Jan Holm

Megalin binds and mediates cellular internalization of folate binding protein

Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol‐linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva and semen. The function and significance of FBPs are unresolved, however, it has been suggested that they may facilitate folate uptake, e.g. during suckling. The present study shows that megalin, a large, multiligand endocytic receptor and member of the low‐density lipoprotein‐receptor family, is able to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of 125I‐labeled folate binding protein (FBP) to sections of kidney proximal tubule, known to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin‐mediated uptake, reveal 125I‐labeled FBP uptake which is inhibited by receptor associated protein and by antimegalin antibodies. Microinjection of 125I‐labeled FBP into renal tubules in vivo shows proximal tubular uptake by endocytosis. Megalin is expressed in several absorptive epithelia, including intestine and kidney proximal tubule, and thus the present findings provide a mechanism for intestinal and renal endocytic uptake of soluble FBP.

Isolation and characterization of the folate-binding protein from cow's milk

The folate-binding protein from cows milk has been purified in milligramme scale by combination of ion-exchange chromatography and affinity chromatography. The molecular weight has been determined by sedimentation equilibrium ultracentrifugation and found to be 30,000±2,000. The amino acid composition is compatible with this value. The molecule contains six disulphide bridges and no free SH-groups. The three per cent carbohydrate content was accounted for by six glucosamine residues per mole of protein. About 50 per cent of the amino acid sequence has been delineated, including the N-terminal sequence and the C-terminal sequence. Isoelectric focusing gave rise to four major peaks with isoelectric points ranging from 8.5 to 7.6, but no heterogeneity was observed in the sequence.

Cooperative binding of folate to a protein isolated from cow's whey.

Abstract Cooperative binding of [3H]folate to a protein (Mr = 35 000) in cows whey was demonstrated in equilibrium dialysis experiments in Tris buffer (pH 7.4) at 37°C. The folate binding protein was isolated from whey by ion-exchange chromatography (DEAE-Sephadex A-50) in a linear NaCl gradient (0.03–0.3 M). The folate binding protein appeared in the effluent before start of the salt gradient. Fractions eluted after start of the salt gradient contained the major whey proteins β-lactoglobulin and α-lactalbumin but were unable to bind [3H]folate. Binding characteristics depended on concentration of folate binding protein. The association constant for folate binding (Kass) was thus inversely proportional to the folate binding protein concentration. That is binding affinity decreased with increasing concentrations of binding protein. A phenomenon of this type may suggest involvement of a polymerising protein system in binding. Changes in the H+ concentration within the interval pH 6–9 did neither decrease maximum bound folate nor the binding affinity. However, lowering pH to 5 produced a 10-fold reduction in Kass. Cooperativity as well as dependence of affinity on the folate binding protein concentration did moreover disappear. Folate binding was virtually insensitive to changes in ionic strength and temperature. Within the interval pH 7.4–5.2 dissociation of [3H]folate from binding protein was slow and incomplete. However, dissociation became rapid and complete at pH 3.5. The physiological significance of the folate binding system in milk is obscure but it may play a role in folate homeostasis.

Aggregation of a folate-binding protein from cow's milk

Sedimentation equilibrium experiments of folate-binding protein from cows milk performed in the analytical ultracentrifuge at pH 5.0 showed a single molecular species with a molecular weight of 30,000. In contrast, at pH 7.4 sedimentation equilibrium indicated molecular weights ranging from about 30,000 to at least 300,000. Sedimentation velocity experiments at pH 5.0 showed a single symmetrical peak with s20,w=2.8 S in agreement with a molecular weight of about 30,000. At pH 6.0 was seen a faster moving boundary (s20,w∼6S) in addition to the slow one. At higher pH two peak maxima were seen: a slow one with sedimentation coefficients of about 5S and a fast one increasing from 9S to 11S with increasing pH. Addition of folate further increased the sedimentation rates. By assuming thatGilberts rapid monomer-n-mer equilibrium theory was valid also for a lower polymer in equilibrium with a higher polymer our data can be interpreted as an equilibrium between a tetramer and a polymer consisting of more than 16 monomers. In the case of folate addition the results may similarly be interpreted as an equilibrium between an octamer and a polymer composed of more than 32 monomers.

High and low affinity binding of folate to proteins in serum of pregnant women

Abstract Binding of [ 3 H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37°C). A Scatchard analysis revealed the presence of high-affinity ( K ass = 10 10 M −1 , N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein ( M r ≈ 30 000–35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/ 30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size ( M r > 200 000) protein, probably α 2 -macroglobulin. High-affinity binding which displayed positive cooperatively was saturated at folate concentrations above 10 −10 M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N 10 -methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10 −10 –10 −7 M. Dissociation of folate was rapid.

A Chemometric Analysis of Ligand-Induced Changes in Intrinsic Fluorescence of Folate Binding Protein Indicates a Link between Altered Conformational Structure and Physico-Chemical Characteristics

Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate) -induced changes in the fluorescence landscape of FBP. Fluorescence excitation and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important finding was the occurrence of two maximum intensity emission wavelengths of tryptophan, 350 nm (component one) and 330 nm (component two). In contrast to the first component, the score of the short wavelength component increased with increasing ligation of FBP. Since the emission wavelengths of indole groups in tryptophan shorten with increasing distance from the solvent surface of proteins, an increasing number of the 11 tryptophan residues seem to reorientate from the solvent surface to the interior of FBP with increasing ligation. The sharp decrease in hydrophobicity at pI=7–8 following binding of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition with radiofolate. This could suggest a rapid and firm complexation of folate to FBP, blocking access of competing ligands.

Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk: Influence of Buffer Type and pH

Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a β-sheet to α-helix transition upon ligation in acetate and phosphate buffers, and the formation of intermolecular β-sheet was indicated at pH 5.0, in agreement with a dimerization of FBP taking place at this pH. The ligand-induced changes in the 2100–2300 nm NIR region were significant for FBP in acetate and phosphate buffers of pH 5.0, and the variations were interpreted as secondary structure changes, based on previous assignments of secondary structures to the combination bands in the NIR region. In the case of acetate buffer, variations in the amide combination bands agreed with the amide I analysis, but for the other buffer types some discrepancies were found and explained by side-chain contributions to the NIR, which could reflect the tertiary and quaternary structure differences. NIR spectra of FBP at pH 7.4 and 5.0 revealed contradictory effects on the side chains, reflecting different polymerization events at the two pH values, whereas the amide I region indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT-IR, the tertiary and quaternary structure changes are reflected in the NIR spectra, although the general influence of the latter changes on the NIR spectra remains to be confirmed.

A High-Affinity Folate Binding Protein in Human Urine. Radioligand Binding Characteristics, Immunological Properties and Molecular Size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 109M−1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand3H-folate. Dissociation of3H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (Mr∼25 000) and a minor one (Mr∼100 000).

Change in binding properties of folate-binding protein in cow's whey due to removal of a cofactor during affinity chromatographic purification

The folate-binding protein in cows whey was purified by affinity chromatography on folate or methotrexate-AH-Sepharose 4B. A change in basic binding properties of the protein occurred probably due to the fact that material removed during affinity chromatography contains a cofactor of great importance to folate binding.

Kinetics of folate-protein binding.

Publisher Summary This chapter discusses the kinetics of folate-protein binding. In mammals, folate-(pteroylglutamate-)binding proteins have been demonstrated in cows milk and human milk, hog kidney, rat liver, rabbit choroid plexus, intestinal brush border membrane from rat, leukemic cells, and in human serum and animal serum as well. Investigations of the kinetics of folate binding to protein need a method that separates protein-bound and free folate, without affecting the equilibrium between them. Adsorption of free folate to the charcoal method is predominant in the competitive radioassay methods for the determination of folate in serum where the folate-binding protein in milk has been used. It has the advantage of being quick and practical. However, it has many disadvantages: (1) in order to ensure that only free folate is bound to charcoal, the particles must be coated with various macromolecular substances, such as dextrane, albumin, or hemoglobin that act as a molecular sieve, (2) by ultrafiltration method, it is possible to obtain a good separation of free and bound folate if neither is bound to the membrane. The method has been used for investigating the binding of folate to milk protein and for an investigation of the binding of folate to serum proteins and to pure albumin preparations.