Genetic variants of placental alkaline phosphatase as detected by a monoclonal antibody

Summary Human placental alkaline phosphatase (PLAP) is a highly polymorphic enzyme. Several common as well as rare allelic forms of PLAP are characterized in this paper in terms of their reactivity with a murine monoclonal antibody (F₁₁). The common type 1 (S) and 3 (I) variants, and the rare type 4 (S₂) and 18 (D) variants were found to react with the F₁₁ antibody, so as did three new electrophoretically defined variants (19, 20, and 21). In contrast, the common type 2 (F₁) variant and the rare type 8 (F₃) and 9 (F₂) variants do not react with the F₁₁ antibody. This selective reactivity of F₁₁ has also allowed the identification of two molecular variants of PLAP with identical electrophoretic mobility. These results establish monoclonal antibodies as invaluable adjuncts in the study of PLAP polymorphism.     

Anti-idiotype induction therapy: anti-CA125 antibodies (Ab3) mediated tumor killing in patients treated with Ovarex mAb B43.13 (Ab1)

 Intravenous injection of the murine monoclonal anti-CA125 antibody B43.13 (Ovarex: Ab₁) into ovarian cancer patients led to the induction of an idiotypic network. Of the 75 patients who received one to ten injections of a 2-mg dose of the antibody, 48 developed anti-(mAb B43.13) antibodies (Ab₂); 18 of these patients also had elevated levels of anti-[anti-(mAb B43.13)] antibodies (Ab₃; = anti-CA125 antibodies) compared to pre-injection values. Characterization of these antibodies revealed that the binding to CA125 could be inhibited by mAb B43.13 in most samples. Human anti-CA125 antibodies or Ab₃ purified from patient serum samples specifically recognized human ovarian tumor cells and tissues expressing CA125. In addition, these anti-CA125 antibodies were able to conduct F_c-mediated tumor cell killing (antibody-dependent cell-mediated cytotoxicity). This raises the possibility of using an Ab₁ for anti-idiotype induction immunotherapy of cancer. Received: 14 October 1997 / Accepted: 9 January 1998     

Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.     

Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells

Certain monoclonal antibodies (mAbs) to human placental alkaline phosphatase (PLAP) block bromelain cleavage of a 2-kDa segment from each of the two polypeptide chains of PLAP. These mAbs also prevent the release of PLAP from cultured cancer cell surfaces by bromelain. Such proteolysis-blocking mAbs serve as tools to specifically modify the molecular topography of cell surfaces by protease treatment.     

Clustered distribution of human placental alkaline phosphatase on the surface of both placental and cancer cells. Electron microscopic observations using gold-labeled antibodies

The cell-surface distribution of human placental alkaline phosphatase (PLAP) on cultured cancer cells, A431 and HeLa TCRC-1, and on normal syncytial cells of placental tissue was examined in immunoelectron transmission microscopy using the gold-labeling technique. Chemically fixed cells were reacted with affinity-purified rabbit polyclonal antibodies to PLAP, and the antibodies were visualized using gold particles tagged with goat antirabbit IgG. On all cells PLAP was observed in clusters distributed throughout the membrane surface, including microvilli, but it was not expressed in desmosomes or along other dense regions on the membrane. Previous histochemical and immunochemical techniques failed to demonstrate clusters. The results show that (1) the gold-labeling technique allows a more precise localization of PLAP on the cell surface than previously employed methods, and (2) the distribution of the enzyme is the same on cultured cancer cells and on normal placental syncytial cells. The clustered distribution of PLAP is thus a general phenomenon and is probably influenced by the physiological function of the enzyme, which has yet to be defined.     

Cloning of the human phospholipase A2 activating protein (hPLAP) gene on the chromosome 9p21 melanoma deleted region

Cutaneous malignant melanoma (CMM) is a common skin cancer. About 50% of CMM sporadic tumours have lost one copy of the chromosome 9p21 region. To identify genes involved in the initiation and/or progression of CMM we have characterised the 9p21 melanoma deleted region and screened the human expressed sequence tag (EST) databases (dbEST) to search for expressed genes. We have identified the gene that encodes the human orthologue of the rat phospholipase A2 activating protein (PLAP). PLAP was considered a potential candidate to be involved in malignant melanoma because it maps to the critical region for CMM and because the PLA2 gene has been identified as a modifier of the APC gene, responsible for the adenomatous polyposis phenotype in the mouse. PLAP encodes a protein of 738 amino acids and has a high DNA (90%) and protein (97%) sequence similarity with the rat and mouse PLAP protein. PLAP has a region of WD40 repeats in the amino-terminus, which allows us to include this protein in the superfamily of beta-transducin proteins. Northern blot hybridisation gave a fragment of 4.5 kb, with higher expression in heart compared to other tissues. PLAP was localised at chromosome 9p21, between marker AFM218xg11 and TEK. SSCP analysis of the coding region of PLAP revealed no variants in the studied samples, but one of six CMM samples analysed by RT-PCR showed specific inactivation of PLAP. Despite PLAP's important role in mediating several cellular responses and its localisation to the chromosome 9p21 region deleted in CMM, it is unlikely that point mutations or deletions in the coding region of PLAP are responsible for the initiation or progression of CMM. Further studies on PLAP inactivation should be performed to clarify its potential involvement in CMM.     

Isolation of B subunit‐specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2

Shiga toxin 2 (Stx2)‐specific mAb‐producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)‐specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat‐labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)‐specific whereas five were Stx2B‐specific antibody‐producing clones. The in vitro neutralization activity of Stx2B‐specific mAbs against Stx2 was greater than that of Stx2A‐specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B‐specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B‐specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the V_H and V_L regions of clones 45 and 75D9 were determined. Our Stx2B‐specific mAbs may be new candidates for the development of mouse‐human chimeric Stx2‐neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.     

Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase.

Placental (PLAP) and germ cell (GCAP) alkaline phosphatases were probed immunologically with a library of 18 murine monoclonal antibodies reacting with different conformational epitopes on PLAP. Three main antigenic domains (I, II and III) were mapped by antibody competition experiments and the relative binding of the antibodies to site-directed PLAP mutants. Relative affinities of each of the antibodies for the wild type (wt) GCAP were 2-3-fold lower than the values found for wt PLAP. Relative affinity was determined for a series of PLAP mutants, in which one, two or three amino acids were substituted for the corresponding wt GCAP residues by site-directed mutagenesis. Substitutions at residues 15, 38, 67, 241 or 254 induced a major decrease in affinity (6-10-fold) primarily for those antibodies reacting within domain I, whereas changes at positions 84 and 297 led to a 2-3-fold enhancement of affinities as measured with antibodies reacting within the three domains. Arg209 was found to constitute the only difference between the S and F allelic phenotypes of PLAP and to structure the epitope for the F/S allotype-discriminating antibodies. Arg241 was found to constitute the epitope for the antibody 17E3 that discriminates between PLAP and GCAP. Mutagenesis at position 68 or 133 had little effect on the overall reactivity with the antibody panel. Substitution in wt PLAP of Glu429 for Gly429 or even for His429 (found at this position in tissue-nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies. The conformational change accompanying mutagenesis of Glu429 in PLAP, is important in view of the recent identification of Gly429 as the major determinant of the unique GCAP inhibition by the uncompetitive inhibitor L-Leu. Relative affinity values determined for the rare L-Leu sensitive heterodimeric FD and SD PLAP phenotypes, suggested that the reactivity pattern of the D homodimer with the antibody panel, would resemble more closely that of wt GCAP than wt PLAP. Our data suggest that the uncompetitive inhibition of GCAP by L-Leu is due to an enzymatically critical conformational change in a loop region proximal to the active site of the enzyme, induced by substitution of a single amino acid residue.     

Attachment of human placental-type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes

Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules. This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C, which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty-acid--PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an O-ester linkage.     

Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY

Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells.     

Production of Monoclonal Antibodies to Guinea Pig Liver Transglutaminase

Monoclonal antibodies are now a powerful tool in biology and medicine. Transglutaminase has been implicated in diverse biological functions, and the characteristics of its catalytic action are suitable for applied enzymology. In this study, we produced hybridoma cells which synthesize monoclonal antibodies against guinea pig liver transglutaminase by fusing mouse myeloma cells with spleen cells of mouse immunized with the enzyme protein. Eight hybridoma clones (coded 2F, 4B, 7C, 8B, 8D, 8E, 9F and 11C) were selected to produce monoclonal antibodies. The subclass of IgG produced by clone 9F was IgG_2a and those from the seven other clones were all IgG₁ The 9F antibody inhibited transglutaminase activity, but the other antibodies did not. A solid-phase antibody-binding assay showed that of these antibodies, 8D antibody has the highest affinity to the antigen. Transglutaminase protein in crude liver extract was identified with Western blotting analysis using 8D antibody as the probe.     

New T cell epitopes identified from an anti-idiotypic antibody mimicking ovarian cancer associated antigen

Anti-idiotype (Id) antibodies can be used to induce specific cellular immune responses against tumor antigens, but the mechanism of antigenicity is not always clear. We previously reported an anti-Id antibody, 6B11, which mimics human ovarian cancer associated antigen OC166-9. To explore the molecular basis of cellular immune response induced by 6B11, a panel of peptides derived from complementarity determining region (CDR) of 6B11 were synthesized. After a series of immunologic experiments, we found that the light chain CDR3 peptide and heavy chain CDR3 peptide were the MHC class I and class II epitopes of 6B11, respectively. The combination of MHC class I and class II epitopes is more effective than 6B11 in inducing specific cellular immune response against ovarian cancer. Our study provided the structural basis of antigenicity of 6B11. The identification of antigen-specific T cell eptitopes in 6B11 should facilitate the design of epitope-based vaccine against human ovarian cancer.     

The novel estrogen receptor GPER regulates the migration and invasion of ovarian cancer cells

G protein-coupled estrogen receptor (GPER) was identified as a new member of the estrogen receptor family in recent years. It has become apparent that GPER mediates the non-genomic signaling of 17β-estradiol (E₂) in a variety of estrogen-related cancers. Our previous study has found that GPER was overexpressed in human epithelial ovarian cancer and was positively correlated with the expression of matrix metalloproteinase 9 (MMP-9), which suggested GPER might promote the metastasis of ovarian cancer. However, the mechanisms underlying GPER-dependent metastasis of ovarian cancer are still not clear. In the present study, estrogen receptor α (ERα)-negative/GPER-positive OVCAR5 ovarian cancer cell line was used to investigate the role of GPER in the migration and invasion of ovarian cancer. Wound healing assay and transwell matrigel invasion assay were performed to determine the potentials of cell migration and invasion, respectively. The production and activity of MMP-9 in OVCAR5 cells were examined by Western blot and gelatin zymography analysis. The results showed that E₂ and selective GPER agonist G-1 increased cell motility and invasiveness, and upregulated the production and proteolytic activity of MMP-9 in OVCAR5 cells. Small interfering RNA (siRNA) targeting GPER and G protein inhibitor pertussin toxin (PTX) inhibited the migration and invasion of OVCAR5 cells, and also reduced the expression and activity of MMP-9. Our data suggested that GPER promoted the migration and invasion of ovarian cancer cells by increasing the expression and activity of MMP-9. GPER might play an important role in the progression of ovarian cancer.     

OVCA1 inhibits the proliferation of epithelial ovarian cancer cells by decreasing cyclin D1 and increasing p16

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.     

Langmuir Films from Human Placental Membranes: Preparation, Rheology, Transfer to Alkylated Glasses, and Sigmoidal Kinetics of Alkaline Phosphatase in the Resultant Langmuir-Blodgett Film

In the present study, we studied the activity of human placental alkaline phosphatase (PLAP) constraint in a planar surface in controlled molecular packing conditions. For the first time, Langmuir films (LFs) were prepared by the spreading of purified placental membranes (PPM) on the air–water interface and their stability and rheological properties were studied. LFs exhibited a collapse pressure π_C = 48 mN/m, hysteresis during the compression–decompression cycle (C–D), indicating a plastic deformation, and a compressibility modulus (K) compatible with liquid-expanded phases. A phase transition point appeared at π_T = 28 mN/m and, following successive C–D, it moved toward lower surface areas and higher K, suggesting the lost of some non-PLAP proteins as components of vesicles that might protrude from the monolayer (confirmed by combining lipid/protein molar ratio analysis, PAGE-SDS and V _max). LFs were transferred at 35 mN/m to alkylated glasses to obtain Langmuir-Blodgett films (LB₃₅) the stability of which was confirmed by AFM. The kinetics of p-nitrophenyl phosphate (pNPP) hydrolysis at 37°C catalyzed by PPM was Michaelian and exhibited the thermostability at 60°C typical of PLAP. In LB₃₅, PLAP exhibited a sigmoidal kinetics which resembled the behavior of the partially metalated enzyme but might become from a cross-talk between protein and membrane structures.     

Cardenolides of Leptadenia madagascariensis from the Madagascar dry forest

Investigation of the endemic Madagascar plant Leptadenia madagascariensis Decne. (Apocynaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the four new cardenolides 1-4. The structure elucidations of these compounds were based on analyzes of their 1D and 2D NMR spectra and mass spectrometric data. The cardenolides were strongly antiproliferative to the A2780 ovarian cancer cell line, with IC₅₀ values of 0.18, 0.21, 0.17, and 0.29 ??M line, and to the H460 human lung cancer cell line, with IC₅₀ values of 0.16, 0.68, 0.37, and 0.48 ??M, respectively.     

Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monoclonal antibodies

Placental alkaline phosphatase (PLAP) in humans shows a high degree of genetic polymorphism as disclosed by electrophoretic analysis. Human testes contain trace amounts of a PLAP-like enzyme, that although immunologically cross-reactive with PLAP, shows unique catalytic properties. As an alternative approach to study enzyme polymorphism we have developed monoclonal antibodies to purified allelic variants of PLAP. Five different monoclonal antibodies are described in this report. The antibodies react with different epitopes on the PLAP molecule. Both conformational dependent and independent determinants are detected. Two epitopes are modified when comparing the S and F allelic variants of PLAP. One epitope is common to PLAP and the intestinal isoenzyme of alkaline phosphatase. The five epitopes appear to be mapped on two rather distant antigenic domains. Combinations of any two antibodies binding to different domains give immunoprecipitates with PLAP on Ouchterlony tests and give good response in sandwich enzyme-linked immunosorbent assays. A study of PLAP-like enzyme in 32 individual testis samples indicates differences in four of the epitopes when compared with PLAP. Four types of testicular enzymes can be distinguished based on their reactivities. These results indicate structural differences between the testicular PLAP-like enzyme and PLAP. These differences are compatible with an underlying genetic mechanism.     

Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: application to placental alkaline phosphatase

An immunological approach to the study of the structure and biogenesis of the phosphatidylinositol glycan (PI-G) membrane anchor at the carboxyl terminus of human placental alkaline phosphatase (PLAP) is described. Based on the protein sequence predicted from full length PLAP cDNA, two epitopes were chosen in the region of the carboxyl terminus for the production of site-directed antibodies. The exo site represents the last nine residues of preproPLAP, (res. 505-513), which is part of the sequence that is expected to be cleaved from the nascent protein during processing and addition of the PI-G tail. A second site, the endo sequence, was selected close to the expected carboxyl terminus in mature PI-G-tailed PLAP (res. 474-484 of proPLAP). The two peptides were synthesized, polyclonal antibodies to the conjugated peptides were prepared, and the antisera were characterized. Analytical methods for both synthetic peptides and proteins are presented. Preliminary applications to the isolation and characterization of the PI-G-linked carboxyl terminus of mature PLAP and to the characterization of nascent PLAP are described. The application of both carboxyl terminal-directed antibodies, and a third antibody directed to the amino terminus of mature PLAP, in studies employing mutant forms of PLAP and to the PI-G tailing process itself are discussed. The immunological approach used here for PLAP should be applicable generally to the study of other PI-G-tailed proteins.