Comparison of bovine serum transferrin A and D2. I. Amino acid residue differences

A comparison is made of single components of the homozygous variants A and D₂ of bovine serum transferrin by tryptic, chymotryptic and cyanogen bromide digestion. It is concluded that there are three substitutions A:D₂ - Glu:Asp, Lys: Arg and Asp:Gly. In the light of the recent work of Brocket al. (1980) it is concluded that all three substitutions occur in the C-terminal sequence of the chain. By homology with the sequence of human serum transferrin (MacGillivray et al., 1982) the Lys:Arg and Asp:Gly substitutions probably occur at residues 527 and 446, respectively, from the N -terminus. The Asp:Gly substitution is considered more likely than our earlier conclusion (Maeda, McKenzie & Shaw, 1977) that there is a deletion in the chain of D₂ (A:D₂, Asp: —). The location of the Glu:Asp substitution is not known.     

Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C

In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid.     

Comparison of bovine serum transferrin A and D2. II. Glycopeptides

Glycopeptides are isolated from subtilisin and pronase digests of whole bovine serum transferrin A and D₂. The two variants yield glycopeptides with identical ami-no acid composition. Hence, there is probably no amino acid substitution in this region of the peptide chain. Amino acid sequence determination of one glycopeptide (subtilisin glycopeptide 8) gives the sequence: (CHO)Asn-Ser-Ser-Leu-Cys. This sequence is identical with that of residues 491–495 of the sequence for human serum transferrin (MacGillivray et al., 1982) except that in the bovine transferrin, Asp is replaced by Asn, enabling carbohydrate attachment. A second glycopeptide sequence Arg-(CHO)Asn-Ala-Thr-Tyr is observed, and the significance discussed in relation to carbohydrate moieties of serum glycoproteins.     

Molecular weight heterogeneity of bovine serum transferrin

Cattle transferrin (Tf) was purified from serum of variant A and four bands were isolated. The peptide patterns of these bands when cleaved by proteases and by cyanogen bromide (BrCN) were compared, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Variant A displays two groups of molecules--large (L) and small (S)--on SDS-PAGE; the molecular weight of the L bands is 78,400 +/- 1700 and that of the S bands is 72,000 +/- 1700. However, S-band molecules could not be produced artificially by heat treatment of L bands in the presence of SDS and 2-mercaptoethanol. Since deglycosylated Tf also showed molecular weight heterogeneity, the sugar moieties of Tf other than sialic acids were not the cause of the heterogeneity. These results suggest that heterogeneity within a given variant is due to the presence of two kinds of molecule of different molecular weight. The peptide patterns of L and S bands produced by proteases and those produced by BrCN were distinctly different from each other. However, the stepwise degradation patterns of L and S bands resembled each other when treated with both chymotrypsin and BrCN. This suggests that L-band molecules differ from S-band molecules only in the presence of an additional carboxyl-terminal peptide.     

Electrophoretic variants of serum transferrin in wild pig populations of Japan

Variants of serum transferrin in Japanese wild pig, Sus scrofa leucomystax, and Ryukyu wild pig, S.s. riukiuanus, of Japan were investigated by using starch gel electrophoresis. Five phenotypes, TfB, BC, C, CX and X, were observed, of which two, TfCX and X, are new variants. Comparison of gene frequency estimates which were calculated for each population showed remarkable geographic differences among several populations of these two subspecies.     

Heterogeneity of horse transferrin: the role of carbohydrate moiety

Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electro-phoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatogra-phy and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N -terminus (glutamic acid followed by glu-tamine) and the C -terminus (valine). Differences were found in carbohydrate composition between components 2a and 4b. Component 2a contained 10 moles of sugar components per mole of protein (4 hexoses, 4 hexosamines and 2 sialic acids), while component 4b contained twice the number of both total carbohydrates and individual sugar components. Carbohydrates were identified as mannose and galac-tose (ratio mannose:galactose = 1.5:l), N -acetylglucosamine and N -acetylneu-raminic acid. At present it is not clear whether the difference between the two components resides solely in the difference of carbohydrate contents. It is proposed that component 2a has one diantennary glycan, while component 4b has two.     

Phylogenetical and ontogenetical studies on the molecular weight heterogeneity of bovine serum transferrin

Antitransferrin (Tf) rabbit serum was highly specific: it reacted with Tfs of ruminants, such as European breeds and Zebu breeds of cattle, Bali cattle, banteng, swamp and river types of water buffalo, anoa, goat, sheep, deer, antelope, camel, and giraffe, but did not react with serum of other non-ruminant species, such as pig, wild boar, hippopotamus, horse, rabbit, rat, chicken, etc. Electrophoresis of Tf and immunoglobulin G (IgG) complexes was carried out using sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). Within ruminants, the following species showed two Tf molecules on SDS-PAGE; European and Zebu cattle, Bali cattle, banteng, two types of water buffalo, and two species of anoa. Other ruminants, sheep, goat, deer, antelope, camel, and giraffe, etc., showed only one Tf molecule. The Tf heterogeneity in molecular weight was, thus, restricted to Bos, Bubalus, and Anoa. The molecular weight of Tf of water buffalo was slightly larger than that of cattle on the gel. The peptide pattern from cyanogen bromide cleavage of Tf of the water buffalo differed clearly from that of cattle. Fetal Tf showed only one molecule during development, but a newborn calf has two Tf molecules, (one large and one small) within 18 hr after birth. We suggest, therefore, that the small molecules formed during the last month of gestation. The peptide patterns of adult and fetal Tfs cleaved by cyanogen bromide differed with regard to the two large peptides; fetal Tf, lacking the second-largest peptide, had twice the amount of the largest peptide compared with adult Tf. From these results, we suggest that a change in peptide sequence occurs from the last month of gestation, when the largest peptide is degraded to the second largest. However, a Tf-like protein detected in the liver microsomal fraction has only one molecular size, both in adult and in fetal livers.     

A new, partially deficient transferrin variant in the pig

A new, partially deficient transferrin variant (TF F) was found in serum samples of a wild boar and his offspring from crossing with Pietrain sows. Family analyses confirmed its genetic control by a codominant allele, TF^F. Finding of this new variant has brought the total number of pig transferrin variants to nine (F, I, A, B, C, X, D', E, D).     

Polymorphism of transferrin in carp (Cyprinus carpio L.): Genetic determination,isolation, and partial characterization

Seven transferrin variants (A, B, C, D, E, F, and G) have been found in carp sera (Cyprinus carpio L.). Genetic analysis involves five variants and agrees with the hypothesis of simple codominant autosomal inheritance at one transferrin (Tf) locus in spite of the fact that the carp is a tetraploid in relation to other species of the same family. Carp populations from three regions were studied which differed in gene frequencies. Individual populations were in Hardy—Weinberg equilibrium. The polymorphism of carp transferrins can be used for the identification of offspring of single parent pairs, stocked in one pond. Transferrins have been isolated and characterized. Homozygous phenotypes comprised four iron-binding components differing in electrophoretic mobility. This heterogeneity is not caused by sialic acid, which is absent. Amino acid composition, content of hexoses (1 mole/mole of protein) and hexosamines (1 mole/mole of protein), molecular weight (70,000), and the isoelectric point (5.0) have been determined. No N-terminal amino acid could be detected.     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.     

Distribution of serum transferrin in spot, Leiostomus xanthurus

Serum transferrin distribution was studied in 128 spot, Leiostomus xanthurus, obtained from the lower Rappahannock River of Virginia. Three phenotypes designated as TfA, TfB and TfAB were observed. These are described as representing two codominant alleles at a single gene locus. Phenotypic distribution did not differ significantly from that predicted by Hardy-Weinberg analysis.     

Identification of genetic variation in the bovine major histocompatibility complex DRß-like genes using sequenced bovine genomic probes

Two bovine genomic clones that crosshybridize with HLA-DR beta cDNA have been isolated. Nucleotide sequence analysis of the beta 1, beta 2 and transmembrane (TM) exon regions for one of these clones revealed 70, 89 and 86% identity with the corresponding HLA-DR beta exons. Stop codons are present in the beta 1 and TM exons and a single base deletion toward the 3' end of the TM exon negates the consensus sequence for exon/intron splicing. Therefore, we conclude this is a bovine DR beta-like pseudogene, BoDR beta I. Exon-containing regions have been used as probes in Southern blot analyses of bovine genomic DNA digested with EcoRI. The beta 2 exon of BoDR beta I results in prominent bands of 18.9, 7.8, 7.2, 6.4, 5.6, 3.6, 3.0 and 2.7 kb. Polymorphisms were observed for all but the 18.9 kb band and at least three of these bands were identified in each of the 185 animals sampled. A probe containing the TM exon of BoDR beta I hybridizes only to the 5.6- and 3.6-kb bands, suggesting that these are allelic bands corresponding to this pseudogene. Results from hybridizations of a TM exon-containing probe of the second bovine DR beta-like clone (BoDR beta II) suggest that the 6.4- and 2.7-kb bands correspond to this second bovine gene. A nonpolymorphic 8.1-kb band results from a probe containing the BoDR beta I beta 1 exon. Major differences in frequency for the 6.4/2.7 alleles were found for the four breeds sampled.     

Two new variants of the bovine PAS-1 glycoprotein

Two new alleles ( A and E ) of the bovine MUC locus which encodes PAS-1 protein, a glycoprotein of the milk fat globule membrane, are reported. The A allele was found in Italian Brown while E was present in the Jersey and the Piedmont breeds.     

Study on the interaction of a cyanine dye with human serum transferrin

Complexation between the primary carrier of ligands in blood plasma, human serum transferrin (Tf), and a cyanine dye, 3,3′‐di(3‐sulfopropyl)‐4,5,4′,5′‐dibenzo‐9‐phenyl‐thiacarbocyanine‐triethylam monium salt (PTC) was investigated using fluorescence spectra, UV/Vis absorption spectra, synchronous fluorescence spectra, circular dichroism (CD) and molecular dynamic docking. The experimental results demonstrate that the formation of PTC–Tf complex is stabilized by van der Waal's interactions and hydrogen bonds, and the binding constants were found to be 8.55 × 10⁶, 8.19 × 10⁶ and 1.75 × 10⁴ M^?1. Moreover, fluorescence experiments prove that the operational mechanism for the fluorescence quenching is static quenching and non‐radiative energy transfer. Structural investigation of the PTC–Tf complexes via synchronous fluorescence spectra and CD showed that the structure of Tf became more stable with a major increase in the α‐helix content and increased polarity around the tryptophan residues after PTC binding. In addition, molecular modeling highlights the residues located in the N‐lobe, which retain high affinity for PTC. The mode of action of the PTC–Tf complex is illustrated by these results, and may provide an effective pathway for the transport and targeted delivery of antitumor agents. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural analysis of seminal and serum human transferrin by second derivative spectrometry and fluorescence measurements

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd·HCl) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturating agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation = 280 nm); (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5–4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0–2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We can conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.