Transferrin polymorphism in Herdwick sheep.

The transferrin system in Herdwick sheep, bred at Compton, was investigated with special reference to the susceptibility of the flock to experimentally produced scrapie. No significant correlations were observed between susceptibility and transferrin phenotypes.     

Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.     

Cholesteryl hemisuccinate's inductive effect on membrane rigidization regarding both, its remodelling of the cells' surface receptor pattern and its decreasing the natural killer susceptibility of K-562 cells.

The relationship of changes in membrane fluidity to natural killer susceptibility of K-562 target cells was investigated. Membrane rigidization was performed by the chemical modulator cholesteryl hemisuccinate. Steady-state fluorescence polarization measurements of the diphenyl hexatriene labelled, modified K-562 cells revealed that cholesteryl hemisuccinate increased the structural order of the hydrophobic region of membranes in a dose dependent way. Investigation of natural killer susceptibility followed by 51Cr release assay indicated that modified cells are less sensitive to natural killer attack. To elucidate whether surface structures such as transferrin and lectin receptors are associated with the altered susceptibility, the surface density of these receptors was followed by (I-125)-transferrin binding assay and quantitative immunofluorescence. We found that the number of transferrin and concanavalin A receptors increased by a factor of 2.44 and 2.00, respectively, whereas that of the wheat germ agglutinin receptor failed to exhibit any changes upon rigidization. From the results we concluded that i the membrane structural order does influence the natural killer susceptibility, ii changes in membrane structural order result in alteration of the number of cell surface transferrin and lectin receptors, iii however, no direct relationship seems to exist between these two events.     

Transformation with SV40 virus prevents retinoic acid inhibition of plasma membrane NADH diferric transferrin reductase in rat liver cells

Retinoic acid inhibits the reduction of diferric transferrin through the transplasma membrane electron transport system on fetal rat liver cells infected with a temperature-sensitive SV40 virus when the cells are in the nontransformed state cultured at 40°C. When the cells are in the transformed state (grown at the permissive 33°C temperature), retinoic acid does not inhibit the diferric transferrin reduction. Inhibition of activity of nontransformed cells is specific for retinoic acid with only slight inhibition by retinol and retinyl acetate at higher concentrations. Isolated rat liver plasma membrane NADH diferric transferrin reductase is also inhibited by retinoic acid. The effect of transformation with SV40 virus to decrease susceptibility to retinoic acid inhibition stands in contrast to much greater adriamycin inhibition of diferric transferrin reduction in the transformed cells than in nontransformed cells.     

Binding and surface exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins.

Neisseria gonorrhoeae is capable of iron utilization from human transferrin in a receptor-mediated event. Transferrin-binding protein 1 (Tbp1) and Tbp2 have been implicated in transferrin receptor function, but their specific roles in transferrin binding and transferrin iron utilization have not yet been defined. We utilized specific gonococcal mutants lacking Tbp1 or Tbp2 to assess the relative transferrin-binding properties of each protein independently of the other. The apparent affinities of the wild-type transferrin receptor and of Tbp1 and Tbp2 individually were much higher than previously estimated for the gonococcal receptor and similar to the estimates for the mammalian transferrin receptor. The binding parameters of both of the mutants were distinct from those of the parent, which expressed two transferrin-binding sites. Tbp2 discriminated between ferrated transferrin and apotransferrin, while Tbp1 did not. Results of transferrin-binding affinity purification, and protease accessibility experiments were consistent with the hypothesis that Tbp1 and Tbp2 interact in the wild-type strain, although both proteins were capable of binding to transferrin independently when separated in the mutants. The presence of Tbp1 partially protected Tbp2 from trypsin proteolysis, and Tbp2 also protected Tbp1 from trypsin exposure. Addition of transferrin to wild-type but not mutant cells protected Tbp1 from trypsin but increased the trypsin susceptibility of Tbp2. These observations indicate that Tbp1 and Tbp2 function together in the wild-type strain to evoke binding conformations that are distinct from those expressed by the mutants lacking either protein.     

Experimental candidiasis associated with liver injury

In an attempt to perform a further investigation on the proposal that an increasing susceptibility to Candida infection in liver injury may be related to unsaturated transferin level (UIBC) and/or to a total amount of transferrin represented by TIBC, we conducted experimental candidiasis using mice with galactosamine-induced liver injury and investigated the effect of preadministration of transferrin prior to inoculation of Candida albicans. Final mortality was 10% in the mice without liver injury and without transferrin (Group 1) and ones with liver injury and with transferrin (Group 3). By contrast a 50% mortality was given in ones only with liver injury (Group 2). The TIBCs in Groups 1 and 3 were significantly higher than that in Group 2. The UIBCs in Groups 2 and 3, although there was no significant difference between them, were significantly lower than that in Group 1. This study confirmed that transferrin (TIBC) may have a direct deterring effect on systemic Candida infection and the decreased TIBC in the liver injury enhances the growth of C. albicans.     

Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake

The human transferrin receptor is post-translationally modified by the addition of a fatty acyl moiety. In earlier studies, transient expression in Cos cells of human transferrin receptors in which Cys62 or Cys67 was altered to serine provided evidence that Cys62 is the major acylation site of the receptor (Jing, S., and Trowbridge, I. S. (1987) EMBO J. 6, 327-331). To determine whether acylation of the receptor is required for high efficiency endocytosis and iron uptake, wild type and mutant human transferrin receptors have been stably expressed in chick embryo fibroblasts using a helper-independent retroviral vector. In marked contrast to Cos cells, both Cys62 and Cys67 of the wild type human transferrin receptor were acylated in chick embryo fibroblasts. Moreover, their modification to serine did not abolish palmitate labeling, implying that one or both of these serine residues could serve as alternative lipid attachment sites in these cells. The relative labeling of mutant receptors with palmitate and the susceptibility of their lipid moieties to cleavage by hydroxylamine were consistent with Ser67 but not Ser62 serving as a lipid attachment site. Consequently, to obtain human transferrin receptors lacking covalently bound lipid in the chick embryo fibroblasts, it was necessary to alter Cys62 and Cys67 to alanine. Functional studies indicated that these non-acylated mutant receptors were internalized efficiently and mediated iron uptake from human transferrin at a similar rate to that of wild type receptors. We conclude, therefore, that acylation of the human transferrin receptor is not essential for endocytosis and recycling.     

Serum protein polymorphisms in patients with primary Sj?gren's syndrome

The distribution of 4 serum proteins, the protease inhibitor alpha-1-antitrypsin, haptoglobin, transferrin and group-specific component or vitamin D-binding protein, were examined in 26 patients with primary Sj?gren's syndrome and 208 healthy donor controls. No significant associations were found between any of the 4 systems and susceptibility to primary Sj?gren's syndrome.     

Differential susceptibility of transferrin glycoforms to chymotrypsin: a proteomics approach to the detection of carbohydrate-deficient transferrin

Transferrin exhibits heterogeneity in glycosylation characteristic of pathological changes in alcohol abuse and congenital disorders in glycosylation. This study investigated an alternative approach in the detection of carbohydrate-deficient transferrin based on the premise that glycosylation may afford some degree of protection to proteolytic action. Differential susceptibility to proteolysis by chymotrypsin was demonstrated for normal glycosylated and nonglycosylated recombinant human transferrin, using reverse-phase (RP) HPLC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and LC-tandem mass spectrometry (MS/MS). Peptide fragmentation profiles were consistent with a predominantly high-specificity cleavage pattern of chymotrypsin. The observed peptide fragmentation profile showed that the C-lobe of recombinant full-length nonglycosylated transferrin (rhTf-NG) appeared to be preferentially cleaved, while cleavage of the N-lobe was restricted to the N-terminal and link sequence regions. Although chymotryptic cleavage sites abound in the N-lobe, their resistance to cleavage was independent of glycosylation. Compared to previous studies of lactoferrin, our data suggest disparity in the role by which glycosylation exerts a protective effect in the siderophilin family. It was clear from the transferrin digestions analyzed by HPLC that N-linked glycosylation did confer protection from proteolysis by chymotrypsin. After fragmentation, a range of peptides representing previously cryptic epitopes were identified as potential candidates for an immunological approach to differentiate between the different transferrin glycoforms. Based on its proximity to the Asn413 glycosylation site, a 15-mer peptide, m/z 1690.472 (NKSDNCEDTPEAGYF), was identified as a suitable candidate for raising anti-peptide antibodies for subsequent immunological detection. This novel approach could form the basis for an alternative assay or reference method for the detection of carbohydrate-deficient transferrin.     

Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells

Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919-1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.     

The interaction of anions with native and phenylglyoxal-modified human serum transferrin

The interaction of various anions with human serum transferrin was investigated due to the concomitant binding of iron and a synergistic anion to form the transferrin-anion-iron complex. Two tetrahedral oxyanion oxidizing agents, periodate and permanganate, were found to partially inactivate transferrin when used at equimolar ratios of oxidizing agent to protein active sites. Hypochlorite, a strong oxidizing agent with little structural similarity to periodate and permanganate, had little effect on iron-binding activity when used at similar low molar ratios of reagent to transferrin active sites. Transferrin treated with a 3:1 molar ratio of periodate or permanganate to active sites lost 74 or 67% of its iron-binding capacity, respectively. The composition of the buffer affected the extent of transferrin inactivation by periodate and permanganate; for example, the extent of inactivation by periodate was threefold greater in a borate buffer than in a phosphate buffer. Comparative oxidations in buffer systems suggest the following order of affinity of three buffer anions for the apotransferrin metal-binding center: phosphate greater than bicarbonate greater than borate. The interaction of phosphate ions with the iron-transferrin complex was also examined due to the increased susceptibility to periodate inactivation of iron-saturated transferrin in phosphate buffer (M. H. Penner, R. B. Yamasaki, D. T. Osuga, D. R. Babin, C. F. Meares, and R. E. Feeney (1983) Arch. Biochem. Biophys. 225, 740-747). The apparent destabilization of the iron-transferrin complex in phosphate buffer was found to be due to the competitive removal of iron by phosphate from the iron-protein complex. We found that phenylglyoxal-modified Fe-transferrin, with no loss of bound iron, was much more resistant to iron removal by phosphate and other competitive chelators.     

Transferrin C subtypes and occupational photodermatosis of the face

In a factory in northern Sweden where 120 workers were uniformly exposed to photoactive substances 73 developed occupational facial eczema while 47 showed no reaction. The workers were examined with respect to 16 genetic marker systems: HLA, blood groups (ABO, Rh, MNSs, P, K, Le and Fy) and serum groups (Hp, Tf, Gc, Pi, Bf, C3, C4 and C6). Between reactors and nonreactors the following differences were found: (1) a significant decrease (p less than 0.05) of HLA A11 among the reactors; (2) a significant increase (p less than 0.05) of the C3 FS type among the reactors; (3) a highly significant increase (p less than 0.001) of the transferrin C2 gene and of the C2 variant among the reactors. The association with Tf C2 remained significant also after correction for number of significance tests. Since transferrin (iron) is known to catalyze the formation of hydroxyl radicals we hypothesize that the Tf C2 variant is more efficient in promoting radical formation and thereby cell damage. Other results supporting the notion that transferrin C2 may be associated with an increased susceptibility to toxic damage are discussed.     

Enzymic synthesis of steroid sulphates. XVII. On the structure of bovine estrogen sulphotransferase

Estrogen sulphotransferase plays a major role in controlling intracellular levels of 17 beta-estradiol in human mammary cancer cells and human endometrium. Bovine estrogen sulphotransferase c-DNA has recently been cloned; the encoded protein having a maximum Mr of 35,000 (Nash, A.R. et al. (1988) Aust. J. Biol. Sci. 41, 507-516). Enzyme of Mr 35,000 by SDS-PAGE has now been isolated and cyanogen bromide-cleaved peptides sequenced. The latter were identified in the c-DNA-predicted amino acid sequence which confirms that the active enzyme (Mr approximately 70,000) exists as a dimer of identical subunits. Sequence data on similar peptides isolated from an enzyme preparation containing a protein of Mr 74,000 as the major species on SDS-PAGE, which was previously thought to represent the enzyme, suggested that this protein was transferrin. This was confirmed by PAGE, SDS-PAGE, susceptibility to neuraminidase and reaction with bovine transferrin antibody. Isoelectric focusing experiments show that active enzyme exists in two or three polymorphic forms (pI values 5.3, 5.7 and possibly 5.9) having similar physicochemical properties of polymorphic forms of transferrin so that they overlap on ion-exchange chromatography and PAGE. The enzyme shows some homology to the amino acid sequence close to the Fe-binding site in lactoferrin and the question is raised as to the possible presence of a tightly bound metal in estrogen sulphotransferase involved in the binding of adenosine 3'-phosphate 5'-phosphosulphate.     

Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.     

Iron requirement and chelator production of staphylococci,Streptococcus faecalis and enterobacteriaceae

The effect of iron deprivation on growth of 101 aerobic strains of gram-positive and gram-negative bacteria was studied on agar media in the presence of various concentrations of the synthetic iron chelator ethylene diamine diorthohydroxyphenyl acetic acid (EDDA) and the iron binding protein transferrin.Growth of Staphylococcus epidermidis was inhibited by 15mm EDDA and 1.5mm transferrin. Staphylococcus aureus was only inhibited by 44mm EDDA and not by transferrin. None of the strains of S. faecalis was inhibited. The majority of the enterobacteriaceae (E. coli, Salmonella spp, Klebsiella spp) was inhibited by 44mm EDDA and 1.5mm transferrin. The relation between susceptibility and concentration of EDDA and transferrin was expressed as S-value for each species. Iron supply with various iron compounds could restore the effects of inhibition.In all species except in S. faecalis iron chelator production could be demonstrated, using indicator plates of media containing EDDA and flooded with 10⁴–10⁵ colony forming units of indicator organisms.The iron chelator of both S. epidermidis and S. aureus could stimulate growth of S. epidermidis, but not that of enterobacteriaceae. Iron chelators from all gram-negative bacteria were functionally interchangeable, but did not stimulate growth of gram-positive bacteria.     

Antioxidant status and lipoprotein peroxidation in chronic fatigue syndrome

The aetiology and pathogenesis of the Chronic Fatigue Syndrome (CFS) are still largely unresolved. Accompanying metabolic disorders such as selective n-6 fatty acid depletion suggest that oxidative stress and more specifically lipid peroxidation might play a role in its pathogenesis. In order to investigate this hypothesis, oxidant-antioxidant status and its impact on lipoprotein peroxidation in vitro was examined in 61 patients with unexplained fatigue lasting more than 1 month. They were subdivided into 2 groups: group CFS+ (33 subjects) fulfilled the 1988 Center of Disease Control criteria for CFS and group CFS- did not but was similar as regards age, sex distribution and clinical characteristics. Antioxidant status was similar in the 2 groups except for lower serum transferrin in the CFS + (mean (95 % CI) 2.41 (2.28-2.54) versus 2.73 (2.54-2.92) g/L in the CFS-, p = 0.009) and higher lipoprotein peroxidation in vitro: 6630 (5949-7312) versus 5581 (4852-6310) nmol MDA/mg LDL and VLDL cholesterol x minutes, p = 0.035). CFS intensified the influence of LDL cholesterol (p = 0.012) and of transferrin (p = 0.045) on peroxidation in vitro, suggesting additional pro-oxidant effects. These results indicate that patients with CFS have increased susceptibility of LDL and VLDL to copper-induced peroxidation and that this is related both to their lower levels of serum transferrin and to other unidentified pro-oxidising effects of CFS.     

Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type-1

To investigate whether transferrin receptor (CD71) expression is affected by acute HIV-1 infection, three different lymphoid cell lines (MT-4, SUPT-1, H9) were infected with HIV-1 and tested for surface CD71 expression after different incubation periods depending on cell survival after infection. We found that expression of surface CD71 was lower in cells infected with HIV-1 than in uninfected controls: the timing and extent of this down-modulation depended apparently on the different susceptibility of the cell lines to HIV-1 infection and cytopathogenicity. Citrate, a molecule capable of chelating iron, dose-dependently prevented down-modulation of surface CD71 in HIV-1 infected cells as well as viral cytopathic effects. We conclude that (i) expression of surface transferrin receptors is down-modulated by acute HIV-1 infection in T lymphoid cells, that (ii) this cell phenotypic modulation is associated with the cytopathic effects of the virus, and that (iii) these phenomena are modulated by iron chelation. These results support the view that iron metabolism may be an important area for interaction between HIV-1 and human cells.     

Effect of glycerol on the molecular properties of cerebrosides, sulphatides and gangliosides in monolayers.

Apolactoferrin and apotransferrin lost their ability to subsequently bind iron when exposed to an excess of either HOCl or myeloperoxidase plus H2O2 and Cl-. Apolactoferrin, however, was more resistant than apotransferrin. By oxidizing a mixture of the two proteins, then separating them by immunoprecipitation, the difference in susceptibility was shown to be due to the greater reactivity of transferrin iron-binding groups, rather than protective groups on the lactoferrin molecule. The iron-saturated proteins were much more resistant to oxidative modification than the apoproteins. The greater resistance of apolactoferrin should be advantageous for maintaining its iron binding capacity when co-released with myeloperoxidase and reactive oxygen species from stimulated neutrophils.     

Transferrin and iron in cultured chick embryonic neurons: a comparison between human and chick transferrins

Transferrin was not required for the short-term survival of cultured chick retinal neurons. Both human and chick transferrin failed to enhance the in vitro survival of 8- or 11-day embryonic chick retinal neurons when cultured in a defined medium. Furthermore, maintenance of neurons in the presence of chick transferrin antibody did not alter in vitro survival. Retinal neurons, however, could bind and internalize human or chick transferrin when assayed for by fluorescence immunohistochemical techniques. Binding and internalization of chick transferrin appeared to be greater than human transferrin. Iron uptake was measured in cultures maintained in the absence of transferrin. After incubation with 59FeCl3, iron uptake was 3.5 +/- 1.1 fmoles/cell. The presence of chick transferrin antibody did not significantly alter the amount of iron uptake occurring in this assay. In a comparison of human and chick transferrin mediated iron uptake, chick transferrin was 50% more effective than human transferrin in transporting iron. This study demonstrates that cultured embryonic retinal neurons are not dependent on transferrin for survival or iron uptake, although they actively bind and internalize transferrin. Results also demonstrate that whereas cultured chick retinal neurons can bind and utilize human transferrin, they do so with less efficiency than chick transferrin.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.