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.     

Correlation between RsaI restriction fragment length polymorphism and electrophoretic types of human placental alkaline phosphatase

Restriction fragment length polymorphism (RFLP) of human alkaline phosphatases was studied in a population sample from northern Sweden using a placental alkaline phosphatase (PLAP) cDNA probe. After digestion of human genomic DNA with RsaI the Southern blots showed DNA fragments most probably derived from three genes: PLAP, germ cell alkaline phosphatase (PLAP-like) and intestinal alkaline phosphatase. In agreement with a previous study, a two-allele polymorphism was found in PLAP with bands at 1.6 kilobases (A1) and 1.8 kilobases (A2). The gene frequencies of A1 and A2 were 0.46 and 0.54, respectively. There was a significant correlation between the RsaI RFLPs and electrophoretic types of PLAP; RSAI A2 showed an association with the ALP2p allele of PLAP.     

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.     

Differentiation of human adult and fetal intestinal alkaline phosphatases with monoclonal antibodies

Two forms of intestinal alkaline phosphatase have been recognized in humans. They are very similar in a number of biochemical and immunologic characteristics, but the exact genetic relationship between them remains unclear. To further study this problem, six monoclonal antibodies and a polyclonal rabbit antiserum to human fetal intestinal alkaline phosphatase have been produced. All of the monoclonal antibodies and the rabbit antiserum crossreact with adult intestinal alkaline phosphatase and with the intestinal-like alkaline phosphatase found in D98/AH-2 human tissue-culture cells. Four of the monoclonal antibodies and the rabbit antiserum crossreact with placental alkaline phosphatase, while none of the antibodies or the antiserum recognize liver or kidney alkaline phosphatase. Four of the monoclonal antibodies can distinguish between adult and fetal intestinal alkaline phosphatase in electrophoretic titration-binding studies, with the relative binding of adult enzyme being significantly greater than that of the fetal enzyme in each case. One of these antibodies, which also reacts with placental alkaline phosphatase, can distinguish the type 3 allelic variant of the placental enzyme from types 1 and 2. This indicates that the antibody detects a structural difference in the protein moiety of one of the allelic forms of the enzyme. These data suggest that adult and fetal intestinal alkaline phosphatases represent structurally distinct proteins, either encoded for by different genes or produced by differential processing of a common precursor molecule determined by a single gene.     

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.     

Two gene duplication events in the evolution of the human heat-stable alkaline phosphatases

There are at least three alkaline phosphatase (AP) isoenzymes in man: a heat-stable placental enzyme (PLAP), a less heat-stable intestinal form (IAP), and the very heat-labile AP enriched in liver, bone and kidney. In addition to these enzymes, there is a heat-stable activity in the thymus and testis that is similar but not identical to the PLAP (the PLAP-like enzyme). Previous work has demonstrated a close structural relatedness among the IAP, PLAP and PLAP-like enzymes. Thus, it is possible that there are three human genes encoding heat-stable AP enzymes. To test this hypothesis, we have used a PLAP cDNA clone to screen a human genomic library cloned into the phage vector 1EMBL-3. Three sets of clones were isolated, each bearing a distinct coding region homologous to the PLAP cDNA probe. Nucleotide sequence analysis of the 5′ ends of these genes allowed comparison of their derived peptide sequences and positive identification of two of the genes. One of the genes encodes the PLAP (the PLAP-1 gene), another encodes the IAP, and a third closely resembles the PLAP-1 gene, but is distinct from it (the PLAP-2 gene). The PLAP-2 gene is highly homologous (> 95%) with the PLAP-1 except in the first exon, where sequences encoding the hydrophobic signal peptide are nearly identical with the same region of the IAP gene. These results demonstrate the existence of a small family of PLAP-related genes which is the result of at least two duplication events during the descent of man.     

Immunoelectron microscopic analysis of the binding of monoclonal antibodies to molecular variants of human placental alkaline phosphatase

Three monoclonal antibodies with distinct antigenic specificities were examined by electron microscopy for their binding to three common genetic variants (SS, FS, and FF) of human placental alkaline phosphatase. In the reaction with the monoclonal antibody H5, all three variants of human placental alkaline phosphatase preferentially formed circular immune complexes composed of two antibodies and two enzyme molecules. In separate reactions with the F11 and B2 monoclonal antibodies, the SS variant formed circular complexes and the FS variant formed Y-shaped complexes composed of one antibody and two enzyme molecules, whereas the FF variant scarcely reacted. These results confirm immunochemical data showing that H5 binds to both S and F subunits with similar affinities, whereas F11 and B2 bind the S subunit with markedly higher affinity than they do the F subunit. Furthermore, the formation of circular complexes in the reaction of the mixture of the two antibodies, F11 and B2, with FS molecules suggests that these two antibodies bind to different sites on the S subunit. Therefore, the F and S subunits differ from one another at more than one site. This is the first indication that alleles of human placental alkaline phosphatase may result from more than just single point mutations in the gene encoding them.     

Discrimination of human placental alkaline phosphatase allelic variants by monoclonal antibodies.

Eighteen monoclonal antibodies were produced by the mouse hybridoma method using purified placental alkaline phosphatase (ALP) as antigen. The ability of the various antibodies to discriminate among allelic variants of the enzyme was tested using a large panel of placental ALPs that had been typed electrophoretically. The panel included sets of samples of each of the six common polymorphic phenotypes as well as a series of rare variants. The reactivity of each antibody with each placental ALP (binding ratio) was determined relative to a single standard placental ALP (type 1) in a quantitative binding assay. The findings for six of the antibodies have already been reported. The results on the other 12 antibodies are presented here, and the combined data on the total series of 18 antibodies are analyzed and discussed. Six of the 18 antibodies showed significantly reduced binding to one or another of the products of the three common alleles. In three cases, the discrimination was reflected by essentially "all-or-none" binding reactions. In the other three cases, the binding differences were less marked but could be demonstrated by quantitative comparisons of the binding ratios. Quantitative binding ratio comparisons also enabled heterozygotes to be differentiated from homozygotes in each case. Some of the antibodies showed reduced binding with certain of the rare variant ALP electrophoretic phenotypes. It is estimated that at a minimum this unselected series of 18 antibodies is directed to at least nine different antigenic determinants on the surface of the placental ALP molecule. The results illustrate the power of monoclonal antibodies to discriminate among allelic variants of enzymes.     

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.     

A possible new locus of alkaline phosphatase expressed in human testis

Summary Human testes contain trace amounts of heat-stable placental-like alkaline phosphatase. Using a recently described allotype-specific monoclonal antibody (F₁₁) toward placental alkaline phosphatase (PLAP), we show that the frequencies of reactivity of the testis enzymes differ greatly from those of the placental phenotypes. By means of the enzyme inhibitors L-Phe, L-Phe-gly-gly, L-Leu, and L-Leu-gly-gly, the testis enzyme can be clearly distinguished in all cases from the placental enzyme. These results argue that the testis enzyme is not a product of the placental gene and suggest the possible existence of a new locus of alkaline phosphatase.     

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.     

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.     

Structural comparisons of two allelic variants of human placental alkaline phosphatase

A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.     

Plasmodium falciparum: intragenic recombination and nonrandom associations between polymorphic domains of the precursor to the major merozoite surface antigens.

Extensive allelic polymorphism in the Plasmodium falciparum major merozoite antigen precursor (MSP1/PMMSA) is partly due to intragenic recombination events within a short region near the 5' end of the gene. Newly described allelic sequences from this region of the gene are compared to those previously published, revealing additional sites of intragenic recombination. Epitopes recognised by monoclonal antibodies on the protein have been assigned on the basis of correlations between serology and amino acid sequence polymorphisms among different allelic types of MSP1. Serological analyses of MSP1 from 567 wild isolates from The Gambia, Nigeria, and Brazil reveal that certain pairs of epitopes, although sited on MSP1 domains separated by known sites of intragenic recombination, are highly significantly associated on parasites in endemic populations. Most associations are similar in the three countries. These associations are discussed with respect to the intragenic recombination hypothesis.     

The mechanism of placental alkaline phosphatase induction in vitro

The mechanism of placental alkaline phosphatase (PLAP) induction by prednisolone in a uterine cervical epidermoid cancer cell line SKG-IIIa was investigated in vitro by enzyme-cytochemistry, enzyme immunoassay, Northern and Southern blot analysis, and in situ hybridization. Enzyme-cytochemical alkaline phosphatase (ALP) staining and immunoassay revealed increased levels of PLAP (heat-stable ALP) in prednisolone-treated cells. Northern blot analysis and in situ hybridization showed increased amounts of PLAP mRNA. Southern blot analysis indicated that PLAP was not a product of an amplified or rearranged gene. These findings suggest that the induction of PLAP mRNA in SKG-IIIa cells by prednisolone in turn increased the levels of PLAP.     

A human monoclonal antibody specific to placental alkaline phosphatase,a marker of ovarian cancer

Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (K_d) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads.     

Enzyme-linked coagulation assay. IV. Sensitive sandwich enzyme-linked immunosorbent assays using Russell's viper venom factor X activator-antibody conjugates

We have applied the enzyme-linked coagulation assay (ELCA) system to the development of an amplified immunoassay using the clotting cascade to enhance sensitivity of detection of immune complexes. The factor X-activating enzyme of Russell's viper venom was detectable using ELCA in amounts as low as 0.25 fg per assay. Monoclonal antibodies to beta-hCG, placental alkaline phosphatase (PLAP), and the P-24 antigen of HTLV-III were labeled with this enzyme or peroxidase and used for "sandwich" immunoassays using another monoclonal antibody (beta-hCG, PLAP) or polyclonal patient IgG (P-24 antigen) bound to a polylysine-glutaraldehyde-coated plate as a "capture" reagent. After the immunobinding step, the plate was washed and substrate consisting of a mixture of factors X, V, and II in buffer containing calcium and lipid was incubated for various lengths of time. The mixture was transferred to another plate coated with fibrinogen and containing peroxidase-fibrinogen in EDTA solution to measure the amount of thrombin generated. Using this protocol, we were able to measure the presence of 2-10 pg/ml of beta-hCG and PLAP (5-30 amol per sample). All three model antigens were detectable at concentrations 2-3 orders of magnitude less using RVV-XA-labeled antibodies and ELCA than they were using peroxidase-labeled antibodies. The assay has considerable potential as a general immunoassay amplification system, yielding a "color test" for antigens of interest with a detection limit not readily attainable using other chromogenic methodologies.     

The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state

Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.     

Molecular cloning and sequence analysis of human placental alkaline phosphatase

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.     

Prediction and molecular modeling of T-cell epitopes derived from placental alkaline phosphatase for use in cancer immunotherapy

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYFPEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.