A novel disorder of N-glycosylation due to phosphomannose isomerase deficiency.

Three siblings suffered from an unusual disorder of cyclic vomiting and congenital hepatic fibrosis. Serum transferrin isoelectric focusing showed increased asialo- and disialotransferrin isoforms as seen in the carbohydrate-deficient glycoprotein (CDG) syndrome type I. Phosphomannomutase, which is deficient in most patients with type I CDG syndrome, was found to be normal in all three patients. Structural analysis of serum transferrin revealed nonglycosylated, hypoglycosylated, and normoglycosylated transferrin molecules. These findings suggested a defect in the early glycosylation pathway. Phosphomannose isomerase was found to be deficient and the defect was present in leucocytes, fibroblasts, and liver tissue. Phosphomannose isomerase deficiency appears to be a novel glycosylation disorder, which is biochemically indistinguishable from CDG syndrome type I. However, the clinical presentation is entirely different.

Influence of transferrin glycans on receptor binding and iron-donation.

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, beta-galactosidase or N-Acetyl-beta-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions.

Binding of human isotransferrin variants to microvillous and basal membrane vesicles from human term placenta.

Transferrin (Tf)-dependent iron transfer from mother to fetus is mediated by Tf receptors (TfRs) which are present on both microvillous and basal membranes of human placental syncytiotrophoblast. We used microvillous and basal membrane vesicles, both isolated from the same human term placenta, to investigate the binding of [125I]-labelled diferric bi-bi antennary tetra-sialo Tf (bb Tf), bi-tri-antennary penta-sialo Tf (bt Tf) and tri-tri-antennary hexa-sialo Tf (tt Tf). To diminish the effect of endogenous Tf, membrane vesicles were washed before binding of [125I]-Tf. The number of TfRs on microvillous membranes was 6.1 +/- 2.4 (mean +/- s.d., n = 15) times higher than that on basal membranes, whereas the affinity of TfRs on basal membranes was 3.9 +/- 0.4 (mean +/- s.d., n = 15) times higher than that of TfRs on microvillous membranes, irrespective the isoTf used. The affinity constants of TfRs on both microvillous and basal membranes were higher for bb Tf than for bt Tf and higher for bt Tf than for tt Tf. However, these latter differences were rather small and probably not of physiological importance.

Isolation and partial characterization of two porcine spleen ferritin fractions with different electrophoretic mobility.

Ferritin isolated from porcine spleen could routinely be separated in two fractions on nondenaturating gradient gels. Both fractions could be isolated with a purity of 96% when applied to two serially linked columns, each 200 cm in length, packed respectively with Sepharose 4B and Sepharose 6B. Both fractions were similar as judged by electron microscopy. Assessed biochemically fractions were equal with respect to subunit composition, iron and phosphorus content, as well as amino acid composition (with the exception of N-acetylglucosamine). Carbohydrate analysis showed that the fraction with an apparent mass of 440 kDa (= FFL) contained 1.8% (w/w) glycans, whereas the fraction with an apparent mass of 670 kDa (= FFH) contained nearly five times as much (neutral) sugar residues (8.9%, w/w) and 10 times as much sialic acid. This difference in amount of carbohydrate side chains might explain the dissimilarity in electrophoretic mobility of the two fractions.

An in vitro study on the binding of Al(III) to human serum transferrin with the isoelectric focusing technique.

Transferrin saturated with Al3+ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF.

Zonal distribution of receptor binding of trypsin-activated alpha 2-macroglobulin, alpha 2-macroglobulin receptor-associated protein, lactoferrin and transferrin on rat liver parenchymal cells.

Periportal and perivenous rat liver parenchymal cells were isolated according to the digitonin-collagenase perfusion method. Affinities and maximal specific binding of a conjugate of glutathione S-transferase and the alpha 2-macroglobulin receptor-associated protein (GST-39kDaP), of lactoferrin and of transferrin to freshly isolated periportal parenchymal cells in vitro were not significantly different from values obtained with perivenous cells. It is concluded that the receptors for these three ligands show a zonally homogeneous expression in rat liver. The zonal homogeneity in binding observed for GST-39kDaP is at variance with the 1.5-fold higher periportal over perivenous binding of trypsin-activated alpha 2-macroglobulin. Since GST-39kDaP as well as trypsin-activated alpha 2-macroglobulin are ligands for the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein, it is suggested that GST-39kDaP can bind to (an) additional receptor(s) with a higher perivenous expression. The zonal homogeneity observed with lactoferrin, an inhibitor of ligand binding to the lipoprotein remnant receptor, may indicate zonal homogeneity of the lipoprotein remnant receptor. The observed zonal homogeneity of the transferrin receptor suggests an equal and essential need for iron by parenchymal cells across the rat liver acinus in vivo.

Transferrin microheterogeneity as a probe in normal and disease states.

Isoelectric focusing of iron saturated serum has been established as a convenient method for showing transferrin glycan microheterogeneity. In a clinical setting, the method is used in the detection of cerebrospinal fluid leakage, the screening for surreptitious alcohol abuse and in the diagnosis of the carbohydrate deficient glycoprotein syndrome. In normal physiological states it can also be used as a tool to probe for changes in N-glycosylation.

Isolation, purification and characterization of porcine serum transferrin and hemopexin.

Two techniques are described for the isolation of porcine serum transferrin and hemopexin, respectively, yielding nearly pure proteins (> 99%) as tested with crossed immunoelectrophoresis. Porcine transferrin has an estimated molecular weight of 79 kDa and porcine hemopexin a molecular weight of 62 kDa. Both purified proteins were subjected to amino acid and carbohydrate analyses. Based on carbohydrate and sialic acid analyses, it is proposed that transferrin contains one bi-antennary glycan chain, whereas hemopexin contains two bi-antennary and one tri-antennary glycan chains.

Purification of isotransferrins by concanavalin A sepharose chromatography and preparative isoelectric focusing.

1. From pooled serum containing genetically homogeneous transferrin C1, transferrin was purified and separated in three fractions (tri-tri, bi-tri- and bi-bi-antennary transferrin C1), using Concanavalin A-Sepharose. 2. Each of these fractions was separated into its sialic acid-dependent subfractions by preparative isoelectric focusing. Sixteen iso-transferrin C1 fractions were obtained, which differed in their degree of glycan branching and/or their sialic acid content. 3. Preliminary carbohydrate analyses suggest that in some iso-transferrins the N-acetylglucosamine and the galactose content is lower than expected.

Adaptation of transferrin protein and glycan synthesis.

We report the patterns of variability in transferrin structure in pregnancy, iron deficiency anemia, women using oral contraceptives, nonanaemic rheumatoid arthritis, iron deficient rheumatoid arthritis and anemia of the chronic diseases. Changes in microheterogeneity were assessed by crossed immuno isoelectric focusing of serum transferrin. Intra-individual variation in the control group was minimal. Equally, inter-individual variation in controls and groups with established stable disease was very limited. In pregnancy an increase in transferrin concentration was accompanied by redirection of glycan synthesis to the highly sialylated and highly branched glycans, an effect also shown in women using oral contraceptives. Iron deficiency anemia was accompanied by increased protein core synthesis without the large shifts in the microheterogeneity pattern as seen in pregnancy at similar transferrin concentration. In contrast to this, rheumatoid arthritis was accompanied by decreased protein synthesis while the microheterogeneity pattern shifted significantly towards the highly branched glycans. Interpreted in the respective pathophysiological contexts results show that: (1) N-linked glycosylation of transferrin is a strictly controlled process, both in the physiological states and in disease. (2) Microheterogeneity is determined independently from transferrin protein synthetic rate. (3) Provisionally observed changes in the glycosylation can modulate the biological activity of the glycoprotein and as a result redirect internal iron fluxes. This proposition can be applied to altered iron metabolism in both pregnancy, oral contraceptives and rheumatoid arthritis. Changes are not operative in iron deficiency because qualitatively iron metabolism is not altered in this state.

The analysis of human serum transferrins with the PhastSystem: quantitation of microheterogeneity.

The PhastSystem was used for three different analyses of human serum transferrins (Tf) by isoelectric focusing: (i) The distribution of iron over the two iron binding sites by separation of the four forms: apoTf, TfFeC, FeNTf and Fe2Tf by using less than 1 microL serum or plasma. The focused bands were visualized by immunoprecipitation and quantified by densitometry. (ii) The relative proportions of the diferic sialo-transferrin fractions in human sera or plasma were determined in a similar way. (iii) The presumed genetic variants can be determined. The methods are easy and reproducible and require much less sample (0.3 microL), time (1 h per run) and antibody (10 microL/sample) than crossed immunoelectrofocusing.

Carbohydrate analysis of transferrin subfractions isolated by preparative isoelectric focusing in immobilized pH gradients.

Analytical isoelectric focusing of human serum transferrin has revealed the presence of up to nine different transferrin subfractions. The less prevalent subfractions have hitherto not been available for analytical work on their composition. Prolonged isoelectric focusing in immobilized pH gradients is capable of effectively separating and concentrating these subfractions from other fractions that are often present in over 70-fold excess in native preparations. Preliminary results of the carbohydrate analysis indicate a heterogeneity extending beyond the present concepts of the transferrin glycan structure.

Isotransferrins and pregnancy: a study in the guinea pig.

During pregnancy the serum isotransferrin pattern changes towards transferrins with more complex carbohydrate chains. The main pregnancy-related isotransferrin (TfFast) and the most common isotransferrin in the non-pregnant guinea pig (TfSlow) were isolated and characterized. TfSlow had one biantennary- and TfFast one triantennary glycan chain. Is there a functional explanation for this pregnancy-related shift towards more complex glycan chain structure? We studied this question in the context of maternal and fetal erythropoiesis. In vitro incubations of maternal bone marrow cells (MBMC) and fetal erythroid liver cells (FELC) with doubly labelled TfSlow and TfFast revealed only slight differences in affinities for the transferrin receptor. Ka(TfSlow) = 0.17 mumol/l; Ka(TfFast) = 0.15 mumol/l. MBMC and FELC had equal Vmax values both for TfSlow and TfFast. Vmax = 100 Fe atoms/transferrin receptor.hour. Irrespective the cell population TfSlow and TfFast showed equal rates for endo- and exocytosis. kendo. = 0.3750 min-1, kexo. = 0.1450 min-1. It is concluded that the described shift in isotransferrin pattern is not functionally related to maternal or fetal erythropoiesis.

Covalent cross-links in oxygen free radical altered human immunoglobulin G.

The damaging effect of an oxygen free radical generating system, i.e. ultraviolet irradiation, on human immunoglobulin G (IgG) was studied. The free radical altered IgG was analysed by a high performance liquid chromatograph equipped with a TSK G 3000 SW-column. Gel filtration of 120 min UV-irradiated IgG resulted in three clearly distinguished peaks corresponding to polymer IgG (MW greater than 500 kD), dimer IgG (MW 300 kD) and monomer IgG (MW 150 kD). Analysis of oxygen free radical altered and aggregated IgG by SDS-PAGE and subsequent silver-staining revealed inter- and intra-molecular reduction (by beta-mercaptoethanol)-resistant cross-links between IgG-molecules were formed. Comparison of amino acid analyses of native IgG with oxygen free radical aggregated polymer IgG showed significant reductions in tyrosine- (7.0%) and histidine- (6.5%) content. These findings suggest that tyrosine and histidine are involved in covalent cross-linking between IgG-molecules caused by oxygen free radicals. These alterations on IgG induced by free radical-activity might render it antigenic, and could initiate the production of rheumatoid factors (RF).

Human serum sialo transferrins in diseases.

Using isoelectric focusing and crossed immunoelectrophoresis on ready-made Immobiline Dry Plates, pH 5-6, we were able to separate human serum transferrin in subfractions with different sialo acid content. The amount of these subfractions is significant different in sera of patients with diseases like CA, RA, haemochromatosis and in sera of pregnant women.

Pregnancy and guinea-pig isotransferrins--isolation and characterization of both isotransferrins.

Microheterogeneity patterns of pregnant and non-pregnant guinea-pig serum transferrin differ significantly. Using preparative isoelectric-focusing 'slow' and 'fast' isotransferrins were separated. Amino-acid and carbohydrate composition of these two fractions are presented.