Steen Ingemann Hansen

The complete amino acid sequence of the folate-binding protein from cow's milk

The complete amino acid sequence of the folate binding protein from cows milk has been determined by automated sequencing of fragments obtained by cleavage with cyanogen bromide, trypsin, S. aureus V8 protease and clostripain. The molecule consists of a single polypeptide chain of 222 amino acid residues and eight disulphide bridges. The molecular weight of the protein part is 25,700. The folate binder contains carbohydrate which gives it a final molecular weight of about 30,000. No sequence homology with other proteins has been observed.

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.

Amino acid sequence homology between human and bovine low molecular weight folate binding protein isolated from milk

The partial amino acid sequence of a low molecular weight folate binding protein from human milk has been determined from N-terminal sequencing of the native protein and isolated cyanogen bromide fragments. A molecular weight of approximately 30,000 is estimated on the basis of amino acid composition and sequence homology with the low-molecular weight folate binder isolated from cows milk. Preliminary studies with a large molecular weight folate binding protein have shown that the N-terminal amino acid sequence is identical with that of the low molecular binder.

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.

Dependence of aggregation and ligand affinity on the concentration of the folate-binding protein from cow's milk

Ultracentrifugation and gel-filtration studies showed that the folate-binding protein from cows milk possessed a remarkable aggregation tendency at pH 7.4. Aggregation was enhanced in the presence of folate which suggested an interrelationship between the mechanisms of ligand binding and polymerization. The degree of polymerization increased with increasing concentrations of binding protein. Thus, while the monomer prevailed at 1 nM, a polymer composed of more thn 32 monomers was formed at 130 microM. Two characteristics of folate binding, i.e., Scatchard plots that were convex upward and a ligand affinity that was inversely proportional to the concentration of binding protein, could be interpreted in terms of ligand binding to a polymerizing system in which the polymerization equilibria affect the ligand affinity.

Folate receptor of human mammary adenocarcinoma

Binding of 3H‐folate to human mammary tumor homogenate was of a high‐affinity type (K= 1010 M−1) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5‐formyltetrahydrofolate acted as a strong inhibitor of radioligand binding. Gel chromatography of radioligand‐labeled homogenate of tumor tissue revealed three peaks: a small 110 kDa peak and two major peaks of folate‐binding activity (Mr∼ 25 kDa and Mr∼ 100 kDa). Mammary tumor tissue showed immunostaining with rabbit antibodies against human milk folate binder. A parallel elevation in the concentrations of folate‐binding protein and triglyceride in tumor tissue as compared to normal tissue adjacent to the tumor was compatible with the localization of folate‐binding protein in the triglyceride‐rich fraction of mammary gland homogenate.

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.

Folate Receptors in Malignant and Benign Tissues of Human Female Genital Tract

We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K=1010M−1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of 3H-folate labelled protein (>=130 and 100kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.

Ligand-induced conformation change in folate-binding protein.

C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cows whey. The c.d. spectrum of unligated FBP predicts the following secondary structure: 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn and 31% random-coil structure. Folate binding to FBP results in significant changes in the c.d. spectrum. Analysis of the spectrum shows a 10% decrease in antiparallel beta-strand as a result of ligand binding. Folate binding also leads to strong quenching of FBP tryptophan fluorescence. The magnitude of the quench is proportional to ligand binding. The guanidinium chloride-induced unfolding of FBP is shown to be a multistate process. Detection by c.d. and fluorescence spectroscopy lead to non-identical transitions. Modelling studies are consistent with the existence of a stable folding intermediate. Ligand binding to FBP increases the apparent folding stability of the molecule. Simultaneous detection by c.d. and fluorescence indicate that the apparent increased folding stability is derived from ligand-induced aggregation of FBP.

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.