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.     

Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.     

The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.     

Preparation of variant-specific anti-human placental phosphatase antisera by immunoabsorption.

Antiserum raised in rabbits sensitized with purified human placental alkaline phosphatase of the rare FD phenotype was absorbed on purified FF phenotype enzyme conjugated to Sepharose. The absorbed antiserum was not able to bind to the F-variant, but was still capable of binding to the D-variant enzyme, determined by electrophoretic retardation and gel filtration. It therefore appears that some allelic variants of placental phosphatase differ in their antigenic structure.     

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.     

A highly sensitive assay method for human placental alkaline phosphatase involving a monoclonal antibody bound to a paper disk

A monoclonal antibody which is specific for human placental alkaline phosphatase and does not cross-react at all with intestinal alkaline phosphatase was prepared, and a procedure for the determination of placental alkaline phosphatase activity in serum was developed involving this monoclonal antibody bound to a paper disk. The minimum amount of placental alkaline phosphatase detectable by this method is 0.0025 King-Armstrong unit. Good correlation with the heat-treatment method was obtained. Therefore this proposed method can be used as a routine clinical test for the determination of serum placental alkaline phosphatase.     

Production of fetal-like alkaline phosphatase by HeLa cells

Alkaline phosphatase produced by HeLa cells differs in its chemical and physical properties from the enzyme found in adult organs and tissues (Cox and Griffin, 1967). In the present study HeLa cell alkaline phosphatase was compared to a fetal form of the enzyme found in human placenta. Both enzymes have approximately the same molecular weight as judged by sucrose density gradients, and the chemical and physical properties of these alkaline phosphatases are similar. The electrophoretic pattern of the HeLa cell enzyme resembles the placental alkaline phosphatase of the heterozygous FS phenotype except that it is slower moving. Double immunodiffusion using an antibody against HeLa cell alkaline phosphatase and placental and HeLa cell enzymes as antigens shows a single line of partial identity between the two enzymes, with a small spur suggesting additional antigenic sites on the HeLa cell enzyme. The data suggest that malignant cells in culture, HeLa, are producing a fetal-like alkaline phosphatase probably by derepression of the genome. However, the electrophoretic and immunological characteristics of the enzyme are altered sufficiently so that it can be distinguished from the normally produced fetal enzyme.This work was supported by U.S. Public Health Service Grant GM 15508 and the Health Research Council of the City of New York.Fourth-year student; Honors Program.Career Scientist Health Research Council of the City of New York.     

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.     

Human placental and intestinal alkaline phosphatase genes map to 2q34-q37

The alkaline phosphatases comprise a multigene enzyme family that hydolyze phosphate esters and are widely distributed in nature. Three main classes have been isolated from humans, the placental, intestinal, and liver/bone/kidney forms. We have mapped the placental and intestinal alkaline phosphatase genes to 2q34-q37 by using chromosomal in situ hybridization and a somatic-cell hybrid panel.     

Developmental changes in the antigenicity and sugar-chain heterogeneity of rabbit alkaline phosphatases

1. Rabbit alkaline phosphatases (APs) clearly fused with the anti-human AP antibodies. In particular, fetal liver and kidney APs reacted slightly less with the anti-intestinal AP antibody as did adult enzymes, suggesting that intestinal AP-like isozyme is expressed at earlier stages of gestation in rabbit liver and kidney. 2. Immunohistochemical data indicated that intestinal AP-like isozyme in the kidney was mainly localized in the distal convoluted tubules and slightly in the proximal straight tubules, whereas liver/bone/kidney AP-like enzyme was found more in the glomeruli and interstitial capillary walls as a major component. 3. The sugar-chain heterogeneity of adult and fetal rabbit APs displayed organ-specificity as did of rat and human APs. Moreover, in fetal development, the expression of high-mannose type or hybrid type sugar chains precedes the expression of complex type sugar chains in fetal development.     

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.     

Leishmania donovani: generation of monospecific antibody reagents to soluble acid phosphatase

Four monoclonal antibodies (McAbs) were generated against the soluble extracellular acid phosphatase (EC 3.1.3.2) (S-AcP) of Leishmania donovani. These were detected in the primary screen using an ELISA with promastigote culture supernatants as antigen. Three of the McAbs demonstrated bound S-AcP from such culture supernatants in an enzyme activity binding assay. All immunoprecipitated metabolically labeled S-AcP but none showed any binding to the promastigote surface by indirect immunofluorescence. Moreover, none reacted with Triton X-100 solubilized plasma membranes by immunoprecipitation or Western blotting. These results demonstrated that the McAbs did not recognize the surface membrane bound acid phosphatase, but were specific for the extracellular soluble enzyme. Further, none of the antibodies immunoprecipitated any of the five human acid phosphatase isozymes or reacted with them in Western blots or the enzyme activity binding assay. Therefore, they are specific for the parasite-derived enzyme. One of these was used to affinity purify sufficient L. donovani S-AcP to immunize a rabbit and generate a specific, polyvalent antiserum. This polyvalent antibody immunoprecipitated S-AcP activity but did not cross-react with the surface membrane acid phosphatase, indicating that these two parasite enzymes are separate gene products.     

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.     

Translation of rat intestinal RNA yields two alkaline phosphatases.

After translation of total rat intestinal RNA, immunoprecipitation using monospecific antiserum against rat intestinal alkaline phosphatase yielded two polypeptides in the adult duodenum and jejunum (molecular masses 62 and 65 kDa). Immunoprecipitation of both bands was blocked by a single purified alkaline phosphatase. In the adult ileum and in the entire small intestine of suckling pups, only the 62 kDa translation product was found. After fat feeding, translated alkaline phosphatase increased by an amount proportionate to the increase in enzyme activity previously seen in the serum. A small fraction of nascent alkaline phosphatase was translocated into microsomal vesicles, producing peptides of 65 and 69 kDa. Tunicamycin-treated membranes demonstrated a different signal peptide for each translation product. N-Terminal sequencing of the translation products showed leucine residues at similar positions, but overlap with the mature protein sequence was not demonstrated. On the basis of these data, we propose the presence of two mRNAs encoding alkaline phosphatase in the rat intestine.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

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.     

Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes.

The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide 'mapping' in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.     

Properties of amphiphilic and hydrophilic forms of alkaline phosphatase from human liver.

Amphiphilic and hydrophilic forms of alkaline phosphatase differed in electrophoretic mobility, sensitivity to heat, activation by phospholipids and albumin, and affinity of monoclonal antibodies, but were similar in substrate Km and inhibitor Ki values, sensitivity to sodium dodecyl sulfate, and electrophoretic behavior on desialylation. Chemical cross-linking experiments failed to conclusively demonstrate an aggregated state of amphiphilic alkaline phosphatase in Triton X-100. Further, attempts to identify a polymeric hybrid between amphiphilic forms of human liver and placental alkaline phosphatase were unsuccessful. We conclude that the covalent attachment of the hydrophobic phosphatidyl-inositol membrane anchor causes the amphiphilic form to behave anomalously on electrophoresis and to affect certain of the enzyme's catalytic and physical properties.     

The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase

The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase.