Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase

Membrane and soluble forms of alkaline phosphatase (ALP) were selectively prepared from human placental microsomes by treatment with 1-butanol at pH 8.5 and 5.5, respectively. The purified membrane (mALP) and soluble (sALP) forms were analyzed for chemical compositions. mALP was found to contain 1 mol each of palmitate, stearate, and glycerol/subunit of ALP, which were absent in sALP. Both the forms contained 1 mol of inositol and 2 mol of ethanolamine/subunit. However, none of these compounds was detectable in another soluble form prepared by treatment with papain, which is known to cleave the carboxyl-terminal region. The results suggest that mALP contains diacylglycerol, the removal of which results in its conversion to sALP. We then prepared [3H]ethanolamine-labeled ALP by incubating choriocarcinoma cells (JEG-3) with the isotope. 3H-Labeled sALP was mixed with unlabeled sALP and treated with papain. A 3H-labeled single component was purified from the digests by sequential chromatography through anti-ALP-IgG-Sepharose, concanavalin A-Sepharose, Bio-Gel P-6, and TSK G-2000 columns. Chemical analyses revealed that the purified sample contains the tripeptide Thr-Thr-Asp, ethanolamine, glucosamine, mannose, inositol, and phosphate. Molar ratios of the latter five compounds were calculated to be 2, 1, 3, 1, and 2, respectively, by taking Asp as 1 mol. The tripeptide sequence was identified at positions 482-484 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 513, containing a hydrophobic amino acid sequence. Taken together, these results suggest that the mature ALP molecule lacks the predicted carboxyl-terminal peptide extension and is attached at Asp484 with a glycosylphospholipid, the components of which are characterized above. The glycosylphospholipid thus attached is considered to function as the membrane anchor of ALP.     

Dephosphorylation of rabbit skeletal muscle glycogen synthase by phosphoprotein phosphatase and human placental alkaline phosphatase

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase results in the incorporation of ³²P into two major tryptic peptides (P-1 and P-2) which are identified by isoelectric focusing on polyacrylamide gel. When ³²P-labeled synthase is incubated with rabbit muscle phosphoprotein phosphatase both P-1 and P-2 are hydrolyzed. Incubation of ³²P-labeled synthase with human placental alkaline phosphatase results in a specific hydrolysis of P-1. Measurement of the increase in synthase activity ratio accompanied by the dephosphorylation of P-1 with human placental alkaline phosphatase and, subsequently, of P-2 with phosphoprotein phosphatase shows that both P-1 and P-2 affect the glucose-6-P dependency of the synthase.     

Purification and partial sequencing of human placental alkaline phosphatase

Two forms of human placental alkaline phosphatase have been purified to homogeneity utilizing high performance liquid chromatography. Both have the same amino acid composition but they differ in their carbohydrate substituents. Sequence data indicate that the two forms are identical for the first forty two residues from the amino terminus are presented.     

Purification and multiple forms of human placental alkaline phosphatase

The commercially available human placental alkaline phosphatase was purified to near homogeneity. Multiple bands of the purified enzyme were resolved in the polyacrylamide gel. The number of bands in the gel was reduced after the enzyme was treated with neuraminidase.     

Stimulation by ATP of alkaline phosphatase in placental plasma membranes.

1. ATP stimulated the p-nitrophenyl phosphatase activity of placental plasma membranes, with an increase in activity of approximately 100% at 5 mM ATP. The stimulation was not dependent on the presence of Mg-2-+. 2. The K-m for p-nitrophenyl phosphate was not changed by the presence of 5 mM ATP. 3. ATP hydrolysis by the plasma membrane preparation under the same assay conditions as for alkaline phosphatase was not influenced by the presence of 5 mM p-nitrophenyl phosphate. 4. Extraction of the plasma membrane preparation with n-butanol abolished the stimulatory effect of ATP, as well as Ca-2-+-activated ATPase activity.     

Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.     

Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Proteolytic modification of Escherichia coli alkaline phosphatase

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.     

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.     

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.