A high-affinity folate binding protein in human amniotic fluid. 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 (M _r 25 000) and a minor one (M _r 100 000).     

High-affinity folate binding in human mammary gland

High-affinity³H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (M_r100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.     

High-affinity folate binding in human prostate

Binding of³H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (M_r100 and 25kDa), but only one single band (M_r65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum³H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n=6).     

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 binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

Characterization of a High-Affinity Folate Receptor in Normal and Malignant Human Testicular Tissue

We have characterized the folate receptor in normal and malignant tissue from male gonads. Radioligand binding displayed characteristics typical of other folate receptors. Those included a high-affinity type of binding (K = 10^{10 M–1}), apparent positive cooperativity changing into non-cooperativity at low receptor concentrations, a tendency to increased binding affinity with decreasing receptor concentrations, a slow dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition by folates, in particular oxidized forms. The gel filtration profile of Triton X-100 solubilized tissue contained a 25 and 100 kDa peak of radioligand-receptor. The latter peak could represent receptor equipped with a hydrophobic membrane anchor that inserts into Triton X-100 micelles. The concentration of radiolabelled receptor ranged from 0.41 nmol/g protein to 1.68 nmol/g protein in specimens of normal testicular tissue from patients with prostatic carcinomas and from 1.54 nmol/g protein to 3.82 nmol/g protein in testicular tissue from young individuals. Compared to normal testicular tissue the concentration of receptor in seminoma tissue was low (0.38–1.27 nmol/g protein) but showed a higher degree of immunoreactivity in the presence of antibodies against human milk folate binding protein as evidenced by ELISA and immunohistochemistry data. Hence a folate receptor isoform homologous to human milk folate binding protein is apparently expressed in seminomas where the total expression of receptor, however, seems to be lower than in normal testicles.     

Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein

The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [³H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [³H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.     

Effect of hydrogen ion concentration and buffer composition on ligand binding characteristics and polymerization of cow's milk folate binding protein

The ligand binding and aggregation behavior of cow's milk folate binding protein depends on hydrogen ion concentration and buffer composition. At pH 5.0, the protein polymerizes in Tris-HCl subsequent to ligand binding. No polymerization occurs in acetate, and binding is markedly weaker in acetate or citrate buffers as compared to Tris-HCl. Polymerization of ligand-bound protein was far more pronounced at pH 7.4 as compared to pH 5.0 regardless of buffer composition. Binding affinity increased with decreasing concentration of protein both at pH 7.4 and 5.0. At pH 5.0 this effect seemed to level off at a protein concentration of 10^–6 M which is 100–1000 fold higher than at pH 7.4. The data can be interpreted in terms of complex models for ligand binding systems polymerizing both in the absence or presence of ligand (pH 7.4) as well as only subsequent to ligand binding (pH 5.0).     

Characterization of the folate receptor in human molar placenta

We have characterized a high-affinity folate receptor in human molar placenta tissue. Radioligand binding exhibited characteristics typical of other high-affinity folate binding proteins. Those included, positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition by folate analogues. The folate receptor cross-reacted with antibodies against human milk folate binding protein, e.g. the syncytothrophoblastic layer of molar placenta tissue sections showed strongly positive immunostaining. The gel filtration profile contained two radioligand-bound peaks (25 and 100 kDa), however, with considerable overlap. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The folate receptor in placental tissue may play a crucial role in the transfer of folate from maternal circulation to the fetus.     

A Folate Binding Protein in Ascitic Fluid, Serum and Ovarian Tissue of Patients with Ovarian Adenocarcinoma Immunoreacts with Antibodies Against Human Milk Folate Binding Protein

The presence of a folate binding protein which immunoreacts with antibodies against human milk folate binding protein was demonstrated in ascitic fluids from seven patients with ovarian adenocarcinoma. Ascitic fluids collected from two patients with other malignancies contained non-immunoreactive FBP. Tumor tissue specimens from five patients with ovarian carcinoma contained immunoreactive FBP. By contrast to normal ovaries ovarian carcinoma tissue showed positive immunostaining on immunohistochemistry. Ascitic fluids from two patients with ovarian carcinoma exhibited single distinct bands on SDS-PAGE immunoblotting. The gel filtration profile of ovarian carcinoma tissue homogenate from two patients contained 25 and 100 kDa peaks of radioligand-bound and immunoreactive folate binding protein, while ascitic fluid from one of the patients exhibited a large 100 kDa immunoreactive peak with no radioligand binding activity. The immunoreactive non-functional 100 kDa FBP could represent unprocessed precursor FBP. Future studies are necessary to evaluate whether determination of immunoreactive FBP in ovarian adenocarcinomatosis is of any diagnostic value.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

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

Binding of [³H]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 = 10¹⁰M^?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 α₂-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 N¹⁰-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.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites: radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity.

High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.     

High-affinity folate binding in human liver membranes

High-affinity binding of³H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel^® AcA 44 chromatography of solubilized membranes saturated with³H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

A high-affinity folate binding protein in human urine. Radioligand binding characteristics,immunological properties and molecular size

High-affinity binding of [³H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (M_r25,000).     

Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 M) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.     

Stopped-Flow Kinetic Studies of the Interaction of Bovine Folate Binding Protein (FBP) and Folate

The kinetics of the interaction of bovine folate binding protein and folate at pH 7.4 and 5.0 were followed by measuring the changes of the intrinsic protein fluorescence intensity using the stopped-flow technique, which enables the study of reactions from the millisecond time-range. Our results immediately reject a simple one-step binding model, which requires a linear dependence of the observed rate constant on the concentration of the ligand. Thus, we are able to conclude that at pH 5.0 the interaction occurs in two steps and at pH 7.4 in three steps. Changes of fluorescence spectra at equilibrium were used to estimate the overall binding constants. Comparative studies on the binding of folate to human albumin are also reported.     

Conversion of an apparent 100 kDa folate binding protein from human milk,choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosly phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.