Biomedical subjects

O A Vanderpuye

Publications and source records attributed to O A Vanderpuye.

17 recordsLinked to original sources

PubMed1993

Predominant expression of the beta subunit of prolyl 4-hydroxylase (disulfide isomerase) in human extravillous trophoblasts.

Prolyl 4-hydroxylase is a heterodimeric enzyme that is crucial in the biosynthesis of collagen. The beta subunit of this enzyme is a multifunctional protein which is also known as protein-disulfide isomerase. Immunofluorescence and monoclonal antibody (Mab) 5B5 were used to localize the beta subunit in human extraembryonic tissues. The strongest sites of 5B5 reactivity were extravillous cytotrophoblasts in the basal plate, uteroplacental arteries and amniochorion, syncytiotrophoblast displayed variable weaker reactivity. Only a small fraction of placental 5B5 antigen was detected as a component of prolyl-4-hydroxylase by affinity chromatography on immobilized polyproline. The results indicate a difference in the expression of an endoplasmic reticulum marker between villous and extravillous trophoblast. The predominance of 5B5 antigen in extravillous trophoblast could be associated with an increased ability to synthesize collagen or other enzymatic reactions associated with prolyl 4-hydroxylase beta subunit.

Antibody Specificity↗

PubMed1993

Expression of CD59, a human complement system regulatory protein, in extraembryonic membranes.

CD59 (leukocyte cluster of differentiation antigen 59), is a phosphatidylinositol glycan-anchored membrane protein that inhibits lysis of cells by terminal complement system components. To further define complement regulatory proteins relevant to pregnancy, this study characterized the expression of CD59 in human extraembryonic membranes. Immunohistology with CD59 monoclonal antibody MEM-43 showed that this molecule was normally present on the apical surface of the syncytiotrophoblast, on extravillous cytotrophoblast, and amniotic epithelium. Immunoblotting confirmed that first and second trimester syncytiotrophoblast microvilli (STM) contained a glycoprotein similar in mass and glycosylation to CD59 from adult cells and tissues. Reactivities of STM with MEM-43 in ELISA were 2- to 6-fold higher than those of kidney, erythrocyte and platelet membranes. Term placental STM from recurrent spontaneous aborting patients after immunotherapy, reacted with MEM-43 in ELISA similarly to STM from normal individuals. Plasmas from pregnant women and umbilical cords had 50% or greater reactivity with MEM-43 than did normal plasmas. CD59 could help protect extraembryonic epithelia from damage by complement in maternal blood and amniotic fluid. The apical location of CD59 reflects the immunological roles and functional polarization of plasma membranes in the syncytiotrophoblast.

Abortion, Spontaneous↗

PubMed1992

Glycosylation of membrane cofactor protein (CD46) in human trophoblast, kidney and platelets.

Many cell surface glycoconjugates are differentiation markers and are involved in cell-cell and intermolecular interactions in development, immunity and cancer. Membrane cofactor protein (MCP) comprises structurally related 65 and 55 kDa glycoproteins that bear O- and N-linked glycans. MCP prevents amplification of autologous complement action on human cells. We used immunoblotting with MCP-specific monoclonal antibody TRA-2-10 to determine lectin-binding properties and glycosidase sensitivities of MCP in a study of cell-specific variation in glycosylation of this protein. The results showed that N-linked glycans on placental syncytiotrophoblast and cytotrophoblast, kidney and platelet MCP are similar in binding to concanavalin A and Lens culinaris lectins, but are not bound by leucophytohemagglutinin. Lectin binding prior to and after neuraminidase digestion indicates that MCP from these sources is highly sialylated. 65 kDa MCP was confirmed to contain more O-linked glycans than 55 kDa MCP. A fraction of platelet 65 kDa MCP is distinct, however, in bearing peripheral fucose residues. Syncytiotrophoblast is unique in containing a 110 kDa form of MCP in non-reducing SDS-PAGE that resembles 65 kDa MCP in glycosylation. Chorion laeve MCP in 4 of 8 preparations was unusually heterogeneous and differed from syncytiotrophoblast MCP after neuraminidase digestion in the forms bound to peanut agglutinin and WGA. The results indicated for the first time, differences in O-linked glycosylation of MCP in chorion laeve cytotrophoblast relative to syncytiotrophoblast, platelet and kidney MCP. We conclude that structures of MCP glycans can differ between trophoblasts and other cell types.

Antigens, CD↗

PubMed1991

A vitronectin-receptor-related molecule in human placental brush border membranes.

The heterodimeric vitronectin receptor (VNR) and platelet glycoprotein IIb/IIIa (GPIIb/IIIa) are two members of the integrin family of cell adhesion receptors that share the same beta subunit (GPIIIa). These proteins are involved in binding to vitronectin, fibrinogen and fibronectin and in cytoskeleton-membrane interactions. The present study shows that the human placental syncytiotrophoblast brush border membrane contains a heterodimer of subunit Mr values of 140,000 and 90,000 (non-reduced) or 125,000 and 100,000 (reduced). This protein was recognized by a monoclonal antibody to GPIIIa, rabbit antisera to the VNR and a human alloantiserum to GPIIIa. Brush border VNR-related protein bound to an immobilized peptide containing the Arg-Gly-Asp sequence and, less avidly, to immobilized fibrinogen. Only a small fraction of brush border VNR was associated with a cytoskeleton fraction. Membrane-bound brush border GPIIIa was distinct from that of platelets in its resistance to digestion by trypsin and Staphylococcus aureus V8 protease, and had a slightly lower mobility on SDS/PAGE. In addition, lectin-binding studies indicate glycosylation differences between microvillar and platelet GPIIIa heterodimers. Thus, although placental syncytiotrophoblast expresses a beta 3 integrin in its apical brush border, differences in protease sensitivity and carbohydrate content suggest that it may lack or mask certain antigenic determinants. This may be beneficial in avoiding harmful maternal alloantibody responses during pregnancy. Immunohistology showed that the VNR was present in syncytiotrophoblast apical but not basal plasma membranes, and was absent from other forms of trophoblast. The brush border VNR could function in localizing Arg-Gly-Asp-sequence-containing plasma proteins to the materno-trophoblastic interface.

Blood Platelets↗

PubMed1990

Lactoferrin binding molecules in human seminal plasma.

During ejaculation, the iron binding protein lactoferrin binds to sperm and forms a major component of sperm-coating antigens. Physicochemical properties of lactoferrin in seminal plasma (SP) and on sperm differ from those of purified lactoferrin. These differences have been attributed to the binding of unknown seminal macromolecules to lactoferrin. We have studied lactoferrin binding molecules in SP. The SP samples were coated onto microtiter plates and tested for binding of biotinylated lactoferrin. SP was found to specifically bind biotinylated lactoferrin. This binding was competitively inhibited by coincubation with unlabeled lactoferrin but was not affected by control incubations done with human IgG or transferrin. Lactoferrin binding molecules in SP were biochemically characterized by using SDS-PAGE and ligand blotting. Biotinylated lactoferrin bound to SP molecules of approximately 120, 60 and 30 kDa. No binding was observed with biotinylated transferrin. The presence of molecules that associate with lactoferrin in SP was further studied by using crossed immunoelectrophoresis. Lactoferrin in SP immunoprecipitated as two peaks, one of which corresponded to purified lactoferrin. These results suggest that some lactoferrin molecules in SP are free and that others are associated with lactoferrin binding molecules. Binding of lactoferrin to lactoferrin binding molecules appears to change its physicochemical properties and thus could influence its biologic activity and its affinity to sperm.

Carrier Proteins↗

PubMed1989

Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli.

Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanavalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.

Actin Cytoskeleton↗

PubMed1988

The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin.

Using immunochemical techniques, we identified forms of erythrocyte membrane proteins in apical and basal plasma membranes of human placental trophoblast. A wheat germ agglutinin-binding intrinsic protein was present in the microvillous (maternal facing) but not the basal (fetal facing) membrane of the syncytiotrophoblast epithelium. Conversely, erythrocyte-related proteins of the basal membrane included two intrinsic membrane proteins, a 95,000 Mr band 3 isoform and a form of spectrin. These four proteins were all absent from the microvillous membrane. The basal membrane spectrin isoform was also present in basal membrane skeletons. A 70,000 Mr polypeptide which reacted with antibodies to band 3 was present in both microvillous and basal plasma membranes. Therefore, certain isoforms of red cell membrane proteins are polarized between the two surfaces of the human placental syncytiotrophoblast. We propose that the localization of spectrin to the basal membrane is related to the less bundled organization of microfilaments at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates for participation in maternofetal anion transport.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1988

Microfilament association of ASGP-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13,762 rat mammary ascites tumor cells.

Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13,762 rat mammary ascites tumor cells. "Phalloidin shift" analyses on velocity sedimentation gradients of Triton X-100 extracts of [3H]-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13,762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. An increase was also observed with sialidase treatment of the microvilli, suggesting that negative charges, probably present on the highly sialated sialomucin ASGP-1 of the ASGP-1/ASGP-2 sialomucin complex, reduce ASGP-2 association with the microfilament core. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Purified ASGP-2 does not bind to microfilaments in sedimentation assays. Since the Triton-insoluble membrane residue is enriched in an actin-containing transmembrane complex, which contains a different glycoprotein, we suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.

Actin Cytoskeleton↗

PubMed1986

Proteins of the human placental microvillar cytoskeleton. alpha-Actinin.

The human placental syncytiotrophoblast microvilli are supported by an underlying cytoskeleton consisting mainly of actin microfilaments. The major proteins associated with the actin have Mr values of 105 000, 80 000 and 68 000. The 105 000-Mr protein is recognized by an antibody preparation raised to purified chicken gizzard alpha-actinin. Electron microscopy has shown that the human placental protein has dimensions similar to those reported for muscle alpha-actinin. About half of the placental microvillar alpha-actinin is released from the cytoskeleton in the presence of Ca2+. This effect occurs at concentrations of Ca2+ greater than 0.3 muM and has been used as the basis of a method for the purification of the placental alpha-actinin. This sensitivity to Ca2+ is not affected by trifluoperazine and is therefore likely to be a property of the alpha-actinin as such rather than being mediated via calmodulin.

Actinin↗

PubMed1983

Structure of human placental microvilli.

Cytoskeletons have been prepared from microvilli isolated from the human placental syncytiotrophoblast. They contain actin and a protein similar to fimbrin. In addition, they contain calmodulin and a protein of relative molecular mass (Mr) 105 000, both of which can be released from the cytoskeletons by treatment with Ca2+. In this respect the 105 000 Mr protein is more similar to non-muscle alpha-actinin than to the intestinal microvillar protein with an Mr of 110 000. Human placental actin displays the anomalous properties of binding to phenyl-Sepharose and wheatgerm agglutinin-Sepharose, suggesting that one or more membrane glycoproteins is associated with the actin. Transferrin, presumably receptor-bound, has been identified in preparations of extensively washed placental microvillar cytoskeletons. These findings are discussed in terms of the earliest events in endocytosis and materno-fetal transfer.

Actins↗

PubMedDate not supplied

Proteins of the apical and basal plasma membranes of the human placental syncytiotrophoblast: immunochemical and electrophoretic studies.

Proteins of the basal and microvillous plasma membranes of the human placental syncytiotrophoblast were compared to elucidate the basis for structural and functional differences in the two membranes. Among the proteins common to both membranes were actin, alkaline phosphatase, albumin, transferrin, immunoglobulin G, proteins of Mr 35,000, 55,000 and 180,000, and five immunochemically detected proteins. Each membrane also contained unique proteins. Major microvillous cytoskeleton proteins of Mr 68,000 80,000 and 105,000 (alpha-actinin) were lacking or absent from basal membrane cytoskeletons which instead contained unique proteins of Mr 14,000, 16,000, 220,000 and 240,000. In addition, immunochemical analyses revealed four glycoprotein antigens unique to microvillous membrane and five unique to basal membrane. Fibronectin was also found to be exclusive to basal membrane. The difference in membrane-associated cytoskeletal proteins correlated with the different organization of actin microfilaments at the two membranes. The protein antigens unique to each of the two membranes provided further evidence for the polarization of membrane proteins and functions in the syncytium.

Cell Membrane↗

PubMedDate not supplied

Syncytiotrophoblast brush border proteins recognized by monoclonal antibody TRA-2-10 and rabbit anti-TLX sera.

Different subsets of placental trophoblast epithelium are directly exposed to the maternal immune system during pregnancy and consequently represent major elements in allogeneic interactions. It has been proposed that the trophoblast--lymphocyte cross-reactive (TLX) alloantigen system is involved in maternal allogeneic recognition during pregnancy. Monoclonal antibody TRA-2-10 putatively recognizes TLX antigens, but its reactivity with trophoblast and normal tissues has not been documented in detail. In this report, immunohistological investigations revealed that TRA-2-10 recognizes all subsets of trophoblast in addition to amniotic and seminal vesicle epithelia. Immunoblotting demonstrated reactivity with glycoproteins of 55,000 and 65,000 mol. mass under non-reducing conditions on various cell types. These proteins displayed tissue-specific size variations and individuals varied in the amounts expressed of the two species. On the basis of blocking and immunoprecipitation experiments, TRA-2-10 reactive antigens are recognized by rabbit anti-TLX sera and are potential TLX antigen candidates. However, TLX antigens are found in seminal plasma whilst TRA-2-10 reactive antigens are not. Both TLX and TRA-2-10 antigens appear related if not identical to membrane cofactor protein (MCP) by virtue of shared molecular characteristics and blocking of lymphocyte binding of monoclonals to MCP by polyclonal anti-TLX. Extra-embryonic membranes are thus richly endowed with a complement regulatory protein which could facilitate their roles in protection of the fetus by avoidance of harmful maternal immune response amplification.

Antibodies, Monoclonal↗

PubMedDate not supplied

Characterization of cofactor activity for factor I: cleavage of complement C4 in human syncytiotrophoblast microvilli.

To coexist with complement, human tissues express membrane-integrated regulatory proteins that inhibit the activity of autologous complement on cell surfaces. Certain of these complement regulatory proteins act as obligatory cofactors for proteolytic inactivation of activated C4(C4b) by factor I. Extraembryonic tissues and in particular trophoblasts constitute an interface at risk from maternal complement during pregnancy. The present study examined syncytiotrophoblast plasma membrane (STM) cofactor activity for cleavage of immobilized methylamine-treated complement component C4(C4ma), a C4b analog by factor I. Membrane cofactor protein (MCP or CD46) provided most of the cofactor activity in STM preparations. Minor cofactor activity was derived from C4 binding protein that was firmly bound to STM. Cofactor activity for cleavage of C4ma at its two sites for factor I was enhanced at higher concentrations of STM and at lower concentrations cleavage at a C terminal site predominated. Soluble cofactor activity was present in STM preparations and was provided by 65 KDa, 55 KDa and 50 KDa soluble species of MCP that lacked amphiphilic properties. These results are consistent with a major role for MCP in regulation of C4 activity on the maternal-facing surfaces of extraembryonic tissues during human development. Soluble MCP may provide additional fluid phase complement regulatory activity in the maternotrophoblastic zone.

Antibodies↗

PubMedDate not supplied

An improved method for the preparation of human placental syncytiotrophoblast microvilli.

A simple procedure is described for the further purification of placental microvillus preparations. Based on previously published methods for the isolation of microvilli from other tissues, it depends on the preferential aggregation of containing structures by Mg2+. In the purified microvillus preparation, the two placental microvillar marker enzymes, alkaline phosphatase and 5'-nucleotidase, were enriched 24-fold and were obtained in 5 per cent yield. Five other microvilla enzymes were also further enriched by the Mg2+-treatment. Marker enzymes for other subcellular components showed that this treatment completely removed contamination by mitochondria and endoplasmic reticulum and contamination by lysosomes was decreased three-fold. (Na+ + K+)-activated ATPase was depleted by the Mg2+-treatment as was beta2-microglobulin.

5'-Nucleotidase↗

PubMedDate not supplied

Transferrin receptors in the basal plasma membrane of the human placental syncytiotrophoblast.

Transferrin was identified in a preparation of human syncytiotrophoblast basal plasma membrane. Such transferrin was shown to be bound to an amphiphilic membrane protein of subunit mol.wt 94 000 by crossed hydrophobic interaction immunoelectrophoresis and immunoprecipitation with antibodies to transferrin. Basal plasma membrane bound 125I-labelled transferrin in a saturable, reversible manner with high affinity (Kd = 2.5 +/- 0.6 X 10(-9) M). The maximum binding capacity (0.9 +/- 0.2 pmol transferrin/mg membrane protein) was approximately a half of that of microvillous membrane. The basal membrane transferrin receptors were similar to microvillous receptors in that their affinity for diferric transferrin was higher than that for apo-transferrin and transferrin dissociation was negligible at pH 5.0 but rapid at pH 7.4. We conclude that syncytiotrophoblast basal plasma membrane possesses a receptor similar, if not identical, to that on the microvillous membrane. These receptors are thus in a position to participate in iron transfer to the fetus or potentially to have alternative functions in the syncytiotrophoblast.

Biological Transport, Active↗

PubMedDate not supplied

The complement system in human reproduction.

Regulation of the complement system in reproduction is unique inasmuch as reproductive tissues represent the only condition where allogeneic interactions occur naturally. Both allogeneic extraembryonic membranes and semen that contact and interact with maternal cells and tissues must avert complement-mediated damage to ensure reproductive success. Several regulators of complement activation exist. Membrane cofactor protein (MCP) and decay accelerating factor (DAF) inactivate C3 and C5 convertases on cell surfaces. In addition, CD59 inhibits the membrane attack complex (MAC) of the complement cascade. Strong expression of these membrane glycoproteins by trophoblast and amniotic epithelium has been observed. MCP, DAF, and CD59 likely safeguard extraembryonic tissues from complement damage originating from maternal and fetal blood or amniotic fluid. Different reproductive tract fluids vary in complement levels. With the exception of ovarian follicular fluid, these levels are generally much less than those in blood. Endometrial and cervical content of C3 appear to be regulated by hormones. These observations suggest that the effects of complement activation may vary in reproductive tissues. MCP is absent from the surfaces of oocytes. Sperm express MCP and DAF in discrete areas that would not be associated with the known complement-regulatory functions of these proteins. Seminal plasma contains MCP and the MAC inhibitor SP-40,40 but not DAF.SP-40,40 may exemplify how complement-regulatory proteins perform alternative functions as it interacts with molecules other than complement components. We have reviewed aspects of the complement system that relate to allogeneic interactions in reproduction and that suggest fruitful areas for further research.

Antigens, CD↗

PubMedDate not supplied

A role for TLX antigens in pregnancy.

Maternal responses to allotypic TLX antigens are proposed as necessary in the immunological maintenance of human pregnancy. The TLX antigens are strategically and strongly expressed in semen and on the extraembryonic tissues which form the anatomical materno-fetal interfaces. Recent findings suggest that CD 46 proteins (a membrane cofactor which protects self tissues from autologous complement damage) in association with the activated complement component, C 3b produce a novel antigenic epitope recognized by both rabbit and human anti-TLX antisera. Control of TLX immunity involves an idiotype-antiidiotype network. We now report the development of rabbit antiidiotypes specific for cross-reactive idiotypes on human anti-TLX IgG antibodies. These antiidiotypes appear to recognize different TLX allotypes and will be useful for gaining additional insight into the immunogenetics of human reproduction.

Animals↗