W.L. van Noort

The microheterogeneity of human transferrins in biological fluids

The serum, cerebrospinal fluid, amniotic fluid and synovial fluid transferrins have been examined by isoelectric focusing in a pH gradient from 5-7. In all biological fluids the presence of transferrins with a varying content of sialic acid has been shown. Differences between the fluids with regard to transferrin have been noticed.

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.

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.

ISOLATION OF RAT TRANSFERRIN USING CNBR-ACTIVATED SEPHAROSE 4B

1. The isolation of transferrin from rat serum by means of affinity chromatography on CNBr-activated Sepharose 4 B is described. -2. Subfractionation by isoelectric focusing yielded two transferrin fractions with identical biological behaviour but with small differences in isoelectric point (6.0 and 5.8) and sialic acid contents.

The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels; no functional difference of the fractions in vitro

Abstract The micro heterogeneity of the four main forms of human transferrin—Tf. TfFe(A), TfFe(B) and Tf.2Fe—as observed with the isoelectric focusing technique can be atrributed to the differences in sialic acid content. Transferrin molecules with the same iron content, but with varying sialic acid content—penta-sialo to asialo transferrins—show the same iron delivery to rat reticulocytes.

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, β-galactosidase or N-Acetyl-β-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.

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.

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.

Optimized separation and quantitation of serum and cerebrospinal fluid transferrin subfractions defined by differences in iron saturation or glycan composition.

The heterogeneity of human serum transferrin and the possible functional significance of transferrin heterogeneity and microheterogeneity in biological systems (bone marrow, liver and placenta) is a topic in transferrin chemistry. For many years it has been recognized that immuno affinity purified transferrin displays a complex electrophoretic behaviour indicating that transferrin is not a homogeneous substance.