, in which it has been demonstrated unambiguously that both lobes are required for binding and iron donation to occur [Brown-Mason and Woodworth (1984) J. Biol. Chem. 259, 1866-1873]. Further support to this contention is added by the ability of both N- and C-domain-specific monoclonal antibodies to block the binding of a solution containing both lobes [Mason, Brown and Church (1987) J. Biol. Chem. 262, 9011-9015]. In the present study a similar conclusion is reached for the binding of human serum transferrin to the transferrin receptor. With the use of recombin...
Proceedings of the National Academy of Sciences, 1981
A radioimmunoassay was developed to directly assay the presence oftransferrin receptors in human tissues. Antisera developed in a goat against purified human placental transferrin binding protein was purified by fractional sodium sulfate precipitation and adsorption against Sepharose-bound transferrin to remove trace anti-transferrin activity. The antisera immunoprecipitates a Mr 94,000 peptide on "'5I-iodinated syncytial trophoblast membranes from placentae. This polypeptide has been identified previously as the transferrin binding protein of the placenta [Wada, H. G., Hass, P. E. & Sussman, H. H. (1979)J. BioL Chem. 254, 12629-12635]. A standard curve using purified 'MIiodinated placental transferrin receptor and various amounts of the purified noniodinated receptor is sensitive from 5 to 900 ng. A reticulocyte-enriched membrane ghost preparation (5% reticulocyte) gives a value of 9.5 jig of receptor per mg of protein. Normal erythrocyte membrane ghosts show binding (0.57 ,.g of receptor per mg of protein) proportional to the amount of reticulocytes normally present in blood (0.5-1.0%). In other tissues in which the transferrin receptor binding has been reported, purified syncytial trophoblastic membranes are found to have 34.5 #g of receptor per mg of protein, and BeWo cells, a choriocarcinoma cell line, are found to have 15.7 Mg of receptor per mg of protein. In contrast, normal breast tissue, which has no demonstrated transferrin binding, contains only 0.18 Mug of receptor per mg of protein by this method.
Identification of Ovotransferrin as an Acute Phase Protein in Chickens
Poultry Science, 2002
Inflammation is homeostatic process associated with a variety of cellular injuries resulting from infections, toxicosis, and physical trauma. The studies on inflammation in avian species are limited. To understand the inflammation-induced changes, 4-wk-old male broiler chickens were subjected to experimental inflammation by a subcutaneous injection of croton oil (inflammatory) with changes in serum measured over time and were compared with birds treated similarly with olive oil (injected control). Croton oil treatment significantly elevated serum interleukin (IL)-6 concentrations and heterophil counts by 6 and 16 h postinjection, respectively, which returned to the basal levels of controls at 16 and 24 h, respectively. Croton oil treatment affected the serum protein profiles of chickens as assessed by SDS-PAGE and densitometric analyses. Compared with olive oil-injected or noninjected chicken sera, there were increases in the density of protein bands corresponding to molecular weights (MW) of 42, 65, 200, and 219 kDa and decreases