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PubMed1991

High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein.

High-affinity [3H]folate binding in solubilized human choroid plexus homogenate displayed characteristics, e.g. apparent positive co-operativity, which are typical of specific folate binding. The highest folate-binding activity per g of protein was associated with the 27000 g membrane pellet where the membrane-marker enzyme gamma-glutamyltransferase had its main localization. Ultrogel AcA 44 chromatography revealed two major folate-binding proteins (molecular masses greater than 110 kDa and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) in the Triton X-100-solubilized membrane pellet. After exposure of the membrane pellet to phosphatidylinositol-specific phospholipase C there was only one large 25 kDa peak of folate binding. This could suggest that the folate-binding protein is anchored to the membrane by a glycosylphosphatidylinositol moiety, which can be inserted into Triton X-100 micelles and thus can give rise to forms of large molecular size on gel filtration. This notion was supported by the identical molecular masses of the greater than 110 kDa and 25 kDa folate-binding peaks determined by SDS/PAGE and immunoblotting. The folate-binding protein in choroid plexus cross-reacted with rabbit antibodies against the 25 kDa human milk folate-binding protein, and paraffin-embedded sections of choroid plexus showed immunostaining after exposure to rabbit anti-(human milk folate-binding protein) serum (1:8000 dilution).

Binding Sites↗
PubMed1992

Immunoreactive folate-binding proteins from human saliva. Isolation and comparison of two distinct species.

Human saliva contains a single 72,000-M(r) species which specifically reacted with rabbit anti-[human placental folate receptor (PFR)] serum on SDS/PAGE and Western blots. Although a specific radioimmunoassay for human PFR and related folate-binding proteins (FBPs) identified 55 ng of cross-reacting material (CRM) per mg of crude salivary proteins, only a minor fraction (1.6 ng) specifically bound radiolabelled folate. The major fraction of CRM did not contain bound endogenous folate and did not bind radiolabelled folates. On the basis of folate binding, salivary CRM species to PFR were designated as either functional (f-FBP) or non-functional (nf-FBP) species respectively. nf-FBPs and f-FBPs were isolated by different purification schemes. Both purified f-FBPs and nf-FBPs migrated as a single apparent 72,000-M(r) species on SDS/PAGE, but on Sephacryl S-200 gel filtration and sucrose-density-gradient ultracentrifugation they were eluted/sedimented with 40,000-M(r) markers. Each microgram of purified f-FBP and nf-FBP was measured in the radioimmunoassay for PFR as being equivalent to 18 ng and 24 ng of CRM respectively, indicating low epitope-relatedness to PFR. The Kd of f-FBPs was 50 pM and 0.94 mol of folate was bound/mol of protein. f-FBPs exhibited an unusual dependence on Triton X-100 for optimal ligand binding, despite the fact that Triton X-100 micelle binding was not demonstrated. The relative order of affinity of f-FBPs for pteroylglutamate greater than methotrexate greater than 5-formyltetrahydrofolate greater than 5-methyltetrahydrofolate was also distinct from that of purified PFR. Whereas amino acid and carbohydrate analysis revealed that nf-FBP (M(r) 51,400) and f-FBP (M(r) 39,200) were distinct glycoproteins with 8 and 13% carbohydrate respectively, isoelectric focusing and immunological studies suggested some structural identity. The presence of f-FBP and nf-FBP in normal saliva raises new questions about their possible role in vivo.

Adult↗
PubMed1981

[3H]Methotrexate as a ligand for the folate receptor of Dictyostelium discoideum.

Studies of the folate chemotactic receptor of vegetative Dictyostelium discoideum cells have been hampered by the presence of the degradative enzyme folate deaminase. The diaminopterin compounds aminopterin and methotrexate (MTX) are chemoattractants but are not attacked by the deaminase. [3',5',7,9-3H]methotrexate ([3H]MTX) is a nondegraded radioligand for the folate receptor. Binding to the receptor is rapid, reaching steady state in less than one min, and reversible in less than 15 s by an excess of unlabeled MTX. A single class of binding sites is found with a Kd of 2 x 10(-8) M, which correlates well with the concentration dependence of chemotaxis. Folate, aminopterin, and MTX all compete for [3H]MTX binding, whereas pterin, p-aminobenzoate, and nucleotides do not. Analysis of the receptor during differentiation indicates a decrease in site number by a factor of 3 with no change in affinity during the first 7 hr. During this time, the directional response (chemotaxis) to MTX and folate is lost, but a nondirectional stimulation of motility rate (chemokinesis) is retained. The response to cyclic AMP displays reciprocal behavior, first appearing as a chemokinetic response and then as a chemotactic response.

Carrier Proteins↗
PubMed1984

Glycine N-methyltransferase is a folate binding protein of rat liver cytosol.

A comparison of the amino acid compositions of one of the folate-binding proteins of rat liver cytosol, folate-binding protein-cytosol II, and that of glycine N-methyltransferase (S-adenosyl-L-methionine:glycine methyltransferase, EC 2.1.1.20) from the same source indicated a great deal of structural homology between the two proteins. Antiserum prepared against the purified folate-binding protein almost completely inactivated the enzyme activity in crude liver cytosol. Purification of glycine N-methyltransferase resulted in the separation of two enzyme species, one that contained bound folate and one that did not. Each species was homogeneous, as judged by NaDodSO4/polyacrylamide gel electrophoresis, and they migrated identically.

Amino Acids↗
PubMed1986

Receptor-mediated folate accumulation is regulated by the cellular folate content.

Cultured monkey kidney cells are shown to possess a cell-surface receptor that binds 5-methyltetrahydro[3',5',7,9-3H4]folic acid (5-methyltetrahydro[3H]folic acid) with high affinity (Kd = 3 nM at 4 degrees C). The cell-surface binding capacity for [3H]folic acid or [3H]methotrexate is equivalent to that for 5-methyltetrahydro[3H]folate, but the Kd values are 0.4 and 20 nM, respectively. These nonlabeled folates also inhibit cellular binding and accumulation of 5-methyltetrahydro[3H]folate, whereas specific moieties of folic acid (i.e., p-aminobenzoylglutamic acid, pteroic acid, and glutamic acid) had no effect. Surface-bound folate(s) can be released by incubation of the cells at pH 3.5. At 37 degrees C the bound material is slowly transferred into cells, where it becomes resistant to acid release. Within 4 hr the cells internalize about 3-4 times as much folate as is bound to the surface in the steady state. The amount of receptor activity increases markedly when cells are depleted of folate through growth in folate-depleted medium. Binding of folate was inhibited by an antibody raised against a soluble plasma folate-binding protein, suggesting that the cell-surface receptor and the circulating folate-binding protein are immunologically related. These data indicate that cells possess a high-affinity, high-specificity folate receptor whose expression is regulated by the folate content of the cell. They also suggest that a small molecule such as folate can enter cells by receptor-mediated endocytosis.

Animals↗
PubMed1987

Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

A membrane-associated folate binding protein (FBP) and a soluble FBP, which is released into the culture medium, have been purified from human KB cells using affinity chromatography. By NaDodSO4/PAGE, both proteins have an apparent Mr of approximately 42,000. However, in the presence of Triton X-100, the soluble FBP eluted from a Sephadex G-150 column with an apparent Mr of approximately 40,000 (similar to NaDodSO4/PAGE) but the membrane-associated FBP eluted with an apparent Mr of approximately 160,000, indicating that this species contains a hydrophobic domain that interacts with the detergent micelles. The amino acid compositions of both forms of FBP were similar, especially with respect to the apolar amino acids. In addition, the 18 amino acids at the amino termini of both proteins were identical. The membrane FBP, following delipidation with chloroform/methanol, contained 7.1 mol of fatty acid per mol of protein, of which 4.7 mol was amide-linked and 2.4 mol was ester-linked. The soluble FBP contained only 0.05 mol of fatty acid per mol of protein. These studies indicate that the membrane FBP of KB cells contains covalently bound fatty acids that may serve to anchor the protein in the cell membrane.

Acylation↗
PubMed1991

Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis.

Difficulties with the nondestructive delivery of macromolecules into living cells have limited the potential applications of antibodies, genes, enzymes, peptides, and antisense oligonucleotides in biology and medicine. We have found, however, that the natural endocytosis pathway for the vitamin folate can be exploited to nondestructively introduce macromolecules into cultured cells if the macromolecule is first covalently linked to folate. Thus, treatment of KB cells with folate-conjugated ribonuclease, horseradish peroxidase, serum albumin, IgG, or ferritin allowed delivery of greater than 10(6) copies of the macromolecules within a 2-hr period. Cytochemical staining using 4-chloro-1-naphthol further demonstrated that the horseradish peroxidase retained activity for at least 6 hr after internalization. Since folate is an essential vitamin required in substantial quantities by virtually all cells, these observations may open the possibility of scientific and medical applications for many of the above macromolecules.

Carrier Proteins↗
PubMed1992

Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate.

The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor increases the efficiency of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.

Animals↗
PubMed1983

Neonatal role of milk folate-binding protein: studies on the course of digestion of goat's milk folate binder in the 6-d-old kid.

Groups of kids were reared from birth to 5 d on goat's milk. On the 6th day five of the kids received by bottle a morning feed of goat's milk with [3H]folic acid added to saturate the folate-binding proteins (FBP) (Expt 1); three kids received raw goat's milk containing only the endogenous folate and hence a large surplus folate-binding capacity (FBC) (Expt 2). The contents of the stomach, duodenum, jejunum and ileum were recovered by washing out 1.5 h after feeding (Expt 1) or at 0.5, 1 and 3.5 h after feeding (Expt 2). Recovery of [3H]folic acid 1.5 h after feeding (Expt 1) in all segments was 58.4%, mainly in a soluble form, most of this being in the stomach (37.0%) and ileum (14.3%). No surplus FBC was found in any gut segment. Sephadex G-75 chromatography of the soluble fractions of the contents of the various gut segments showed that [3H]folic acid remained bound to FBP throughout the stomach and small intestine. The bound [3H]folic acid exhibited a molecular weight of 81 000 in stomach contents, similar to that in the milk feed, presumably representing an aggregated form of the FBP, whereas in the intestinal contents its molecular weight was 39 000 indicating dissociation to monomer due to dilution in the recovery process. In Expt 2, the total recovery of free FBP in all four gut segments was 67, 54 and 23% respectively at 0.5, 1 and 3.5 h after the milk feed, and the distribution of FBP along the gut at 1 h was similar to that of [3H]folic acid-labelled FBP at 1.5 h in Expt 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Influence of goat's-milk folate-binding protein on transport of 5-methyltetrahydrofolate in neonatal-goat small intestinal brush-border-membrane vesicles.

1. The influence of goat's-milk folate-binding protein (FBP) on the uptake of 5-methyltetrahydrofolate (MTHF) by brush-border-membrane vesicles prepared from the small intestine of the 6-d-old goat was investigated using a rapid-filtration assay. 2. Uptake of MTHF by the membrane vesicles was strongly enhanced by FBP within the pH range 4.5-6.5, with an optimum at pH 5-5.5. 3. Both the initial rate of MTHF uptake and uptake of MTHF at equilibrium were markedly increased in the presence of FBP. 4. Uptake of MTHF by brush-border-membrane vesicles was maximal when the molar ratio FBP:MTHF was 1.0-2.5. 5. The relation between pH and 125I-labelled FBP binding to the membranes was similar to that for uptake of MTHF, with an optimum at pH 5. 6. In experiments in which the osmotic pressure of the incubation medium was progressively increased with cellobiose, 125I-labelled FBP was found to be taken up primarily by binding to the brush-border-membrane surface. 7. Uptake of 125I-labelled FBP was time-dependent and saturable, with a Km of 0.39 (SE 0.07) microM and Vmax of 6.73 (SE 0.92) micrograms/mg protein. 8. Experiments in which various milk proteins (cow FBP, goat FBP, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin and lactoferrin) were allowed to compete in turn with 125I-labelled FBP for uptake by brush-border-membrane vesicles indicated that high-affinity binding was probably specific to FBP, although lactoferrin reduced uptake possibly by non-specific coating of the mucosal surface. 9. It was concluded that a folate transport mechanism mediated by the FBP in milk exists at the intestinal brush border of neonatal goats. It is suggested that this may reinforce the developing endogenous transport system.

Animals↗
PubMed1990

Secondary structure of the human membrane-associated folate binding protein using a joint prediction approach.

The secondary structures of the human membrane-associated folate binding protein (FBP) and bovine soluble FBP are assessed by a joint prediction approach that combines neural network models, information theory, homology modeling and the Chou-Fasman methods. Two new profile maps are used to characterize the non-regular secondary structure and to assist in assigning buried and exposed parts of secondary structure: (i) the loop potential profile and (ii) the long range contact profile. Approximately half of human FBP is predicted to form regular secondary structure (alpha-helices-35% or beta-sheets - 12%, excluding the transmembrane helices) and the rest is predicted to form coil, turns or loops. The bovine milk soluble FBP is predicted to have a similar secondary structure as expected because of the high degree of homology between the FBP's. Discriminant analysis predicts two transmembrane segments for the human FBP sequence, one at the amino terminus (a leader sequence) and the other at the carboxy terminus. These predicted transmembrane domains are absent in the bovine milk soluble FBP, further supporting these predictions. The present set of secondary structural predictions for human FBP is obtained by 'consensus' to aid in modeling the super-secondary structure of the protein.

Amino Acid Sequence↗
PubMed1990

The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.

Animals↗
PubMed1990

Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate.

The folate receptor is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that mediates the delivery of 5-methyltetrahydrofolate to the cytoplasm of MA104 cells. Ordinarily the receptor is sequestered into numerous discrete clusters that are associated with an uncoated pit membrane specialization called a caveola. By using two different methodological approaches, we found that the maintenance of both receptor clusters and caveolae depends upon the presence of cholesterol in the membrane. These results suggest that cholesterol plays a critical role in maintaining the caveola membrane domain and modulates the interaction of GPI-anchored membrane proteins via their phospholipid anchors.

Animals↗
PubMed1991

A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors.

Amebae of Dictyostelium exhibit a transient uptake of extracellular Ca2+ approximately 5 s after activation of surface folate or cAMP receptors (Bumann, J., B. Wurster, and D. Malchow. 1984. J. Cell Biol. 98:173-178). To further characterize these Ca2+ entry systems, we analyzed 45Ca2+ uptake by resting and activated amebae. Like the surface chemoreceptors, folate- and cAMP-induced Ca2+ uptake responses were developmentally regulated; the former response was evident in vegetative but not aggregation-competent cells, whereas the latter response displayed the opposite pattern of expression. In contrast, other characteristics of these Ca2(+)-uptake pathways were remarkably similar. Both systems (a) exhibited comparable kinetic properties, (b) displayed a high specificity for Ca2+, and (c) were inhibited effectively by Ruthenium Red, sodium azide, and carbonylcyanide m-chlorophenyl-hydrazone. These results, together with the finding that vegetative cells transformed with a plasmid expressing the surface cAMP receptor exhibit a cAMP-induced Ca2+ uptake, suggest that different chemoreceptors activate a single Ca2+ entry pathway. Additional pharmacological and ion competition studies indicated that receptor-mediated Ca2+ entry probably does not involve a Na+/Ca2+ exchanger or voltage-activated channels. Chemoattractant binding appears to generate intracellular signals that induce activation and adaption of the Ca2(+)-uptake response. Analysis of putative signaling mutants suggests that Ca2+ entry is not regulated by the guanine nucleotide-binding (G) protein subunits G alpha 1 or G alpha 2, or by G protein-mediated changes in intracellular cAMP or guanosine 3,'5'-cyclic monophosphate (cGMP).

Biological Transport↗
PubMed1992

Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.

The folate receptor is clustered on the surface of MA104 cells in association with caveolae. This relationship is thought to be essential for the proper internalization and recycling of the receptor during the delivery of 5-methyltetrahydrofolate to the cytoplasm of folate-depleted cells. Both the clustered organization of the receptor and the integrity of caveolae are disrupted when cells are deprived of cholesterol. We now show that cholesterol depletion of MA104 cells markedly reduces the rate of 5-methyltetrahydrofolate internalization and causes a 70% decline in the number of receptors present in the internal, recycling compartment. This effect is consistent with morphologic data showing that cholesterol-depleted MA104 cells have a reduced number of caveolae as well as fewer receptors per caveolae.

Animals↗
PubMed1985

Biochemistry and physiological function of human milk proteins.

Human milk has many unique properties that benefit the breast-fed infant. Several of these properties reside in the protein fraction of human milk; eg, host defense factors (such as immunoglobulins, lysozyme, and lactoferrin), digestive enzymes (lipases, proteases, amylase), specific binding proteins, and growth factors. Increased knowledge of human milk proteins and their biochemistry will aid our understanding of their physiological significance in the infant.

Carrier Proteins↗
PubMed1988

Folate-binding protein and the absorption of folic acid in the small intestine of the suckling rat.

The folate in milk is largely bound to high-affinity folate-binding protein (FBP). With an in vivo intestinal loop technique, we examined the absorption of folic acid bound to FBP (FA-FBP) in the small intestine of the suckling rat. In contrast to unbound folic acid (FA), FA-FBP is absorbed more avidly in the ileum than in the jejunum (p less than 0.025) and its absorption is not inhibited by 1 mmol sulfasalazine/L. Folate-binding activities in the mucosa of the proximal (duodenum and jejunum combined) and distal (ileum) small intestine were also examined and found to be 0.32 and 1.31 pmol/mg protein, respectively (p less than 0.001). A 6-h fast produced a 42% decrease in folate-binding activity in the distal small intestine (p less than 0.01) but did not change activity in the proximal portion. Collectively, these observations suggest that FA-FBP is absorbed by a mechanism that is distinct from that responsible for the absorption of FA and that absorption does not require prior dissociation of the vitamin-binding protein complex.

Animals↗