Human Placental Microvilli Research Articles

Cofactor Activity in Placental Microvilli for the Cleavage of Complement Component 3 by Factor I

Human placental microvilli are in direct contact with maternal blood and the activity of complement components of the immune system must be regulated on this plasma membrane to prevent adverse effects to the fetus. A key point for regulation of the complement cascade is the proteolytic inactivation of complement component 3 which has become fixed on cell membranes by Factor I which requires a membrane bound cofactor. CD46 is the most widely distributed integral membrane protein cofactor for Factor I and is abundant in placental microvilli. An assay was developed for cofactor activity for Factor I that involved coupling of biotinylated methylamine‐ activated C3 (C3ma) to thiolpropyl agarose as a substrate. Microvilli from the surface of placental trophoblast cells are readily isolated by subcellular fractionation. The immobilized C3ma substrate was incubated with Triton X‐100 solubilized microvilli or other potential cofactor sources in the presence of Factor I. Cleavage of the C3ma was detected by SDS gel electrophoresis followed by electrophoretic transfer to nitrocellulose, blotting with alkaline phosphatase‐conjugated avidin and detection with an alkaline phosphatase substrate. It was found that cofactor activity was detectable in Triton X‐100 solubilized placental microvilli and platelets. However, a soluble form of CD46 that was not sedimented by centrifugation at 100,000g was associated with microvilli and also had co‐factor activity. In addition it was found that either CD46 or Factor I could first be bound to the immobilized C3ma and that cleavage could then be initiated upon addition of the missing component. The cofactor activity in placental microvilli could be removed by immunoabsorption with polyclonal or monoclonal antibodies to CD46 suggesting that this was the main source of cofactor activity for intrinsic C3 inactivation on placental microvilli.In conclusion, CD46 was found to be the major if not only cofactor on placental microvilli for Factor I inactivation and existed in membrane bound and soluble forms associated with microvilli. Either of Factor I or its cofactor CD46 can bind to C3ma and the missing component can be added to form an active ternary complex.

Placental sulphate transport: A review of functional and molecular studies

Sulphate is required by the feto-placental unit for a number of important conjugation and biosynthetic pathways. Functional studies performed several decades ago established that sulphate transport in human placental microvillus and basal membrane vesicles was mainly via a DIDS-sensitive anion-exchange mechanism. In contrast, no evidence was found for Na+-dependent transport. Studies performed using isolated human placental tissue confirmed anion-exchange as the main mechanism. More recently, molecular studies have established the presence of anion-exchange proteins which could play a role in transplacental sulphate movement. However, the presence of transcripts for NaS2 has been reported and has prompted the suggestion that Na+-sulphate cotransport may play an important role in maternal-fetal sulphate transport. This article reviews our present knowledge of placental sulphate transport, both functional and molecular, and attempts to form a model based on the available evidence.

Review: Differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes

Human endothelial dysfunction is a common feature in many diseases of pregnancy, such as gestational diabetes (GD). Metabolic changes include abnormal synthesis of nitric oxide (NO) and abnormal membrane transport of l-arginine and adenosine in primary cultures of human umbilical vein (HUVEC, macrovascular) and placental microvillus (hPMEC, microvascular) endothelial cells. These alterations are associated with modifications in the expression and activity of endothelial (eNOS) and inducible (iNOS) NO synthases, respectively, an effect that is maintained at least up to passage 5 in culture. HUVEC and hPMEC exhibit expression and activity of the human cationic amino acid transporter 1 (hCAT-1), equilibrative nucleoside transporters 1 (hENT1) and hENT2, as well as the corresponding SLC7A1, SLC29A1 and SLC29A2 gene promoter activities. Altered gene expression results from increased NO level, protein kinase C, mitogen-activated protein kinases, and hCHOP-C/EBPα transcription factor activation. Reduced ENT-mediated adenosine transport in GD is associated with stimulation of the l-arginine/NO pathway, and mainly due to reduced expression and activity of hENT1. In addition, hENT2 activity seems able to restore the reduced adenosine transport in GD. Additionally, insulin exerts a differential modulation of endothelial cells from macrocirculation compared with microcirculation, possibly due to expression of different insulin receptor isoforms. It is suggested that a common functional characteristic leading to changes in the bioavailability of adenosine and metabolism of l-arginine is evidenced by human fetal micro and macrovascular endothelium in GD.

CLIC-5A Functions as a Chloride Channel in Vitro and Associates with the Cortical Actin Cytoskeleton in Vitro and in Vivo

CLIC-5A is a member of the chloride intracellular channel protein family, which is comprised of six related human genes encoding putative chloride channels. In this study, we found that reconstitution of purified recombinant CLIC-5A into artificial liposomes resulted in a dose-dependent chloride efflux that was sensitive to the chloride channel blocker IAA-94. CLIC-5A was originally isolated as a component of an ezrin-containing cytoskeletal complex from human placental microvilli. Here we show that similar protein complexes can be isolated using either immobilized CLIC-5A or the C-terminal F-actin-binding domain of ezrin and that actin polymerization is required for de novo assembly of these complexes. To investigate the behavior of CLIC-5A in vivo, JEG-3 placental choriocarcinoma cells were stably transfected with epitope-tagged CLIC-5A. In fixed cells, CLIC-5A displayed a polarized distribution and colocalized with ezrin in apical microvilli. Microvillar localization of CLIC-5A was retained after Triton X-100 extraction and was disrupted by treatment with latrunculin B. In transient transfections assays, we mapped a region between residues 20 and 54 of CLIC-5A that is required for targeting of CLIC-5A to microvilli in JEG-3 cells. Interestingly, expression of CLIC-5A in JEG-3 cells did not enhance the rate of iodide efflux in intact cells, suggesting that if CLIC-5A is a chloride channel, its channel activity may be restricted to intracellular membrane compartments in these cells. Regardless of its role in ion transport, CLIC-5A, like ezrin, may play an important role in the assembly or maintenance of F-actin-based structures at the cell cortex.

Single-channel characterization of a nonselective cation channel from human placental microvillus membranes. Large conductance, multiplicity of conductance states, and inhibition by lanthanides.

The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance, multisubstate Cl- channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions), to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance at the single-channel level. The channel has a large (>200 pS) and variable conductance, is cation selective (P(Cl)/P(K) approximately or approximately equal 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (>10) of current sublevels with a "promiscuous" connectivity pattern. The occurrence of both "staircaselike" and "all-or-nothing" transitions between the minimum and maximum current levels was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during syncytiotrophoblast-volume regulation.

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

Glutamine transport in human and rat placenta

Glutamine plays an important role in fetal nutrition. This study explored the transport of [3H]glutamine into apical and basal predominant membrane vesicles derived from rat and human placenta. Na+-dependent glutamine transport was present in both apical and basal predominant vesicles derived from 20- and, to a lesser degree, 14-day gestation rat placenta. Amino-acid transport systems A, ASC-like, B(o,+) (in apical membrane vesicles) and, perhaps, y+L were involved in Na+-dependent glutamine transport. Na+-dependent glutamine uptake into human placental microvillus and basolateral membrane vesicles also occurred via several distinct transport activities. Glutamine transport via system N was not detected in either rat or human placental preparations. Na+-dependent glutamine transport in the rat was more pronounced in basal as compared to apical membrane vesicles. Conversely, in the human preparations, activity was significantly higher in microvillus as compared to basolateral membrane vesicles. It is concluded that Na+-dependent glutamine transport occurs through a variety of transport agencies in both the rat and human placenta. Transport varies with ontogeny and between species.

In vitro exposure to mercury and cadmium alters term human placental membrane fluidity

The effect of incubation with mercury (Hg) as HgCl 2 and cadmium (Cd) as CdCl 2, at levels of 6 or 12 μg/ml of medium, on explants of term human placental microvillus membrane fluidity were studied. After incubation for 6 or 24 hr explants for each dose level were pooled and washed with fresh medium to remove any unbound metal. Placental membranes were separated by differential centrifugation and fluidity was studied by steady-state fluorescence polarization, expressed as the fluorescence anisotropy, r, with 1,6-diphenyl-1,3,5-hexatriene as a probe. The results show that membranes derived from explants incubated for 24 hr with either 6 or 12 gmg/ml medium of either metal showed fluorescence anisotropy values (i.e., decreased fluidity) significantly higher than that of their respective controls. With 6 μg/ml of either metal the decrease in fluidity was highly significant for both metals and with 12 μg/ml a further decrease in membrane fluidity was observed with either metal. Both metals accumulated in placental membranes in proportion to their level in the medium. Membrane accumulation of Cd was higher than that of Hg. The cholesterol, phospholipid, and cholesterol-to-phospholipid mole ratios in membranes derived from metal-treated explants were unchanged, compared to their respective controls. However, no changes in membrane fluidity were observed in the samples incubated for 6 hr. In conclusion, exposure of placental cells to Hg and Cd caused accumulation of the metals in the membranes and lowered the membrane fluidity, which may affect membrane function and cause damage to the developing fetus.

Uptake of zinc by human placental microvillus border membranes and characterization of the effects of cadmium on this process.

The uptake of Zinc (Zn) by microvillus border membrane vesicles formed from the trophoblast of term human placentae is markedly increased over brief periods of incubation with much slower increases persisting for up to 2 h of incubation. Zinc is both bound to membrane components and transported into intravesicular osmotically active space. Uptake is saturable, temperature dependent from 4 to 37 degrees C with a Q10 of 1.7, and is inhibited by the sulphydryl agent DTNB. About 20 per cent of the uptake is susceptible to inhibition by Cadmium (Cd) at concentrations from 5 to 50 microM, a significant part of the action of this metal being on the transmembrane component of Zn uptake.

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

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 DNP-lysine Sepharose. Three placental IgG-binding proteins coprecipitated with immune complexes (Mr = 68,000, 52,000-56,000, 40,000) 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)-I, 3-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 110,000-120,000 Mr. The findings suggest that pIBP68, pIBP56 and pIBP68 are responsible for IgG binding activity of placental StMPM.

Human placental microvilli as a source of antigen for the preparation of a polyclonal antibody directed against the LDL receptor

Polyclonal antibodies were prepared by immunization of rabbits with partially purified LDL receptor obtained from human placental microvilli. The antiserum reacted with membranes from human placental microvilli and human fibroblasts, as assessed by immunobinding studies. It also reacted with purified LDL receptors of both origins. The antiserum markedly inhibited 125I-labeled LDL binding to cultured human fibroblasts.

Human carboxypeptidase M: Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic A or B, human plasma N, and H (enkephalin convertase). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name carboxypeptidase M. Because of its localization on the plasma membrane and optimal activity at neutral pH, M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.

Monoclonal antibodies (GB16, GB18, GB19, GB22) raised against human placental microvilli recognize the transferrin receptor

The placental syncytial trophoblasts perform a variety of transport and secretory functions during pregnancy. It has long been established that these trophoblasts possess specific membrane receptors for transferrin (Faulk and Galbraith, 1979; Seligman, Schleicher and Allen, 1979; Wada, Hass and Sussman, 1979), an iron-binding plasma protein. This liganddreceptor interaction is similar to that of reticulocytes in which it serves the function for haemoglobin synthesis (Jandl and Katz, 1963). Transferrin is an indispensable nutrient for the maintenance of cell growth in serum-free media (Iscove and Melchers, 1978). However, the mechanism(s) by which transferrin exerts its effect on cell growth has yet to be determined; it has been suggested that iron transport may represent one of its complex functions (Faulk and Galbraith, 1979; Ekblom et al, 1983; Hill et al, 1985). A few polyclonal antibodies raised against placental syncytiotrophoblastic microvillous membrane were found to react with the transferrin receptor (Enns et al, 1981; Booth and Booth, 1982). However, to date, none of the monoclonal antibodies produced in the same fashion were directed against the receptor for transferrin (Bulmer and Johnson, 1985). During our programme of screening monoclonal antibodies to human extraembryonic membranes, we have found that four antibodies (GB16, GBI~, GBI~, GBaa), raised against syncytiotrophoblastic microvilli, immunoprecipitated a r8o-kilodalton (K) dimer. This molecule had the ability to bind human transferrin, and it could not be detected on terminally differentiated HI,-60 cells. The epitope of GBI~ was different from that recognized by GBI~, GBI~ and GBaa. In addition, GBaa inhibited the cell growth of activated lymphocytes and Daudi cells, but not that of HL-60 or Jurkat cells.

Purification and Na + uptake by human placental microvillus membrane vesicles prepared by three different methods

Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca 2+ (Method 1) or Mg 2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg 2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 ± 1.9, 25.3 ± 2.7) and ouabain binding (5.9 ± 2.6, 5.3 ± 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly ( P < 0.01) lower alkaline phosphatase enrichment (18.1 ± 1.2), but ouabain binding enrichment (6.3 ± 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na + uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na + uptake was 3–6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2′,7′-bis(car☐yethyl)-5(6)-car☐yfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.

Monoclonal Antibody GB24 Recognizes a Trophoblast‐Lymphocyte Cross‐Reactive Antigen

GB24 is a mouse monoclonal antibody (IgG1) raised against human term placental microvilli. This antibody displayed pan-trophoblast reactivity pattern. In addition, GB24 recognized normal peripheral leukocytes and transformed cell lines (Daudi, HL-60, Jurkat, AV3, BeWo, HT-29) by using membrane immunofluorescence and flow cytometry. By SDS-polyacrylamide gel electrophoresis, this antibody immunoprecipitated two proteins of 62 kilodaltons (kDa) and 75 kDa from placental microvilli. Isoelectrofocusing analysis revealed that these two bands exhibited different isoelectric points: 4.7 for the 62 kDa protein, 4.6 and 4.4 for the two spots corresponding to the 75 kDa protein. Microvilli from different placentae revealed substantial differences regarding the intensity of the labeling of these two bands, suggesting that the antigens recognized by GB24 are probably allotypic.

Selenium (selenate) transport by human placental brush border membrane vesicles.

1. Selenate uptake by human placental brush-border-membrane vesicles was studied in order to establish whether this anion shares a pathway with sulphate. 2. Selenate uptake was found to be saturable with respect to medium selenate and was inhibited by the anion exchange inhibitor 4,4'-diisothiocyano-stilbene-2,2'-disulphonate (DIDS). 3. Anions which have a similar tetrahedral shape to selenate, e.g. chromate, molybdate, tungstate and sulphate, were effective inhibitors of selenate uptake when added to the incubation medium. 4. Sulphate inhibited selenate influx in a dose-dependent fashion; moreover sulphate was found to be a competitive inhibitor of selenate uptake. 5. It is concluded that selenate and sulphate share a pathway for transport in the human placental microvillus membrane.

Monoclonal antibodies (GB16, GB18, GB19, GB22) raised against human placental microvilli recognize the transferrin receptor.

GB16, GB18, GB19 and GB22 are mouse monoclonal antibodies produced against full-term human placental microvilli. These antibodies reacted predominantly with the apical surface of the syncytiotrophoblast from first-trimester and full-term placentae, and also reacted with several cell lines derived from non-haematopoietic tissues by immunofluorescence. The radioiodinated BeWo (choriocarcinoma) cell surface proteins were immunoprecipitated with these four antibodies and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results demonstrated that the immunoprecipitates migrated at 180 and 90 kilodaltons under non-reducing and reducing conditions, respectively. Using enzyme-linked immunosorbent assay, the antigens recognized by GB16, GB18, GB19, and GB22 were able to bind human transferrin. Immunoabsorption studies showed that these four antibodies bound to the same molecule as OKT9, an antibody to the transferrin receptor. Moreover, the reactivities of these antibodies with HL-60 (promyelocytic leukaemia) cells diminished following dimethyl sulphoxide-induced differentiation by flow cytometric analysis. These data indicate that these four antibodies recognize the transferrin receptor. By competition assay, GB18 bound to an epitope different from those recognized by GB16, GB19 and GB22. In addition, GB22 displayed significant growth inhibition against the activated lymphocytes and Daudi cells, but not against HL-60 or Jurkat cells. These data suggest that these four monoclonal anti-transferrin receptor antibodies will provide additional means to investigate the physiological roles played by the transferrin receptor.

A study of selenate efflux from human placental microvillus membrane vesicles.

Selenate efflux from human placental brush border membrane vesicles was studied using an ion-exchange column assay. Selenate efflux was found to be mediated almost exclusively by a temperature dependent DIDS-sensitive pathway. Chromate markedly inhibited selenate efflux: in contrast medium selenate had no effect. It is concluded that selenate and sulphate share a common pathway for transport across the human placental microvillus membrane.

External anions relieve DIDS inhibition of SO4 efflux from placental membrane vesicles.

Sulphate efflux from human placental microvillus membrane vesicles was inhibited by external DIDS (KI congruent to 10(-6) M). This inhibition was partially reversed on addition of the translocated substrates sulphate or selenate to the external medium: selenite which is not translocated does not protect against DIDS inhibition. These findings show that the mechanism responsible for sulphate efflux can be modified by substrate in the external medium.

Detection of distinct receptors for native and acetylated low-density lipoproteins in human placental microvilli by ligand-immunoblotting

Ligand-immunoblotting was used to detect distinct receptors for native low-density lipoprotein and for acetylated low-density lipoprotein on microvillous membranes from human term placentas. Antisera directed against native and modified low-density lipoproteins were prepared in rabbits and their specificities were assessed by immunodiffusion and immunoelectrophoresis. The receptor for low-density lipoprotein was detected as a 160 kDa protein and that for acetylated low-density lipoprotein as a 200 kDa protein. These receptors were compared with their counterparts in cultured human skin fibroblasts, bovine adrenal cortex and J774 macrophage-like cells. This is the first investigation that visualizes the presence of receptors for both native and modified low-density lipoproteins in a steroidogenic tissue.