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1 Department of Clinical Chemistry, Horsens Hospital, Horsens, DK-8700, Denmark. .... 6, (A-C). A: Diffuse cytoplasmic staining of the trophobol ast cell lining of.
Bioscience Reports, Vol. 16, No. 5, 1996

Characterization of the Folate Receptor in Human Molar Placenta Jan H o l m , 1'6 Steen Ingemann Hansen, 2 Carl W. Nichols, 3 Mimi Hoier-Madsen, 4 and Poul Erik Helkjaer 5 Received February 15, 1996; accepted May 19, 1996 We have characterized a high-alfinity 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 70kDa 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. KEY WORDS: Folate; receptor; mola; placenta.

INTRODUCTION

High-affinity folate binding proteins (FBPs) exist in two forms, a membranebound folate receptor (FR) and a soluble FBP, sFBP (4). The glycosylphosphatidyl inositol (GPI)--anchored FR resides in the plasmalemmal vesicles of many epithelial cell membranes and internalizes folates by a process known as potocytosis (1, 6, 21). The sFBP purified from bovine milk has been characterized as to primary and secondary structure, polymerization phenomena and ligand binding mechanism (8,18,22,27). Extensive homology existed between the 1 Department of Clinical Chemistry, Horsens Hospital, Horsens, DK-8700, Denmark. 2 Department of Clinical Chemistry, Central Hospital, Hillerod, DK-3400, Denmark. 3 Department of Pathology, West Paces Medical Center, 3200 Howell Mill Rd., NW, Atlanta, Georgia 30327, U.S.A. 4 Laboratory for Autoimmune Serology, State Serum Institute, Copenhagen, DK-2300 S, Denmark. 5 Department of Obstetrics and Gynaecology, Central Hospital, N~estved, DK-4700 Denmark. 6 To whom correspondence should be addressed. 379 0144-8463/96/1000-0379509.50/09 1996PlenumPublishingCorporation

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amino acid sequence of bovine sFBP and FBP in human milk (26), as well as in membranes of human cancer cells and placenta (5, 19, 23, 25). The developing fetus obtains its supply of folate from the mother via transfer across the placenta. The importance of the placental transfer of folate in the growth and development of the fetus is emphasized by the observation that supplementation with this vitamin during the periconceptional period of pregnancy can reduce the incidence of neural tube defects including spina bifida and anencephaly in developing fetuses (29). A high-affinity FBP localized in the maternal-facing brush-border membrane of the human placenta has been isolated and characterized (2, 7). Furthermore a high-affinity s-FBP was shown to be present in human amniotic fluid (11). In the present study we have characterized a high-affinity FR in human molar placenta as to its ligand binding mechanism, molecular size and immunological properties. Immunohistochemical studies were performed to establish features of the cellular localization of FBP.

MATERIALS AND METHODS

Tissue Preparation

Molar placenta tissue was minced and homogenized with a Braun homogenizer (5 strokes at 1000 rev./min) in 5 mM Tris/HC1 buffer (pH 7.4, 4~ containing 5 mM mannitol (5 ml of buffer/g of tissue). Homogenate solubilized (2 h, 4~ with Triton X-100 (20 g/l) was centrifuged (1000 g, 30 min) and the supernatant analyzed. The proteinase inhibitor, phenylmethane sulphonyl fluoride, PMSF (1 mM; BDH) was added to the preparation.

Radioligand Binding, Inhibition and Dissociation

The tissue sample was dialyzed against 0.2 M acetate buffer of pH 3.5 at 4~ to remove endogenous folate. Equilibrium dialysis experiments were performed as described previously (12) in 0.17 M Tris/HC1 buffer of pH 7.4 (37~ with the sample in the internal (1000/zl) and 3H-folate (3H-pteroylglutamate with a specific activity of 44 Ci/mmol from Amersham International Ltd., Amersham, UK) in the external solution (1000 ml). Triton X-100 at a concentration of 10 g/l was added to both sides of the dialysis membrane. In inhibition experiments, the folate analogues, 5-formyltetrahydrofolate supplied by Sigma and methotrexate (Lederle, 4587-24) purified as previously described (10) were added to the external solution (cf. above) together with the radio-label. Portions (1000/zl) of tissue predialysed to equilibrium (pH 7.4, 7~ against i nM 3H-folate were dialyzed against 1000ml volumes of folate-free buffer according to a previously published procedure (12).

Folate Receptor of Human Molar Placenta

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Enzyme-linked Immunosorbent Assay (ELISA) for FBP Rabbit antisera against human milk FBP were pooled, the immunoglobulins precipitated and employed in a previously described ELISA for quantitation of human FBP (12).

Gel Filtration The molecular size of FBP was estimated by chromatography on a column (5.3 cm2 • 94cm) of UltrogelR (IBF) as previously reported (12). Triton X100 (lg/1) was added to the elution buffer 0.17 M Tris/HCL of pH 7.4 (5~ Samples were incubated with 10 nM 3H-folate for 3 h (25~ prior to column application.

SDS-PAGE and Immunoblotting Homogenized molar placenta tissue was analyzed by polyacrylamide gel (12%) electrophoresis in the presence of sodium dodecylsulphate (SDS-PAGE) according to Laemmli (20) and Kyhse-Andersen (17) as described in a previous report (12). Anti-human milk FBP rabbit immunoglobulin was used as primary antibody, non-immune rabbit immunoglobulin as control and peroxidase conjugated swine-anti-rabbit (total) immunoglobulin (Dakopatts) as second antibody.

Immunohistochemistry Paraffin embedded tissues used in FBP immunostaining were selected from the histology files of West Paces Medical Center, Atlanta, GA. FBP immunostaining was demonstrated by avidin-biotin complex methodology (16) using reagents of the Stravigen Multilink Detection System from Biogenex (San Ramon, CA). Four micron histologic sections were used throughout and mounted on silanized glass slides (Fisher, Pittsburgh, PA). All incubations and washes were carried out in phosphate buffered saline, pH 7.4. The sections, after dewaxing and before immunostaining, were subjected to trypsin (0.1%) pretreatment for 20 min at 37~ (Bovine Trypsin Type III from Sigma, St. Louis, MO). After blocking of endogenous peroxidase, the sections were incubated 5 min with 5% horse serum then 100/~1 of rabbit anti-human FBP antiserum diluted 1:500 was added and incubated 10 min at 37~ After development of aminoethylcarbazole chromogen the sections were counterstained with aqueous hematoxylin from Biomeda Corp. (Forest City, CA). Sections of kidney were used as positive control tissues in all immunostain experiments. Negative control slides were prepared by omitting the addition of primary antibody.

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homogenate undiluted (0) and 5-fold diluted (O). Equilibrium dialysis experiments in 0.17 M Tris-HCl buffer (pH 7.4, 37~ Each value is a single determination. Scatchard plots of binding data (abscissa, bound folate nM; ordinate, bound/free) are given as insets.

RESULTS M e c h a n i s m o f R a d i o l i g a n d B i n d i n g , D i s s o c i a t i o n and I n h i b i t i o n in Molar Placental Tissues

Saturation curves for radioligand binding in h o m o g e n i z e d m o l a r placental tissue is shown in Fig. 1 (Scatchard plots inserted). Binding data analyzed in Scatchard and Hill plots (not shown) are summarized in Table 1. T h e binding displayed a p p a r e n t positive cooperativity as indicated by a Hill coefficient significantly higher than 1.00 and u p w a r d convex Scatchard plots (Fig. 1). The binding affinity s h o w e d a t e n d e n c y to increase with decreasing r e c e p t o r concentration (Table 1). N o dissociation of 3H-folate f r o m the tissue o c c u r r e d at p H 7.4 even after dialysis for several days while an almost complete dissociation occurred at p H 3.5 within 24 h of dialysis (Fig. 2). T h e effect of the folate

Table 1. Parameters of High-affinity Binding of 3H-folate to Molar Tissue Homogenate

in Equilibrium Dialysis Experiments (pH 7.4, 37~ Binding data were analyzed by Scatchard and Hill plots. N, maximum 3H-folate bound; So.s, the concentration of free folate at half saturation of folate binding. "Overall" affinity expressed as 1/S0.5 N (nM) So.5 (nM) 1/So.5 (M-1) h (S.D.) Undiluted 6.0 0.157 6.4 • 109 1.35 (0.063)* Five-fold diluted 1.1 0.059 1.7 x 10a~ 1.20 (0.067)** The Hill coefficient (h) is significantly higher than 1.00: P