Translocation of different forms of transferrin from blood to bile in the rat.

Five different forms of transferrin (rat apo [iron-free], rat diferric, diferric rat asialo, human diferric, and diferric human asialotransferrin type 3) were used to monitor the passage of this protein and its metal to the bile. Cumulative biliary excretion of the dose over 3 hours was determined. In addition, an excretion profile was constructed from the concentration of tracer in bile samples collected over 10-minute intervals. The profile obtained with apotransferrin was very similar to that found in an earlier study with albumin, the implication being that the apo form is transferred passively (e.g., by diffusion). Behavior of rat diferric transferrin, however, was consistent with the assumption that this form is transferred both passively and actively (i.e., in vesicles). The three other transferrins were investigated with the intent of broadening the spectrum of ligand affinities for the plasmalemma of hepatocyte. The higher this attraction was, the larger fraction of the dose appeared in bile. When transferrin was targeted to lysosomes, the bile contained several intermediate proteolytic fragments. Double-labeled (125I, 59Fe) transferrin was used to measure recovery of iron (Fe) relative to the protein (P) in bile. With rat diferric transferrin, the Fe/P ratio was 0.72. Lower values were recorded with transferrins (human or asialo) that had higher affinities for the plasmalemma and therefore were expected to be transported to a larger extent in vesicles. Of the biliary 59Fe, 85% to 92% was protein bound. The proportion of the protein-bound fraction was essentially independent of the magnitude of Fe/P ratios.

Effect of transferrin on the degradation of glycoproteins bearing a hybrid or high-mannose glycan by alveolar macrophages.

A subfraction of hen ovalbumin and a special form of rat transferrin, possessing a hybrid glycan, were studied with respect to their binding to, and degradation by macrophages isolated from the rat lung. Both ligands were found to be degraded partly through the mannose receptor pathway and partly by another mechanism, presumably adsorptive pinocytosis. Catabolism of both proteins was markedly increased by adding standard (i.e., normally glycosylated) transferrin from various species to the medium. This increase was not diminished, or even augmented, when Ca2+ was depleted in the medium, thus implying involvement of the pinocytic pathway rather than the mannose receptor. Binding to the cell surface of both ligands was altered in the presence of transferrin. A hypothesis is advanced to suggest that when transferrin is bound to a component of macrophage plasmalemma, thought to be the glycosaminoglycan of heparan sulfate, its conformation may change in such a way that it attracts other proteins. Protein molecules temporarily captured by adsorbed transferrin would then be taken up by a "piggyback" process and degraded.

Observations with a residualizing label in use to map the catabolic sites of plasma proteins.

Rat albumin, immunoglobulin G, transferrin, and aglycotransferrin were prepared for the comparison of their sites of degradation in rats. Iodotyramine-cellobiose was used as the residualizing label and a tyrosine-iodinated portion of the corresponding protein was used as the marker of extracellular undegraded protein. Each protein yielded a distinct distribution (or map) of catabolic activity throughout the body when expressed as percent dose accumulated per gram of tissue. The maps for albumin and transferrin were broadly comparable, whereas those for immunoglobulin G and aglycotransferrin were markedly different. As a whole entity, the liver appeared to top the list of organs/tissues contributing to the degradation of albumin and transferrin. Additional experiments aimed at facilitating the interpretation of results with residualizing labels were carried out with denatured albumin, asialofetuin, and human asialotransferrin type 3. These showed that various types of cells retained the label for markedly different periods of time. We feel, therefore, that the technique is more suited for making comparative measurements than for obtaining degradation rates as absolute values in a given anatomical location.

Transport of lactoferrin from blood to bile in the rat.

The bile contains small quantities of lactoferrin, the origin of which is uncertain. For this reason, we studied the liver's capability of transferring lactoferrin from the plasma to the bile by injecting a dose (10 to 20 micrograms/100 gm) of labeled bovine lactoferrin intravenously and following its appearance in bile over 3 hr. Whether diferric or iron free, lactoferrin peaked in the bile 35 min after administration (i.e., the same time as bovine lactoperoxidase and diferric rat transferrin). However, only a small portion of the lactoferrin dose (approximately 1%) was recovered with the bile in 3 hr. On the basis of autoradiographic evidence, the excreted lactoferrin appeared intact. The biliary excretion profile of albumin, a protein thought to reach the canaliculus by paracellular diffusion, was notably devoid of a peak. This, together with competition observed between lactoferrin and lactoperoxidase on one hand and 2Fe-transferrin and lactoferrin on the other for transfer to bile, suggests that lactoferrin is routed through the hepatocyte in vesicles. The process is initiated by binding to a plasma membrane component to which lactoperoxidase and 2Fe-transferrin can also bind. Most 59Fe bound to lactoferrin accompanied the protein carrier to the bile. We conclude that under normal circumstances (i.e., when concentration of lactoferrin in the plasma is very low), lactoferrin transferred from plasma by the liver is probably not the major source of this protein in bile.

Interaction of transferrin and its iron-binding fragments with heparin.

The interaction of heparin with transferrin (Tf; bovine and rat) and the isolated iron-binding lobes of bovine Tf were investigated. Affinity chromatography of rat Tf on heparin-agarose showed that interaction depended on both the iron content of Tf and the pH of the medium. Both the iron-free and iron-saturated forms of Tf were strongly bound by the column at pH 5.6, but only the iron-free form revealed significant affinity at pH 7.4. Desialylation of Tf moderately promoted interaction, treatment with cyclohexanedione moderately reduced interaction, and succinylation abolished it altogether. In the presence of heparin, iron release from the N-terminal lobe of native bovine Tf was accelerated and from the C-terminal lobe it was slightly reduced. The heparin effect remained qualitatively the same on each lobe after their separation by tryptic digestion and DEAE-cellulose chromatography. The affinity of native bovine Tf for heparin was very close to that of its isolated N-terminal lobe, thus suggesting that it is this portion of the molecule that binds to the glycosaminoglycan. It is concluded that the consequences for iron-binding strength of the two transferrin lobes are diagonally opposite when Tf is bound to heparin as opposed to its natural cell-surface receptor.

Lactoferrin interferes with uptake of iron from transferrin and asialotransferrin by the rat liver.

Intravenous injection of bovine or human lactoferrin (6.25 x 10(-2) mumol/100 g body wt) in rats resulted in marked reduction of hepatic iron uptake from transferrin and asialotransferrin. The effect was dose dependent, saturable at approximately 5 mg/100 g body wt, and independent of lactoferrin's iron content. At this dose level, iron uptake from transferrin was reduced by 28% and from asialotransferrin by 43% in experiments lasting 90 min. Bovine lactoperoxidase, another basic protein, was similarly effective. The clearance of asialofetuin and pinocytosis of polyvinylpyrrolidone remained unaffected. Perfusion of isolated rat livers at 4 degrees C showed a strong reduction in asialotransferrin binding in the presence of lactoferrin. Chromatography of hepatic heparan sulfate proteoglycan on immobilized lactoferrin, lactoperoxidase, asialotransferrin, and transferrin showed that it possessed affinity for each of these proteins, more for the first two than the latter two. Heparan sulfate proteoglycan binding and efficacy in reducing hepatic iron uptake were also studied after selective modifications of positively charged amino acids in these proteins. The data obtained are compatible with the hypothesis that lactoferrin and other proteins with similarly high affinity for hepatic heparan sulfate exert their negative effect on iron uptake by preventing transferrin binding to the proteoglycan. The possibility is thus raised that the large number of low-affinity transferrin binding sites reported by earlier investigators for the liver may be heparan sulfate molecules.

The perfused liver is capable of producing all transferrin glycan variants found in the sera of intact rats.

The single oligosaccharide attachment in rat transferrin exhibits marked structural microheterogeneity. In this study we examined whether all microheterogeneous forms of rat transferrin found in plasma are derived from a single organ, such as the liver. To this end we analyzed the glycans of rat transferrin synthesized by the isolated perfused rat liver by a method established earlier for rat transferrin isolated from rat plasma. Our observations provide evidence that the liver can and does produce all variant rat transferrin glycans present in plasma. However, this discovery does not preclude the possibility that extrahepatic sources with an active rat transferrin gene may contribute to the circulation rat transferrin molecules, which bear glycan variants identical to those made by the liver. The glycan spectra of rat transferrin in plasma and in liver perfusate compared closely with each other in a quantitative sense. Nevertheless, rat transferrin in the perfusate was sialylated to a lesser extent and fucosylated to a greater extent than rat transferrin in plasma. These differences could not be eliminated by supplementation of the medium with insulin, dexamethasone, pyruvate and adenine or adenosine either alone or in combinations, nor could it be eliminated by use of a fluorocarbon O2 carrier. In contrast, epidermal growth factor normalized both parameters. The pH of the perfusing medium also influenced sialylation and fucosylation in such a way that higher pH brought these parameters closer to their values in plasma rat transferrin. Lower pH, on the other hand, reduced sialylation and left the fucosylation index unchanged.

In vivo behaviour of rat transferrin bearing a hybrid glycan and its interaction with macrophages.

Production of rat transferrin containing a single hybrid glycan was induced by treating rats with swainsonine, an inhibitor of alpha-mannosidase II. The principal component of this variant transferrin containing one sialic acid residue per mole of protein was separated from other forms of transferrin by anion-exchange chromatography, followed by lectin affinity chromatography. Transferrin bearing the hybrid glycan was degraded in vivo with a half-life of 14 h as compared with 40 h for transferrin containing a standard diantennary glycan. By using 125I-labelled tyramine-cellobiose, a label whose discharge from lysosomes is strongly retarded, organs rich in reticuloendothelial elements (liver, bone marrow, lungs, and spleen) were identified as the major sites of catabolism of the transferrin variant. The liver took up more 59Fe from the variant (26% of the dose in 90 min) than from control rat transferrin (12%). The excess iron uptake was reduced by the intravenous injection of either human transferrin or ovalbumin, and it was abolished by administering both. Macrophages from bone marrow and lungs degraded the transferrin variant in vitro. The degradation was significantly enhanced when transferrin receptors were blocked by human transferrin, and it was significantly reduced by ovalbumin and methyl glucopyranoside.

Transferrin glycosylation in hypoxia.

The aim of this study was to examine the effect of reduced O2 tension on the glycosylation of transferrin. Rats were placed in a hypobaric chamber (380 mmHg) that corresponded to an altitude of 5486 m above sea level for 21 days. The animals responded with marked increases in hematocrit (from 44 to 76%) and cardiac weight, and with reductions in the concentration of plasma transferrin averaging 15%. Analyses of their plasma transferrin by serial anion-exchange and lectin affinity chromatography revealed no changes in the extent of glycan branching. However, there was a moderate rise in the proportion of fucosylated transferrin molecules (fucosylation index) and a slight decrease in the transferrin fraction bearing a tetrasialylated biantennary glycan. The fucosylation index correlated positively with plasma transferrin concentrations in the test animals, but not in the controls. In contradistinction to the situation with transferrin, hypoxic rats exhibited a reduced fucosylation index of immunoglobulin G.

Carbohydrate microheterogeneity of rat serotransferrin. Determination of glycan primary structures and characterization of a new type of trisialylated diantennary glycan.

A previously established procedure [Regoeczi, E., Chindemi, P.A., Rudolph, J. R., Spik, G. & Montreuil, J. (1987) Biochem. Cell Biol. 65, 948-954] was used to isolate from three DEAE-cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf-1 (not retarded by ConA column), rTf-2 (retarded) and rTf-3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400-MHz 1H-NMR spectroscopy. All glycans possessed a common, trimannosyl-N,N'-diacetylchitobiose core with or without one L-fucose alpha-1,6-linked to the Asn-linked GlcNAc. However, there were differences in the antennae. Thus, in rTf-3, both antennae were of the disialylated diantennary N-acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the alpha-1,3-Man-linked antenna in rTf-1 as well as rTf-2 had the sequence: Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GlcNAc(beta 1-2)Man. In addition, the alpha-1,6-Man-linked antenna deviated in rTf-2 from the standard structure by having the sequence: Neu5Ac(alpha 2-3)Gal(beta 1-3)GlcNAc(beta 1-2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE-cellulose fraction (peak V) or rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.

Reduced hepatic iron uptake from rat aglycotransferrin.

Rat aglycotransferrin (rAgTf) was produced from the disialosyl diantennary fraction of rat transferrin (rTf) by treatment with peptide: N-glycosidase F. Following removal of the enzyme by gel filtration and isolation of the deglycosylated protein by lectin chromatography, rAgTf was compared to rTf both in vitro and in vivo. No significant differences were found between the two proteins with respect to affinity for iron and kinetics of Fe release from the N-lobe and C-lobe. The fluorescence emission spectrum of apo-rTf was red-shifted by approximately 3 nm relative to diferric rTf; however, no spectral difference was detected between rTf and rAgTf when the analogous forms (apo or diferric) were compared. Plasma clearance of radioactive iron administered to rats as either rTf or rAgTf was comparable. Reticulocytes took up iron from rAgTf slightly faster than from rTf. In contrast, Fe acquisition by the liver from rAgTf was significantly reduced relative to rTf. This finding contrasts sharply with earlier observations with asialotransferrin (rAsTf) and provides a basis for discounting charge loss as the mechanism of enhanced hepatic Fe uptake from rAsTf. It is suggested that the glycan complement of rTf, while unimportant for interaction of the protein with specific receptors, probably plays a role in the interaction with low-affinity hepatic binding sites.

Absorption of plasma proteins from peritoneal cavity of normal rats.

The present study was undertaken to examine whether the uptake of plasma proteins from the peritoneal cavity is quantitative so that tracers could be introduced that way for measuring their turnover. To this end, the metabolic behavior of seven homologous plasma proteins, labeled with 125I, was compared in rats after intravenous or intraperitoneal administration. The animals were maintained under physiological conditions. Total body radiation measurements showed that the degradation rates of albumin, immunoglobulins A and G, alpha 1-macroglobulin, and transferrin were the same regardless of the route of injection. This implies that these proteins are quantitatively absorbed from the peritoneum without undergoing modifications. The half-life of intraperitoneally injected alpha 1-acid glycoprotein was consistently shorter by an average 9%, thus suggesting that this protein becomes slightly altered if introduced that way. Only one-half of intraperitoneally injected fibrinogen survived normally, whereas the other underwent rapid degradation. The surviving molecules had the same half-life as fibrinogen injected intravenously. The fraction of surviving fibrinogen could be augmented by mixing the dose with serum. Within a wide range of concentrations and quantities injected, the degradation rate of transferrin remained the same. Analysis by deconvolution of the plasma curves of albumin and alpha 1-macroglobulin absorbed from the peritoneum showed that the transport process was independent of protein size and, at least up to 35 mg, of the amount injected. According to the same technique, intraperitoneally administered diferric transferrin retained its iron during passage into the circulation.

Rat aglycotransferrin and human monoglycotransferrin: production and metabolic properties.

Rat transferrin (rTf), containing one complex glycan, and human transferrin (hTf), containing two complex glycans, were treated with peptide:N-glycosidase F (PNGase) under nondenaturing conditions. Apo rTf with a nonfucosylated standard biantennary glycan, but not its diferric counterpart, yielded satisfactory amounts (approximately 55% in 7 h) of aglyco Tf (AgTf). The presence of a chitobiose core fucose reduced yields to approximately 30%, whereas an additional NeuAc on the GlcNAc in the Man(alpha 1-3) branch had no adverse effect. Triton X-100 impaired deglycosylation. The main product (approximately 65%) obtained from apo hTf was monoglyco Tf (MgTf). Analysis of the cyanogen bromide fragments of MgTf revealed that PNGase did not discriminate between the two glycosylation sites of hTf. A negligible portion (2-4%) of AgTf, that was also obtained during the reaction, probably resulted from PNGase action on denatured hTf molecules. Modified Tfs were separated by affinity chromatography, radiolabeled, mixed with another preparation of interest, and injected in rats. Total-body radiation measurements showed that the half-life of rat AgTf was 19-20% shorter than that of rTf but 9% longer than that of asialo Tf. This suggests that close to 76% of the change in the degradative rate observed after desialylating rTf is referable to charge loss rather than the exposure of Gal residues. Human MgTf was catabolized by rats 8-9% faster than the parent protein, while human AgTf behaved in vivo like a denatured protein. It is concluded that sialyl carboxyls are a codeterminant of the normal lifetime of transferrins.

The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin.

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92-94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60-63% of plasma transferrin, was bound by concanavalin A - Sepharose, while the smaller one (30-32% of plasma transferrin) resolved into an unbound (25-27% of plasma transferrin) and a retarded (4-5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20-29%) and an unbound (71-80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.

Metabolic stability of the fucose in rat transferrin.

The metabolic behaviour of the chitobiose core fucose that is a natural constituent of a large proportion of rat transferrin molecules was studied in rats comparatively to that of the polypeptide portion of the glycoprotein by using appropriate labels ([3H]fucose and 125I) and affinity chromatographic techniques (lentil-Sepharose). No evidence was obtained to suggest that this residue is cleaved from the glycan in significant amounts before removal of the entire glycoprotein for catabolism. Similarly, [14C]fucose linked to GlcNAc residues in the antennae of human asialotransferrin was being eliminated in pigeons at the same rate as the polypeptide itself. It is concluded that in spite of transferrin's exposure to the cellular milieu, the fate of its fucose is distinctly different from that of the same in plasma membrane glycoproteins.

Preferential hepatic uptake of iron from rat asialotransferrin: possible engagement of two receptors.

Hepatic iron uptake from and degradation of rat asialotransferrin prepared from the least anionic (major) component of rat transferrin were studied in intact rats. In experiments lasting 60-90 min, rat asialotransferrin delivered a three to four times larger fraction of the Fe dose to the liver than rat transferrin. Variations in the concentration of endogenous circulating rat 2Fe-transferrin by up to 300% failed to affect the enhanced hepatic delivery of Fe from rat asialotransferrin. However, pretreating the animals with a large dose of asialomucin, or fully sialylated human transferrin, or a combination of both did affect the delivery. In all cases, rat asialotransferrin delivered Fe to the liver at rates comparable with those seen with rat transferrin. The reason for the efficacy of human transferrin was clarified in competitive binding studies on rat hepatocytes and reticulocytes, which showed that human transferrin possessed an approximately sevenfold higher affinity for rat transferrin receptors than the homologous protein. These findings suggest that the enhanced hepatic uptake of Fe from rat asialotransferrin is mediated by simultaneous binding of the ligand both through its glycan and transferrin receptor affinity site. Pretreatment with asialomucin and human transferrin had no suppressing effect on basal hepatic delivery of iron from rat 2Fe-transferrin. The data suggest that deposition of a significant fraction of Fe in rat liver from rat transferrin is likely to take place by a mechanism not involving transferrin receptors. Desialylation shortened the metabolic half-life of rat transferrin from 33 to 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of cytotropic compounds on the resialylation of human asialotransferrin type 3 in the rat.

Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented.

Lactoferrin catabolism in the rat liver.

The hepatic uptake and degradation of human diferric 125I-lactoferrin by the liver of the intact rat were studied. After intravenous injection, the tracer was rapidly cleared by the liver, probably by adsorptive pinocytosis, as inferred from observations with a 3,470-fold dose range. Endocytosed lactoferrin was transferred, with a delay, from a light-density subcellular particle to an organelle that had a density similar to lysosomes. The loss of protein bound 125I from the liver was very slow (half-life 2.7 h), and its rate matched closely that of human asialotransferrin type 3. Lactoferrin was found to be a poor substrate for lysosomal hydrolases in vitro. Fucoidin effected the release of a portion of lactoferrin from the liver back into the plasma. By using this agent, indirect evidence was obtained suggesting that a fraction of lactoferrin is being repeatedly endo- and exocytosed (diacytosed) by the liver over prolonged periods of time. Fucosylation failed to impart lactoferrinlike properties on human asialotransferrin type 1, although the derivatized protein exhibited a less than or equal to 10-fold increase in affinity for the liver relative to the parent molecule.

Synthesis and turnover of prothrombin during experimental inflammation in rats.

The response of prothrombin to inflammatory reactions was investigated in rats. Inflammation was induced by the administration of either subcutaneous turpentine or intraperitoneal endotoxin, and its effects were studied 24 h and 48 h later. Albumin and alpha 1-acute-phase globulin served as the controls. There were only insignificant changes in plasma prothrombin concentration during inflammation which contrasts sharply with a decrease in circulating albumin by approximately 25% and an increase in alpha 1-acute-phase globulin by 300-400%. These changes were paralleled by similar changes in the incorporation of [3H]lysine into these proteins during the incubation of liver slices from rats that had been pretreated with the phlogistic agents. Prothrombin catabolism, studied using 131I-prothrombin, was increased by approximately 20%; albumin turnover, studied simultaneously with 125I-albumin, was not significantly affected, though the capillary transfer rate of albumin was significantly elevated 48 h after the induction of inflammation. It is concluded that rat prothrombin is not an acute-phase protein.