Proteins associated with activity of Fc receptors on isolated human placental syncytiotrophoblast microvillous plasma membranes

Placenta (1989), IO, 227-246

Proteins Associated with Activity of Fc Receptors on Isolated Human Placental Syncytiotrophoblast Microvillous Plasma Membranes

JAMES A. DOWN”, MICHIKO KAWAKAMI, MICHEL H. KLEIN & KEITH J. DORRINGTONb Departments of Immunology and Biochemistry, Toronto, Toronto, Canada MgS I A8

timversi!)~

of

u Present address, Department of Biochemistry and Molecular Biology, Harvard Universi
Paper accepted

25.

I I. I

988

INTRODUCTION The immune system of newborns is incompletely developed and immunity to infection is derived from maternal immunoglobulin G (IgG) that is transferred to the fetus via the placenta. Materno-fetal transfer of passive immunity has been thought to be mediated by receptors recognizing the Fc portion of IgG (FcR) that are expressed on fetal syncytiotrophoblast cells on the maternal face of the human placenta (reviewed by Johnson and Brown, 1981). Early studies suggested that FcR mediate IgG transfer across the rabbit yolk sac placenta (Brambell, Hemmings and Oakely, 1958; Brambell et al, 1959), implying a role in human prenatal transfer of passive immunity from the mother to the fetus. While this remains a working hypothesis in humans, FcR may also protect the fetus from maternal immune responses (Balfour and Jones, 1978). At present the function of placental FcR, the mechanism of IgG transport across the human placenta and their possible relatedness have not been elucidated. Immunochemical data suggest that human placental FcRs are expressed on microvilli (Matre and Haugen, 1978), capillary endothelium (Matre, Kleppe and Tonder, 1981; Matre and Haugen, 1978; Johnson, Trenchev and Faulk, 1975) and HolIbauer cells (Moskalewski, Ptak and Czarnik, 1975). Binding studies using monomeric human IgG have detected FcR on placental plasma membranes (Balfour and Jones, 1978; Watanabe, Gitlin and Gitlin, 1980; Neizgodka et al, 1981; Van der Meulen et al, 1980; McNabb et al, 1976) and isolated syncytiotrophoblast microvillous plasma membranes (StMPM) (B rown and Johnson, 1981). Each site may have specific ligand preferences (Matre and Haugen, 1978; Johnson, Trenchev and Faulk, 1975; Matre and Johnson, 1977; Johnson, Faulk and Wang, 1976). In efforts to understand the role of placental immunoglobulin receptors during pregnancy, 0x43-4004/89/030227

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0 1989 Bailliire Tindall Ltd

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there have been numerous attempts to identify IgG-binding proteins from human placenta (Balfour and Jones, 1978; Mikulska et al, 1982, Fernandez-Botran and Suzuki, 1982; Matre, Kleppe and Tonder, 1981; Brown and Johnson, 1981; Watanabe, Gitlin and Gitlin, 1980) and several contradictory reports have described putative FcR proteins isolated from placental sources. Mikulska et al (1982) reported a 60 ooo M, species in glycoprotein fractions of crude placental membranes which bound to immune complexes containing IgG. Matre, Kleppe and Tonder (1981) isolated a 47 ooo M, protein from aqueous extracts of whole trophoblast tissue by ligand affinity chromatography on heat-aggregated IgG-Sepharose. A putative FcR that demonstrated phospholipase-A, enzymatic activity was also isolated from detergent extracts of human amnionic membranes (Fernadez-Botran and Suzuki, 1982). Though a monoclonal antibody was prepared against the 47 ooo M, protein (Matre, Haaheim and Tonder, 1984), to date no further corroboration of biochemical analysis of these species has been reported. To help define the functions of the placental FcRs, we sought to characterize them biochemically. After solubilization of isolated StMPM by non-denaturing detergents, FcR were coprecipitated with immune-complexed IgG or isolated by affinity chromatography over antigen-bound monomeric IgG. Photoaffinity labelling of the FcR by crosslinked-derivatized IgG was used to corroborate the previous observations. In the course of these experiments, it was found that three proteins (M, = 68 ooo; 5200o-56000; and 40000) were isolated from StMPM detergent lysates by their ability to bind IgG and were discriminated by their binding to immune complexed IgG versus immobilized monomeric IgG. We present evidence to suggest that the 52 000-56 ooo M, StMPM protein was possibly identical to the 60 ooo M, glycoprotein Fc receptor previously isolated from extracts of semipure placental membranes by Mikulsa et al (1982) and that the 40 ooo and 68 ooo M, StMPM proteins bound IgG in a manner distinct from the glycoprotein FcR. . MATERIALS

AND METHODS

Preparation of placental syncytiotrophoblast microvillous plasma membranes and plasma membrane extracts Exfoliated placental syncytiotrophoblast microvillous plasma membranes (StMPM) were isolated from fresh full term placentae by the method of Booth, Olaniyan and Vanderpuye (1980) and were used immediately. Prior to FcR isolation, FcR activity of each StMPM preparation was confirmed by an agglutination assay (see below). Glycoprotein extracts of crude placental membranes were prepared by lithium diiodosalicylate (LDS)/phenol extraction as described by Neizgodka et al (1981) and detergent extracts of StMPM were prepared as follows. Approximately I mg of StMPM in a volume of 50 PL was suspended in I mL of phosphate-buffered saline (PBS: 20 mM NaH,PO,, pH 7.4, 0.15 M NaCl) containing I per cent detergent and IO mM EDTA (Aldrich), IO pg soybean trypsin inhibitor (Sigma), IO pg pepstatin (Worthington), 20 pg phenylmethylsulfonyl fluoride (PMSF, Sigma) and I mg c-amino caproic acid (Sigma). After 30 min incubation on ice, the mixtures were centrifuged at IOOooog for 30 min and the supernatants were used immediately. The following detergents were individually used: Triton X IOO (TXIOO, Shell), Nonidet P 40 (NP40, Particle Data Laboratories), Cholate (Sigma), Deoxycholate (DOC, Sigma), Taurocholate (Sigma) and 3-[(-cholamidopropyl)dimethylammonioll-r-propane sulfonate (Hjelmeland, 1980) (CHAPS, Biorad). All procedures were done at 4°C or on ice. Fc receptor assay Agglutination of StMPM

by IgG-coated

sheep erythrocytes

(EA) was modified from a pre-

Damn er al: Human Placental

Syncrtiotrophoblast

FcR proterns

229

vious procedure (Van der Meulen et al, 1980). EA were produced by incubating one volume of packed, saline-washed SRBC (Woodlawn) with a 1:800 dilution of rabbit anti-SRBC IgG (Cordis) in normal saline for I h at 37°C. The EA were washed four times with normal saline and used immediately. For each agglutination assay, 15 PL of EA were mixed with IOOPL dilutions of StMPM in round bottomed microtitre plates (Cooke). Optimal concentrations of StMPM produced agglutination patterns of 6 mm diameter. Inhibition experiments were performed under the same conditions using optimal concentrations of StMPM in the presence of serial titrations of inhibitor proteins.

Radioiodination of proteins Placental StMPM were radioiodinated by chloramine T (Hunter and Greenwood, 1962). A typical reaction mixture contained I mg of StMPM in a 50 PL volume buffered with o. IM sodium phosphate, pH 7. The mixture was put on ice, then 0.5 to 2.0 mCi of carrier-free Na[ * z51] (Amersham) and I PL of a solution of 0.22 M chloramine T (BDH) were added. After 20 min, the reaction was stopped by dilution into I mL of Tris-buffered saline (TBS: IO mM Tris-HCl, pH 7.4, 0.15 M NaCl) containing 0.2 per cent sodium azide. Unbound [125I] was removed by dialysis against TBS buffer. After dialysis, 90 to 95 per cent of counts were precipitated by IO per cent TCA. The specific activity was approximately 10~ cpm mgg i when I mCi of[ 125I] was used. Lactoperoxidase was used to incorporate [ 125I] onto IgG because chloramine-T catalyzed iodination produced IgG breakdown (Down and Kawakami, personal communication). Lactoperoxidase (Sigma) was added to a 50 PL volume of o. I 1M sodium phosphate containing IOO to 1000 pg of IgG in a weight ratio of 1:50 (LP: IgG). 0.5-2 mCi of Na[‘251] (Amersham) were added and incorporation was started by addition of 5 PL of a I : 4000 dilution of 30 per cent H,O, (Fisher). Two further additions of H,O, were made at IO min intervals and then the reaction was stopped with 5 ~1 of 20 per cent sodium azide. Free [’251] was removed from the IgG by gel exclusion chromatography on Sephacryl S200 (Pharmacia). Approximately 99 per cent of the counts contained in the purified IgG were precipitated by IO per cent TCA.

Isolation of placental IgG-binding proteins Coprecipitation of FcR molecules with immune complexes formed between sheep or goat antihuman IgG antibodies and human myeloma IgG, (He) was performed according to a protocol established for isolation of a placental FcR from glycoprotein extracts of crude placental plasma membranes (Mikulska et al, 1982). Ligand affinity chromatography on immune complexed IgG was performed in batches after the method of Kulczycki (1983) for FcR affinity chromatography on IgG Sepharose. Approximately 400 pug of rabbit anti-dinitrophenol (DNP) IgG or F(ab’), were added to 250 PL of packed DNP-lysine Sepharose, which was previously washed with TBS buffer containing o. I per cent NP40. After rotation overnight at 4°C the beads were washed three times at room temperature with TBS buffer, and then non-specific sites were saturated by incubation with 500 pg of human myeloma IgG, (He) or rabbit non-immune IgG or respective F(ab’), fragments, for 6 h at 4°C. After ten washes with TBS buffer, IOO PL of a 50 per cent suspension of complexed matrix was mixed with 0.25 mL of radiolabelled StMPM detergent lysate or LDS/ phenol extract and the mixture was rotated overnight at 4°C. Following four washes with 1.3mL ice-chilled TBS buffer containing 0.1 per cent detergent (described below), bound proteins were eluted from the beads by boiling in SDS-PAGE sample buffer and analysed on SDS-PAGE.

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Photoaffinity crosslinking of IgG to FcR Approximately 400 pg of rabbit anti-DNP IgG was reacted for I h in darkness with a ten-fold molar excess of N-succinimidyl(4-azidophenyl)-I,3-dithiopropionate (SADP, Pierce) in PBS buffer containing 10mM iodoacetamide (Kodak). The reaction was z ml volume and was stopped by addition of 0.2 ml of I M Tris-HCr, pH 7.4. Unbound azidophenyldithiopropionate (APDTP) was removed by overnight dialysis at 4“C against 2 1 of PBS buffer containing IO mM iodoacetamide. BSA (I mg) was included to reduce losses on the dialysis bag due to non-specific adsorption. Care was taken to exclude ambient light during all steps and manipulations were performed under a red safety lamp. Optimal photoactivation conditions were determined for each batch of APDTP-IgG by crosslinking to Staphylococcus aureus protein A (Pharmacia). Thirty ~1 mixtures containing 1-2 pg of [iZ51]-APDTP-IgG, IOOpg protein-A and 3.3 mM iodoacetamide were set up in 12 x 80 mm borosilicate culture tubes and were incubated in darkness at 20% for 30 min. The tubes were immersed in ice to prevent heating and the mixtures were illuminated with an ultraviolet lamp for between 30 set and 15 min. Extent of crosslinking was analysed by SDS-PAGE and subsequent autoradiography. To crosslink APDTP-IgG to FcR, 2-3 mg of StMPM in a volume of 150 ~1 were incubated (either unlabelled or [‘251]-labelled as overnight at 4°C with IO ,ug (IOO ~1) of APDTP-IgG described above) in PBS buffer containing 4 per cent BSA, 2 mM iodoacetamide and protease inhibitors (see above). The reaction mixture was irradiated with UV light for a time predetermined by control experiments as previously described. StMPM-complexed APDTP-IgG was separated from the unbound ligand by centrifugation in PBS buffer containing IO mM iodoacetamide and 0.5 M NaCl at IOOooog for 15 min. The StMPM FcR-IgG complexes were then solubilized by 30 min incubation on ice in PBS containing IO mM CHAPS and protease inhibitors. The mixture was centrifuged at IOOooog for 30 min and the resulting supernatant was rotated overnight at 4°C with 0.5 ml of a 50 per cent solution of DNP-lysine Sepharose and IO mg BSA. The Sepharose beads and bound anti-DNP-IgG-FcR complexes were washed three times by microcentrifugation in PBS containing I per cent cholate and I mM iodoacetamide, then packed in a I ml syringe and further washed with 1000 volumes of PBS containing 0.1 per cent cholate. FcR-IgG complexes were eluted with IO mM DNP-lysine (Sigma) and were analysed directly on SDS PAGE, or were subjected to further rounds of labelling and DNP-lysine affinity chromatography. For the latter, final elution of FcR from the complex was accomplished with 25 tIIM DTE after removal of iodoacetamide during washing. The final eluates were adsorbed with insoluble protein-A prior to SDS-PAGE analysis. Enzyme assays To assay NADH dehydrogenase activity, 50 ~1 of placental isolate (4-20 pg of protein) were added to a reaction mixture composed of 0.4 ml of 20 mM Tris-HCr, pH 7.4,0.2 ml of 3.3 mM potassium ferricyanide and 0.4 ml of 0.3 ItIM NADH in 20 mM Tris-HCr, pH 7.4. The reaction was quickly mixed and allowed to react at room temperature while optical density was monitored at 340 nm in a Cary 219 spectrophotometer. Alkaline phosphatase activity was monitored by mixing 50 ~1 of placental preparation with I mL of I mM p-nitrophenyl phosphate in I M Tris-HCr, pH 8.0. The reaction was kept at 27°C while optical density was measured at 420 nm. Purification and assay of protein reagents Purification of IgG and production of proteolytic fragments was performed as previously described (Ellerson et al, 1976). Purity was assessed by SDS PAGE under reduced and non-

lkmn CIaI, Human Placental Synritrotrophoblast FcR proteins

231

reduced conditions. Protein concentrations of membranes were estimated according to Lowry et al (1951) using BSA as a standard. Immunoglobulin concentrations were estimated from absorbance at 280 nm using A280( I per cent) = 14.0. SDS polyacrylamide gel electrophoresis and western blot analysis Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE) was performed according to Laemmli (1970). The following molecular weight standards (Biorad) were run under reducing conditions: myosin heavy chain (200 ooo), B-galactosidase (I 16 OOO), phosphorylase-B (92 LOO),bovine serum albumin (68 ooo; reduced), ovalbumin (43 ooo), carbonic anhydrase (30000). The fixed, dried gels were autoradiographed at -7oC on Kodak XAR film over DuPont Lightning enhancing screens. Western transfer to nitrocellulose (Biorad) was done according to an established protocol (Towbin, Staehlin and Gordon, 1979) using filtered 3 per cent BSA as a blocking reagent and [ ‘251]-protein-A (labelled as described for IgG) as a reporter. Electron microscopy Membranes were pelleted by centrifugation and fixed in normal saline containing 2.5 per cent glutaraldehyde. After extensive washing, samples were stained, imbedded and viewed using standard protocols by the Electron Microscopy Services at the University of Toronto.

RESULTS Isolation of a previously described glycoprotein FcR from lithium diiodosalicylate extracts of placental membranes Precipitation of placental plasma membrane glycoproteins with immune complexes was performed as described by Mikulska et al (1982) and yielded a glycoprotein of approximately 52 000-56 ooo M, (Figure I). Proteins of approximately 200 ooo and 240 ooo were also very prominent on non-reduced SDS-PAGE. They migrated near the molecular weight of nonreduced immunoglobulins and after reduction, were replaced by a protein of approximately 70 ooo M, (shown later in Figure 6) suggesting they were maternal immunoglobulins that were found by anti-immunoglobulin antibodies in the immune complexes. Though maternal IgG was identified in detergent lysates of placental plasma membranes by western blotting (Down, 1986), blots containing the glycoprotein extract reacted with antisera against human IgG, IgD, IgM, or IgA did not detect reactivity at 240 ooo or zoo ooo non-reduced M,. Consequently, the identity of these higher M, IgG-binding proteins is uncertain. The 56 ooo M, protein isolated here under identical conditions as described by Mikulska et al (1982) was similar in size and thus possibly identical to the 60000 M, FcR they described but contrary to their findings, it did not migrate at 30 ooo M, after disulfide reduction. Its binding specificity for Fc was further characterized (see further). Characterization of exfoliated placental microvilli as a source for immunoglobulin receptors Most previous biochemical reports used crude membranes or whole tissue as sources for placental FcRs (Balfour and Jones, 1978; Mikulska et al, 1982; Fernandez-Botran and Suzuki, 1982; Matre, Kleppe and Tonder, 1981; Watanabe, 1980). In our next experiments, placental syncytiotrophoblast microvillous plasma membranes (StMPM) were utilized as a source for FcR isolation because they represent a source of placental plasma membranes of much higher

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Placenta (1989), Vol. IO

F&UP I. Autoradiogram of nonreduced SDS PAGE of labelled placental plasma membrane glycoproteins that were coprecipitated with immune complexes formed between human IgG and sheep anti-human IgG antibodies. Numbers to left indicate migration distance of molecular weight markers ( x IO “) and the arrow on the right indicates the position of the glycoprotein FcR reported previously by Mikulska et al (1982).

purity (Booth, Olaniyan and Vanderpuye, 1980). As previously reported by Booth, Olaniyan and Vanderpuye (1980), the method produced a preparation free of other contaminating organelles (Figure 2) and loo-fold enriched in alkaline phosphatase (Table I), a reliable marker of placental plasma membranes (Booth, Olaniyan and Vanderpuye, 1980). We confirmed Johnson and colleagues’ finding, that purified syncitiotrophoblast microvilli exhibited FcR activity. (Figure 3) (Brown and Johnson, 1981; Johnson and Brown, 1981; Ogbimi et al, 1979). StMPM FcR activity was not abolished by the chloramine-T catalysed iodination used for FcR labelling (Down, 1986). In vitro assay of placental FcR activity StMPM ligand binding specificity was tested using an in vitro agglutination assay similar to that described previously (Van der Meulen et al, 1980). As expected from previous reports (Brown and Johnson, 1981; Van der Meulen et al 1980), competing ligands, such as human IgG or Fc fragments displaced the erythrocyte-bound IgG, producing smaller agglutinations while human Fab or F(ab’), fragments did not inhibit the StMPM-EA agglutination (Figure 3). Surprisingly, goat or sheep IgG enhanced agglutination when added to the reaction, due apparently to anti-IgG reactivity, since pre-absorption of the immunoglobulin fractions on columns of IgG-Sepharose before addition to the FcR assays removed the enhancement (Figure 3). Nonetheless, after absorption the sheep and goat IgG still inhibited FcR reactivity,

Down et al: Human Placental

.Tynritiotrophoblast

FcR proteins

233

Fqu~r a. Electron micrographs of placental StMPM. EM analyses were performed asses\ the purity of StMPM used for FcR isolation. x ao ooo.

as described in order to further

though less effectively than human IgG,. Figure 3 depicts an example of equal inhibition of FcR activity by absorbed sheep or rabbit anti-DNP specific IgG (done as controls for the immune coprecipitation experiments). This result contrasted with previous findings and contradicts the notion that IgG of species which lack transplacental IgG transport (e.g. sheep, goats, cows) do not bind human placental Fc receptors (Matre and Haugen, 1978; Van der Meulen et al, 1980). isolation of proteins associated with StMPM FcR activity Isolation of murine and human FcR have been achieved after solubilization from plasma membranes by conventional nonionic detergents (e.g. Kulzycki, 1983). Since this had not been reported for the placental FcR, immune precipitations were carried out as described above using StMPM detergent lysates. Because FcR solubility may differ with detergent (e.g. Rivnay et al, 1982). five were tested: TXIOO. NP40, CHAPS, DOC, Cholate. However, all the detergents tested gave a similar result: labelled proteins of approximately 68 ooo; 52 00~56000 and Tub/,, I

Fnrichment

of marker enzymes during

Protein Preparation

(mg)

Initial Filtrate Pure microvilli Enrichment

3200 35

purification of human placental membranes Specific activity Alkaline Phosphatase 3.2 270.4 85.6

(PM

syncitiotrophoblast

min

’ mg

microvillous

’protein)

NADH Dehydrogenase 4ro.o 22.5 0.05

Placenta

5

(rgbp), Vol. 10

(e)

4

5

(f)

4 3

0

200

400

600

800

2L 0

1000 Inhibitor

200

400

600

800

(+g)

FQW~ 3.Assay of StMPM FcR activity by agglutination

of placental StMPM and EA. To determine that placental StMPM were an effective source of FcR, analysis of Fc-binding activity was tested as previously described (Van der Meulen et al, 1980). Purified StMPM were incubated with sheep erythrocytes coated with rabbit anti-sheep erythrocyte IgG. The reactions were performed in U-shaped microtitre wells and binding of the IgG-coated erythrocytes to placental FcR was assessed. Competitive inhibition by homologous and heterologous IgGs produced a reduction in the size of the agglutination pellets as compared to control reactions done in the absence of competing ligand.(a) The binding was specific for the Fc portion of IgG as demonstrated by the observation that intact myeloma IgG (He) inhibited agglutination ( W), as judged by a reduction in the extent of the agglutination, whereas F(ab’), (He) fragments (0) had no effect. (b) Since goat (@) or rabbit (B) anti-DNP IgG reacted with erythrocyte-bound IgG, as judged by the enhanced agglutination they produced, the antisera were absorbed on human IgG Sepharose prior to experiments. (c)Absorbed goat (Cl) and rabbit (b) anti-DNP IgG inhibited FcR binding. (d) Likewise, absorbed goat nonimmune IgG (0) competively displaced EA-IgG but to a lesser extent than human IgG ( ?? ). (e) Absorbed rabbit and, (f) mouse non-immune IgG also inhibited microvilli FcR activity. At the dilutions of antisera used, natural antierythrocyte reactivity was not observed as evidenced by a lack of agglutination of unsensitized erythrocytes. The agglutination diameters were read in millimeters to a precision of 0.5 mm.

40 ooo M, coprecipitated with immune complexes of human IgG and sheep or goat anti-IgG (Figure 4). For convenience, they are referred to as pIBP68, pIBP56 and pIBBP4o (pIBP: placental immunoglobulin binding protein). Our previous result suggested that placental FcRs bound sheep or goat IgG. However, Neizgodka et al (1981) demonstrated that FcR activity in situ in placental membranes was not inhibited by bovine, sheep, pig or horse immunoglobulins. complexes of sheep or goat anti-IgG antibodies and human

This would suggest that immune F(ab’), fragments could not bind

Down et al: Human Placenral S,yncitlotrophoblasr FcR proteins

NONlDET

P40

a35

TRlTON

X 100 RA

116-

45-

Frpm 4, Autoradiograms of detergent solubilized, radiolabelled StMPM proteins that were coprecipitated with immune complexes. Immune coprecipitations, SDS PAGE and autoradiography were performed as described. NONIDET and TRITON indicate results obtained using Nonidet P 40 or Triton X too, respectively, for StMPM solubilization. The gels were 1.5 mm thick and sample wells were 2.5 cm wide in order to minimize distortion by the IgG in the pellets. The samples were run under nonreducing (NR) conditions or were reduced by boiling for a min in IO rnM DTE followed by alkylation by addition of IM iodoacetamide to a final concentration of ao rnM (RA). In each pair of experiments, proteins were coprecipitated with human F(ab’), (I) or IgG (2) and sheep anti-human IgG. Numbers on left indicate relative molecular weights ( x IO “). On right are indicated positions of proteins referred to in the text.

Table 2. Relative molecular

weights of immunoglobulin-bound proteins isolated from StMPM LDS/phenol extracts of placental membranes Immune precipitation

Method

Extract

StMPM Detergent lysate

67.7 f 1.6 (29) 53.7 * 3.9 (13) 38.9 f 0.9 (14)

lysates or

Immune complex affinity chromatography

Glycoprotein extract

StMPM Detergent lysate

237.4 * rr.s(6) ‘94.4 f 7.2 (6) pIBP68 pIBP56 pIBP40

detergent

54.7 * a.6 (9)

Glycoprotein extract 233.0 * 5.9 (7) r&a f 7.6 (7)

51.2 * 1.7 (22)

54.3 f 0.6 (8)

Relative molecular weights are the sizes observed for respective M, on SDS PAGE under non-reducing conditions and are presented x IO 3. The values represent means f s.d. of observations made by SDS-PAGE and subsequent autoradiography. Numbers of observations are indicated by within brackets. Names of the proteins referred to in the text are presented at left.

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FcRs. We repeated the immune precipitations using labelled StMPM proteins and immune complexes containing sheep anti-human IgG antibodies with human F(ab’), and indeed observed that the numbers of cpm in the pellets were greater in complexes containing human IgG rather than F(ab’),. This indicated a preference of the IgG-binding proteins for intact human IgG but absolute specificity for Fc was not observed and, as shown in Figure 4, when approximately equal numbers of cpm were applied to SDS-PAGE the autoradiograms indicated that pIBP68, pIBP56 and pIBP40 were seen in complexes containing sheep anti-IgG antibodies with either human IgG or F(ab’),. This follows our previous finding that StMPM FcRs bound sheep IgG in situ and thus isolated putative FcRs should have bound immune complexes containing intact sheep IgG. Finally, pIBP56 was not observed on SDS-PAGE after reduction and alkylation. Possible explanations for this are that after reduction, pIBP56 formed a complex which did not enter the gel, or reduced to subunits which migrated at the dye front of the gels in possible agreement with the findings of Mikulska et al (1982), or comigrated with pIBP68 and/or pIBP40. Figure 5 presents these observations quantitatively in histograms, showing the number of experiments in which each of the respective pIBPs were detected. It can be seen that the pIBPs were not detected in IOO per cent of the autoradigrams. Also they did not appear to bind to the same extent, with pIBP68 observed more often than pIBP56, in turn more often observed than pIBP40. Finally, the upper panel of Figure 5 shows data pooled from experiments in which the nonionic detergents NP40 or TXIOO were used for StMPM solubilization. It shows apparent Fc-specific binding of pIBP40. We were unable to produce immune pellets of human F(ab’), and F(ab’), precipitating antibody and thus could not do control precipitations with immune complexes lacking Fc. Instead, affinity chromatography was used to investigate StMPM FcR-ligand specificity. Our previous attempts to isolate StMPM FcRs by IgG ligand affinity chromatography, utilizing IgG covalently crosslinked to Sepharose, were unsuccessful (in contrast to Matre, Kleppe and Tonder, 1981; Fernandez-Botran and Suzuki, 1982). This was perhaps due to chemical modification or steric constraint imposed on the IgG ligand by crosslinking to the Sepharose, or due alternatively to dissociation of FcR-IgG complexes during isolation. To counteract these possibilities, affinity chromatography was performed batchwise in small volumes (Kulczycki, 1983) over immobilized immune complexes of rabbit anti-DNP IgG bound to DNPSepharose, with as few washing steps as feasible carried out as quickly as possible on ice. This method allowed us to compare the specificity for Fc of the putative FcR proteins by binding them to intact IgG or F(ab’), fragments, themselves bound to the hapten-Sepharose. Under these conditions, the gel profiles revealed apparently irrelevant high molecular weight proteins and the surprising omission of pIBP68 and pIBP40 previously found to bind immune complexes in solution (Figure 6). In contrast, pIBP56 was predominant in eluates from immobilized IgG and appeared to show Fc-specific binding activity since it was hardly detectable or absent in eluates from F(ab’), Sepharose. For comparison, binding specificity of the pIBP56 isolated from crude placental membranes by LDS glycoprotein fractionation (Mikulska et al, 1982) was tested by the same method described above. When the binding reaction was performed in the absence of detergent, there was no apparent specificity for Fc since pIBP56 bound F(ab’), equally well, as shown in Figure 7. In the presence of NP40 or TXIOO, there was binding preference of pIBP56 for intact IgG but it was not absolute because binding to F(ab’), was also observed and the difference in binding was only seen after short-term autoradiographic exposure (not shown). Figure 7 illustrates two further points. As previously mentioned, it can be seen that the unidentified 240 ooo and 200 ooo M, IgG-binding glycoproteins were replaced by a 70 000 M, protein after

a37 100

100

r

pl BP68

plBP56

plBP40

r

FiRwe 5. Summary of observations of pIBP68, pIBP56 and pIBP40 bound to pelleted immune complexes. Labelled protein bands observed on exposed autoradiograms within the ranges of 68 000; 54 ooo and 40 ooo M, ( f 5 per cent) were tallied and expressed as a percentage of the total number of experiments (N). The histograms summarize the autoradiographic data by the following groupings. Upper: The gels were run under non-reduced conditions and only experiments utilizing the non-ionic detergents (TXroo, NP 40) are presented. Fc specific binding of pIBP40 is seen. Middle: The gels were run under non-reduced conditions and all experiments were counted, regardless of detergent used during the binding reaction (i.e. CHAPS, NP 40, TXIOO, taurocholate or cholate). Bottom: Observations from gels run after reduction and alkylation of the samples from binding reactions utilizing the detergents CHAPS, NP40, TXtoo, taurocholate or cholate. Open bars indicate results of experiments using immune complexes containing human IgG,, black bars indicate results obtained when immune complexes contained human F(ab’), fragments. Each respective detergent was used for both initial solubilization and to retain solubility during the immune coprecipitations (i.e. detergent combinations were not used).

disullide

reduction.

Also, the protein

at the position

of 56 ooo M,, which we interpret

as being

pIBP56, retained the same mobility after disulfide reduction. This was in contrast to detergent-solubilized StMPM pIBP56. A facile explanation for these observations would be that we observed

two different

56000 M, proteins

in the two preparations.

We doubt

this because

seems too coincidental that two dissimilar placental integral membrane proteins identical molecular weights and also bind IgG. The difference in SDS-PAGE

it

would have mobility of

Placenta (1989), Vol. 10

238 1

2

3

4

5

6

-116

-43

-Fi&re 6. Autoradiograms of labelled, detergent solubilized StMPM proteins &ted from affinity chromatography on immune complexed IgG. Examples of results obtained with three detergents are shown: Nonidet-P 40 (lanes I,z), Triton X roe (lanes 3,4) or CHAPS (lanes 56). The putative FcR is visible in eluates from IgG-bound Sepharose (lanes 2,4,6) (indicated by an arrow) and not in controls of immobilized F(ab’), (lanes 1,3,5). Nonidet and Triton samples were run in IO per cent polacrylamide and CHAPS samples were run in 7.5 per cent polyacryalmide gels. Arrows to left of lanes I to 4 indicate relative molecular weights of same protein standards ( x IO “) adjacent to lanes 5 and 6.

reduced IBP56 from either source may arise from proteolysis; either in StMPM pIBP56 accessibility to protease attack or in the presence of proteases in the StMPM preparations that were not present in the glycoprotein extracts. Photoaffinity crosslinking of IgG to StMPM proteins In an attempt to address the problem of disparity between proteins which bound IgG in immune complexes (M, = 68 ooo; 52 000-56 ooo; 40 ooo) or immobilized on affinity columns (M, = 52 00~54000), monomeric IgG was crosslinked to StMPM FcR in situ. For this purpose, the heterobifunctional photoaffinity crosslinker, N-succinimidyl(4-azidophenyl)r,3dithiopropionate (Vanin and Ji, 1981), was derivatized to rabbit anti-DNP IgG. Control experiments demonstrated that the derivatized APDTP-IgG was able to crosslink to S. aureus protein-A, (Figure 8) indicating that the APDTP moiety was functional and the derivatized IgG retained Fc structure required for binding to protein-A. Covalent bonding of APDTP-IgG to FcR was produced with photolysis conditions deter-

Damn 01al: Human Placental $yncittotrophoblast FcR proteins

2

1

239

3

4

-FcR

-43 Ft~urr 7. Autoradiogram of labelled glycoproteins eluted by SDS from immune complex affinity columns. SDS PAGE analyses of eluates from rabbit anti-DNP IgG (lanes 2,4) or F(ab’), (lanes t,3) affinity chromatography batches. The ligands were bound to DNP-Scpharosc as described in the methods section. The samples were run in 7.5 per cent polyacrylamide gel under reducing (lanes 1,~) or nonreducing (lanes 3,4) conditions. The position of the glycoprotein FcR is indicated. Numbers indicate Mr x IO '.

mined

in the control

of unbound

experiments

APDTP-IgG,

anti-DNP

IgG were affinity

periments,

the crosslinking

rabbit

or human

(Steiner

M, (i.e. uncomplexed), that crosslinked

purified

(Figure

with CHAPS

9). The StMPM

and the complexes

on DNP-lysine

was performed

IgG. To determine

eluted from a single round sions: under non-reducing dimension

using protein-A

solubilized

Sepharose.

in the presence

the approximate

were washed

of StMPM-FcR

In simultaneous

of a roe-fold

size of the IgG-FcR

1986). By this method,

the APDTP-IgG

282 ooo M, and the top of the gel (not shown).

to the IgG

was deduced

from

the difference

166 ooo species, which indicated that the latter species proteins with a total M, of approximately I 16 ooo.

resulted

and ex-

excess of underivatized complex,

of affinity purification were subjected to SDS-PAGE conditions in the first dimension, and after reduction

and Luscher,

control

free

proteins

in two dimenin the second

migrated

at 166000

The size of the protein(s)

in the M, of the 282000 from crosslinking

and

of the IgG to

Placenta (1989), Vol. IO

240

200-

92.5Figure 8. Control crosslinking of radiolabelled APDTP-IgG to S. aurtvs protein-A. To determine the optimal amount of illumination required for APDTP-IgG photolysis and subsequent crosslinking to FcR, control experiments were preformed using S. a~etl~ protein A as an FcR model. t-a pg of [ ’* SI]-labelled, APDTP-IgG was incubated with IO pg of unlabellcd SpA in a total volume of ao pL. The mixture was incubated in darkness for IO mitt, then illuminated with a UV light. The samples were immediately diluted t:t in SDS-PAGE sample buffer containing IO rnst iodoacetamide, then run on SDS-PAGE and processed as previously described. Times of illumination (from left to right) are o, I, a, 5, to, and t5 min. The crosslinked SpA-IgG complex was detected as a band of about 40 ooo mass higher than IgG (arrow). After reduction (not shown) the conjugated IgG heavy and light chains ran with the same mobilities as the underivatized species.

Proteins crosslinked to IgG could not be identified in eluates from a single round of affinity purification because non-specific binding of other irrelevant proteins obscured the analyses. Therefore, the proteins eluted from DNP-lysine Sepharose were subjected to two more rounds of relabelling and repurification in DNP-lysine Sepharose. To minimize IgG contamination in the final eluate, FcR were eluted by cleaving the APDTP disulfide bond with DTE and passing the eluate over formalin-fxed S. aweus (Pansorbin) prior to SDS-PAGE analysis. Autoradiograms of control and test experiments showed a labelled protein of approximately 50 ooo to 55 ooo M, (distorted because of BSA, which was added to reduce nonspecific binding). Proteins of I 16 ooo, 40 ooo, and 30 ooo M, bound specifically to the IgG since they were not seen when the reaction was performed in the presence of excess unlabelled IgG (Figure 9).

DISCUSSION Discrepancies exist in the literature regarding the biochemical characteristics of placental Fc receptors. Their size, affinity for IgG, and indeed, the number of placental FcR types and their sites of expression remain somewhat uncertain (for reviews see Johnson and Brown, 1981; Burton, 1985). The studies described here were performed to corroborate previous observations of placental FcRs and extend their biochemical characterization. A major drawback to biochemical analyses of proteins associated with placental plasma membranes arises from the difficulty in excluding membrane contaminants derived from cell types other than syncytiotrophoblast. Circulating lymphocytes and macrophages express FcR (e.g. Burton, 1985; Dorrington, 1984; Unkeless, Fleit and Mellman, 1981), and thus membrane contamination from these cell types must be excluded in studies of placental FcRs. To address this problem, exfoliated placental StMPM (Booth, Olaniyan and Vanderpuye, 1980) have been used as a source for placental FcR (Brown and Johnson, 1981) and their purity assessed by enzymatic assay and electron microscopy as previous (Brown and Johnson, 1981;

Dmn

ct ul: Human

Placental

.Synritiotrophoblast FcR proteins ia)

200

(b)

_

30 e

.

-_

A

Fgurv 9. Autoradiogram of labelled StMPM proteins crosslinked to APDTP-IgG. The FcR-IgG complex was purified by three successive rounds of DNP-lysine affinity chromatography, elution, dialysis and relabelhng. The putative FcR proteins were eluted by reduction of the APDTP disulfide bond and the eluate was incubated with insolubilized SpA to remove any possible IgG contaminants. The samples were diluted I: I in SDS PAGE buffer and run in SDS PAGE slabs. (a) Crosslinking in the presence of excess unlabelled IgG produced a labelled protein of approximately 50000 Mr (distorted because of unlabelled BSA). (b) When the experiment was performed in the absence of competing ligand, the same 50 ooo55 ooo Mr protein was seen in greater amount and proteins of I 16000, .+oooc and 30 ooo were also observed (arrows). Numbers indicate apparent molecular weights of standard proteins

(x IO “).

Booth, Olaniyan and Vanderpuye, 1980; Ogbimi et al, 1979). Because [Iz51] was used to label microvillous membrane proteins, from which FcRs were to be isolated, it was tested and confirmed that iodination did not abolish microvilli FcR activity (Down, 1986). The protocol of Mikulska et al (1982) was used to isolate labelled IgG-binding proteins from detergent lysates of placental microvilli. In contrast to previous studies (Balfour and Jones, 1978; Mikulska et al, 1982; Fernandez-Botran, 1982; Matre et al, 1981), proteolytic inhibitors were introduced since other human FcR are known to be sensitive to proteolysis by endo-

242

Placenta (1989), Vol. IO

genous

proteases

globulin

(Kulczycki,

binding

(‘pIBP40’)

Solanki

proteins

of

M, reproducibly

or goat anti-human

IgG. Similar antibody

results

were obtained

reasons.

1984). StMPM

52 000-56000

with immune

(not shown).

for the following

1981; Kulczycki,

(‘pIBP68’),

coprecipitated

as the precipitating this method

and Cohen,

68000

Specificity Isolation

(‘pIBP56’),

immunoand

40000

complexes

of human

IgG and sheep

when rabbit

anti-human

IgG was used

for Fc could not be demonstrated

of placental

FcR

using

by coprecipitation

with

immune complexes of human IgG and sheep or goat anti-human IgG (Mikulska et al, 1982) was based on the premise that placental FcR interact with the human IgG in the complexes and do not bind

IgG

from these

1980). Using this method, a putative

FcR does not bind immune

Fc portion

[i.e. Fab or F(ab’),].

to goat or sheep IgG appeared tal FcR

activity.

heterospecific example,

This

see Matre

formed

F(ab’),

ive binding

To counteract

Sepharose.

previous

of placental

of a putative fragments.

F(ab’),

variant

to remove results

(for

et al, 1980).

FcR to immune However,

in these experiments,

of small immune

FcR

in vitro placen-

et al, 1981; Van der Muelen

and anti-human

possibilities,

we isolated

hapten-specific

com-

complexes

an apparent

precipitates

in this manner

by its lack of binding

suggest

that they are the same molecule (Hames,

or

rather

of

negat-

than lack

specificity

F(ab’),.

The

of StMPM

immobilized proteins,

on

pIBP56

of pIBP56

difference

60 ooo M, FcR of Mikulska and might

chromatography

F(ab’),

IgG-binding

and the binding

and the previously

It is noteworthy

IgG

observed

to immobilized

reported

FcR by affinity

rabbit

Of the previously

pIBP56

hapten-

bound

IgG

for Fc was con-

between

the size of

et al (1982) is small enough

be accounted

for by different

to

SDS-PAGE

1986) or proteolysis. that pIBP.56,

branes

rather

bound

than StMPM

to immobilized

isolated

detergent

F(ab’),

function

(Anderson,

findings

since the glycoprotein

removed

the FcR-associated

Mikulska

for

that

lacking the

of the IgG fractions

accounting

for Fc is lack of binding

F(ab’),

over

that was immobilized

by detergent

of non-binding

after absorption

form poorly and, as observed

these

conjugated

could explain

IgG fragments

since both species’ IgG inhibited

perhaps

et al,

is to demonstrate

1986).

lysates

conditions

human

the initial premise

result may be due to the production (Down,

1978; Van der Meulen

of binding

containing

1978; Neizgodka

specificity

of human

detergent

firmed

However,

to be incorrect

and Haugen,

and Haugen,

Fc specificity

complexes

reactivity,

and anti-F(ab’),

of binding

(Matre

control

was only discovered

anti-IgG

Proof of FcR binding plexes

species

the necessary

from glycoprotein

lysates,

fragments.

Reports

1980; Aida and Onoue, isolation

protocol

specificity

of placental

of other

FcRs

found

1983). Such a requirement

lipids whose function

why the Fc binding

extracts

did not bind with specificity

(Marchesi

lipids

and Andrews, by pIBP56

necessary

might account

may have been replaced

was retained

plasma mem-

for Fc since it also for

for our

1971) would have by detergents.

liberated

This

from StMPM

solubilization. et al (1982) concluded

that the 60 ooo M, glycoprotein

FcR was a disulfide-linked

homodimer of 30000 M, subunits. We observed that pIBP56 isolated from glycoprotein extracts migrated on reducing SDS-gells with unchanged M,. In contrast, pIBP56 isolated from detergent lysates was not observed on SDS gels run under reducing conditions suggesting that it migrated too dispersely or too small to be resolved on our gels. Proteolysis of the FcR at a site within

a loop closed by an intrachain

disulfide

bond might

account

for this dis-

crepancy (see the model by Bourgois, Kahn-Perles and Sire, 1983). In this study, the presence of protease inhibitors during the production of placental glycoprotein extracts would have limited proteolysis and possibly allowed the observation of intact p56 after disulfide reduction. Although pIBP68 and pIBP40 coprecipitated with immune complexes of human IgG and sheep or goat anti-human IgG, they did not bind to immobilized rabbit IgG, thus raising the

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suywd 113~lsnlqoydulloilt.luAg /“IU”.‘?llduinunf{:I” 1.’ udM[j

Placenta (rgbp), Vol. I*

244

human StMPM with the added possibility that there may be specific ligand preferences since pIBP56 bound IgG immune-complexed to hapten Sepharose or to IgG in immune complexes in solution whereas plBP40 and pIBP68 only bound to the latter. The observation that pIBP68 and pIBP40 were not isolated from placental glycoprotein extracts either in these or previous experiments (Neizgodka et al, 1981; Mikulska et al, 1982) may be accounted for by several obvious possibilities. The lithium diiodosalicylate/phenol fractionation method of Marchesi and Andrews (1971) which was used to extract glycoproteins from placental plasma membranes may have denatured the pIBP40 and pIBP68, destroying IgG binding properties. Alternatively, pIBP40 and pIBP68 may not separate into the included phase during the glycoprotein extraction protocol. This would suggest that they were not or were poorly glycosylated. Results of immune precipitation experiments utilizing NP40 or TXIOO extracts of StMPM suggested that pIBP40 showed specificity for Fc in the presence of these detergents. However, rigorous testing of the Fc binding specificity of pIBP40 and pIBP68 could not be absolutely demonstrated by the methods used in this study and it remains to be determined whether they are true Fc receptors or possibly accessory IgG-binding proteins. The sizes of pIBP68 and pIBP40 resemble FcRI and FcRII expressed on human leukocytes (for review see Anderson and Looney, 1986) but it seems unlikely that they represented FcRI and FcRII arising from membrane contamination by lymphomyeloid cells because, under the conditions of ligand affinity chromatography on anti-DNP IgG bound to DNPSepharose in which pIBP40 and pIBP68 were not seen to bind at all, FcRI and FcRII from myelomonocytic cell lines HL60 and U937 were observed to bind well (Down, 1986). Thus, with regard to binding immobilized hapten-specific IgG, pIBP68 and pIBP40 appear to be operationally different from monocytic FcRs.

SUMMARY To identify Fc receptors from human placental microvilli, proteins that were liberated by detergents from human placental synctiotrophoblast microvillous membranes (StMPM) were characterized by their abilities to bind human IgG in immune complexes with sheep or goat anti-human IgG and to monomeric rabbit anti-dinitrophenol (DNP) IgG bound to DNPlysine Sepharose. Three placental IgG-binding proteins coprecipitated with immune complexes (M, = 68 ooo, 52 000-56000, 40000) and were designated pIBP68, pIBP56 and pIBP40, respectively. Of the three proteins only pIBP56 bound to immobilized monomeric rabbit IgG. It was isolated from detergent lysates of StMPM and LDS/phenol glycoprotein extracts of placental plasma membranes suggesting that pIBP56 was a glycoprotein FcR previously reported (Mikulska et al, 1982). The binding specificities of pIBP56 and pIBP40 appeared to be detergent dependent. Photoaffinity crosslinking of StMPM surface proteins in situ to monomeric rabbit derivatized with N-succinimidyl(4-azidophenyl)-r,g-dithiopropionate identified IgG-binding proteins identical in size to pIBP56 and pIBP40. Crosslinking further suggested that monomeric IgG covalently bound to a complex of StMPM proteins with a total size of I 1ooo~12oooo M,. The findings suggest that pIBP68, pIBP56 and pIBP68 are responsible for IgG binding activity of placental StMPM.

ACKNOWLEDGEMENTS Some of the data were presented at the 6th International

Congress of Immunology

held in Toronto in August, 1986.

I)own et al: Human Placental Syncttiotrophoblast FcR proterns

245

At that time helpful discussions were had with Dr Roald Mane, Dr Josef Lisowski and Dr Clark Anderson. Our thanks to Dr Peter Johnson who alerted us to the possibilities of the StMPM method. Within the Dept. of Biochemistry we thank MS Cathy Horne, Dr David Iseman, Dr Robert Painter, Dr Douglas Romans, Dr Trudy McNabb and Dr David Kells for their advice and criticisms, Thanks to Mr Batista Calvieri for performing the electron microscopy and Dr Paul Hamel for providing DNP-lysine Sepharose. Special thanks to the nursing staffs at the delivery suites of Toronto General Hospital, Womens College Hospital and Mount Sinai Hospital in Toronto for their help in obtaining placentas. This work was supported by grant 4259 from the Medical Research Council of Canada.

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C. L. (1980) The murine macrophage

of

Fc receptor for IgG2b is lipid dependent. _?‘ournnZ ZmmunoZo~y,

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