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.     

Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding 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. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.     

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).     

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.     

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.     

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.     

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).     

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.     

Ligand Binding and Polymerization Characteristics of Human Milk Folate Binding Protein Depend on Concentration of Purified Protein and Presence of Amphiphatic Substances

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.     

叶酸受体基因转染细胞诱导后的类脂改变对叶酸摄取的影响

用胆固醇合成抑制剂Ｌｏｖａｓｔａｔｉｎ（洛伐他汀,暂译名）和神经鞘脂类合成抑制剂ＦｕｍｏｎｉｓｉｎＢ１（抑鞘脂素Ｂ１,暂译名）处理能表达糖基化磷脂酰肌醇锚定叶酸受体（ＧＰＩＦＲ）的基因转染细胞（ＦＲα·ＴＲＶＢ－１）３ｄ,发现前者可使细胞的胆固醇含量减少约３５％,而后者可使神经鞘脂类减少４４％以上;同时发现,处理组细胞结合叶酸的能力减少约４０％,其对５－甲基四氢叶酸的摄入率减少逾８０％．这主要是由于细胞质膜中胆固醇和神经鞘脂类含量减少,从而导致膜内ＧＰＩＦＲ结合叶酸功能降低及ＧＰＩＦＲ摄入叶酸的内化过程障碍所致．其详细作用机制尚待进一步研究     

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.     

叶酸白蛋白靶向纳米粒的肿瘤细胞吸收及叶酸受体相关基因表达研究

实验选用叶酸受体表达阳性FR(+)卵巢癌细胞株SKOV3与表达阴性的FR(-)肺癌细胞株A549,通过肿瘤细胞对叶酸白蛋白靶向纳米粒的吸收及叶酸受体相关基因的表达,研究分析叶酸白蛋白靶向纳米粒叶酸受体吸收特征及叶酸受体胞吞途径的参与机制。结果表明:随着叶酸白蛋白靶向纳米粒给药时间的增加,SKOV3细胞FR1基因的表达显著增强,而A549细胞未见FR1基因表达。同时SKOV3细胞FR1基因的表达程度与细胞摄取叶酸白蛋白靶向纳米粒的量成正相关。叶酸代谢通路相关的基因(RFC、FPGS、GGH)表达,研究显示叶酸白蛋白靶向纳米粒能够启动细胞膜叶酸通道相关蛋白的表达。     

SYNTHESIS OF FOLATE RECEPTOR-TARGETED AND DOXORUBICIN-COUPLED CHEMOTHERAPEUTIC NANOCONJUGATE AND RESEARCH INTO ITS MEDICAL APPLICATIONS

Folate-targeted drug delivery has become an alternative therapy for the treatment of various cancers. Folate receptors are known to be responsible for cellular accumulation of folate and folate analogs with high binding affinity. The anthracycline antibiotic doxorubicin has a broad spectrum of antineoplastic action and a correspondingly widespread degree of clinical use. In this work, we aimed to prepare a folate receptor-targeted doxorubicin delivery system to achieve minimal effect of doxorubicin on healthy cells and more cytotoxicity of it on tumor cells. Folate–poly(ethylene glycol)–doxorubicin (FOL-PEG-DOX) nanoconjugate was synthesized through this aim and characterized with nuclear magnetic resonance (NMR), zetasizer, and atomic force microscopy (AFM). Doxorubicin release studies were also performed in vitro. The size of FOL-PEG-DOX was 78.84 nm. The results indicated that doxorubicin release rate from the conjugate was faster at pH 5.0 than pH 7.4 and the amide bond between DOX and PEG was more stable at pH 7.4 than pH 5.0. As a consequence, FOL-PEG-DOX nanoconjugate could be a potentially useful delivery system for folate receptor-positive cancer cells.     

Decreased Expression of Transporters Reduces Folate Uptake across Renal Absorptive Surfaces in Experimental Alcoholism

In this study, we examined the mechanistic insights of folate reabsorption during alcoholism, considering enhanced renal excretion as one of the major contributing factors to alcohol-induced folate deficiency. Male Wistar rats were fed 1g/kg body weight/day ethanol (20% solution) orally for 3 months. The results on characterization of the folate transport system in renal basolateral membrane (BLM) suggested it to be a carrier-mediated, acidic pH-dependent and saturable one. Chronic ethanol feeding decreased the uptake mainly by increasing the K _m and decreasing the V _max of the transport process at the BLM surface. At the molecular level, reduced folate transport activity in renal tissue during chronic ethanol ingestion was attributable to decreased expression of reduced folate carrier (RFC) and folate binding protein (FBP). Antibodies against RFC protein revealed a parallel change in RFC expression in both brush border and BLM surfaces during chronic alcoholism. Such findings highlight the role of downregulation of RFC and FBP expression and provide mechanistic insight into the observed reduced folate transport efficiency at renal absorptive surfaces in alcoholism, which may result in low blood folate levels commonly observed in alcoholics.     

Folate Transport by Prawn Hepatopancreas Brush-Border Membrane Vesicles

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn (Penaeus japonicm) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 M and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.     

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.     

Bimane-labelled pepstatin, a fluorescent probe for the subcellular location of cathepsin D.

1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.     

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.