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.     

Structural and functional analysis of human germ cell alkaline phosphatase by site-specific mutagenesis.

Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function assignment to conserved residues in mammalian alkaline phosphatases

We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.     

DNA polymorphism of alkaline phosphatase isozyme genes: linkage disequilibria between placental and germ-cell alkaline phosphatase alleles.

The use of human placental alkaline phosphatase (PLAP) cDNA as a probe allows the detection and identification of restriction DNA fragments derived from three homologous genes, i.e., intestinal alkaline phosphatase (AP), germ-cell AP (GCAP), and PLAP. In previous RFLP studies we have reported linkage disequilibria between an RsaI and two PstI (a and b) polymorphic restriction sites and electrophoretic types of PLAP. In this report we present evidence that, in spite of the strong correlation with PLAP types, PstI(b) is an RFLP of GCAP. The data indicate close linkage between the PLAP and GCAP loci.     

Crystal structure of alkaline phosphatase from human placenta at 1.8 A resolution. Implication for a substrate specificity

Human placental alkaline phosphatase (PLAP) is one of three tissue-specific human APs extensively studied because of its ectopic expression in tumors. The crystal structure, determined at 1.8-A resolution, reveals that during evolution, only the overall features of the enzyme have been conserved with respect to Escherichia coli. The surface is deeply mutated with 8% residues in common, and in the active site, only residues strictly necessary to perform the catalysis have been preserved. Additional structural elements aid an understanding of the allosteric property that is specific for the mammalian enzyme (Hoylaerts, M. F., Manes, T., and Millán, J. L. (1997) J. Biol. Chem. 272, 22781-22787). Allostery is probably favored by the quality of the dimer interface, by a long N-terminal alpha-helix from one monomer that embraces the other one, and similarly by the exchange of a residue from one monomer in the active site of the other. In the neighborhood of the catalytic serine, the orientation of Glu-429, a residue unique to PLAP, and the presence of a hydrophobic pocket close to the phosphate product, account for the specific uncompetitive inhibition of PLAP by l-amino acids, consistent with the acquisition of substrate specificity. The location of the active site at the bottom of a large valley flanked by an interfacial crown-shaped domain and a domain containing an extra metal ion on the other side suggest that the substrate of PLAP could be a specific phosphorylated protein.     

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.     

Structural studies of human placental alkaline phosphatase in complex with functional ligands

The activity of human placental alkaline phosphatase (PLAP) is downregulated by a number of effectors such as l-phenylalanine, an uncompetitive inhibitor, 5'-AMP, an antagonist of the effects of PLAP on fibroblast proliferation and by p-nitrophenyl-phosphonate (PNPPate), a non-hydrolysable substrate analogue. For the first two, such regulation may be linked to its biological function that requires a reduced and better-regulated hydrolytic rate. To understand how such disparate ligands are able to inhibit the enzyme, we solved the structure of the complexes at 1.6A, 1.9A and 1.9A resolution, respectively. These crystal structures are the first of an alkaline phosphatase in complex with organic inhibitors. Of the three inhibitors, only l-Phe and PNPPate bind at the active site hydrophobic pocket, providing structural data on the uncompetitive inhibition process. In contrast, all three ligands interact at a remote peripheral site located 28A from the active site. In order to extend these observations to the other members of the human alkaline phosphatase family, we have modelled the structures of the other human isozymes and compared them to PLAP. This comparison highlights the crucial role played by position 429 at the active site in the modulation of the catalytic process, and suggests that the peripheral binding site may be involved in the functional specialization of the PLAP isozyme.     

Some distinctive characteristics of the alkaline phosphatase of Serratia marcescens.

A mutant strain of Serratia marcescens produces a constitutive enzyme (phosphatase F), which differs from the alkaline phosphatase of Escherichia coli in the following characteristics: one enzyme species with higher mobility on electrophoresis, less heat stability, no rapid reactivation following exposure to high hydrogen ion concentrations, no hybridization with E. coli enzyme in vitro, little activation at increased ionic strength, greater sensitivity to EDTA inhibition, and no cross reaction of rabbit anti-serum with the E. coli enzyme.     

A comparative study of 5’nucleotidase and alkaline phosphatase in human placenta during development

Activities and a few properties of alkaline phosphatase and 5’-nucleotidase were compared in the developing human placenta. Both the enzymes were mostly membrane-bound and displayed similar developmental patterns with the highest activities at 24/26 weeks of the placenta. L-Phenylalanine, L-tryptophan and L-leucine were inhibitors of alkaline phosphatase, whereas they had no effect on the 5’-nucleotidase. Alkaline phosphatase from a late stage of gestation appeared to be almost heat-stable. An appreciable part of 5’-nucleotidase was also resistant to heat inactivation and this fraction varied with gestational age of the tissue. For both the enzymes, V_max changed without alteringK _m values with periods of gestation. Ca²⁺, Mg²⁺ and Mn²⁺ ions stimulated the alkaline phosphatase activity and Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ were inhibitory. 5’-Nucleotidase was not activated by any of these cations. EDTA and Concanavalin A inhibited both the enzymes, although the extent of inhibition was different and also varied with gestation.     

Similarities between alkaline phosphatase and Ca2+ ATPase activities in HeLa cells

Parallel changes in the enzyme activities of CA2+ATPase and alkaline phosphatase were observed in HeLa cells. Both enzymes were inhibited to a similar degree by L-phenylalanine, L-tryptophan, and L-leucine, while being relatively resistant to L-homoarginine. Exposure to heat (56 degrees C, 60 degrees C, and 65 degrees C) resulted in a loss of both enzyme activities. Both alkaline phosphatase and Ca2+ ATPase, when treated with EGTA, required Ca2+ for the restoration of activity. Cells grown in the presence of agents that affect alkaline phosphatase (dexamethasone, butyric acid, and hyperosmolar NaCl) showed similar changes in the activities of both enzymes.     

Structural evidence of functional divergence in human alkaline phosphatases

The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes, i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-structure of PLAP(1) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family.     

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.     

Studies on alkaline phosphatase in normal and nitrofurantoin resistant Vibrio el tor.

Activity of alkaline phosphatase as well as its substrate specificity in Vibrio el tor and in nitrofurantoin resistant Vibrio el tor have been studied. A lower level of activity is observed in Vibrio el tor after its acquisition of resistance towards nitrofurantoin. The enzyme activity in both the strains is significantly inhibited by EDTA, and also by metal ions like Mg++, Zn++ and Mn++. The normal strain is found to possess two isoenzymes for alkaline phosphatase whereas the resistant strain has only one isoenzyme.     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.     

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.     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Specific amino acid residues in both the PstB and PstC proteins are required for phosphate transport by the Escherichia coli Pst system.

Three mutant alleles of the pstC gene and one mutant allele of the pstB gene were produced by site-directed mutagenesis. The pstC gene encodes an integral membrane protein of the phosphate-specific transport (Pst) system of Escherichia coli. The amino acid substitutions resulting from the pstC gene mutations, Arg-237----Gln, Glu-240----Gln, or a combination of both, caused the loss of phosphate transport through the Pst system, but the alkaline phosphatase activity remained repressed. The pstB gene encodes a peripheral membrane protein of the Pst system which carries a putative nucleotide-binding site. The amino acid substitutions Gly-48----Ile and Lys-49----Gln, resulting from the pstB mutations, caused the loss of phosphate transport through the Pst system and the derepression of alkaline phosphatase activity. The residues Gly-48 and Lys-49 are key residues in the putative nucleotide-binding site.     

Factors affecting the zinc content of E. coli alkaline phosphatase

Through experiments with radioactively labeled EDTA, it has been shown that alkaline phosphatasc from E. coli has a high affinity for binding EDTA, requiring extensive dialysis for removal. This paper reviews the results of zinc analyses of E. coli alkaline phosphatase prepared in the presence and absence of EDTA. The presence of EDTA in most preparations of alkaline phosphatase accounts for previous overestimates of the zine content of the enzyme.With radioactively labeled EDTA, direct evidence for the binding of EDTA to the metal-free alkaline phosphatase is presented. It has been shown that the apoprotein binds two EDTA molecules rather strongly. Addition of four metal ions are necessary for reactivation of this EDTA-contaminated apoenzyme. However, when the EDTA-contaminated apoenzyme is subject for extensive dialysis and EDTA is removed, the addition of two zinc ions restores the enzyme activity completely.