Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Study of fibroblast origins and lineages is complicated by the lack of unambiguous markers that could be used to identify discrete subpopulations on the basis of functional attributes. We have studied the role of the membrane-anchored hydrolytic enzyme tissue-nonspecific alkaline phosphatase (TN-AP) and the placental alkaline phosphatase (PL-AP) in collagen phagocytosis and in the deletion of cells by apoptosis. Rat-2 cells, which do not constitutively express AP, were transfected with full-length rat TN-AP or PL-AP cDNAs to determine the impact of the TN-AP collagen-binding domain on cell function. Various levels of expression were driven by early (strong) or late (weak) SV40 promoters in the plasmid construct. Controls were transfected with plasmids that did not contain AP cDNA. AP expression in transfected cells was confirmed by Northern blotting, histochemical analysis, and SDS-PAGE analysis of membrane-anchored enzyme released by phosphatidyl inositol phospholipase C. Low levels of TN-AP expression increased cell spreading slightly, nearly doubled the percentage of collagen phagocytic cells (up to 80%), and increased the number of internalized collagen-coated fluorescence beads per cell. In cells transfected with PL-AP (i.e., no collagen-binding domain), collagen phagocytosis was not affected. Internalization of BSA beads was also not affected by either AP isozyme, indicating that AP was selective for integrin-mediated phagocytosis. In single cells, histochemically demonstrable TN-AP activity on cell membranes was colocalized with the binding of collagen beads, but this colocalization was not detected in cells transfected with PL-AP. Phagocytosis was inhibited by antibodies to the alpha 2 integrin and to AP but not by levamisole, an inhibitor of AP phosphohydrolytic activity. High-level TN-AP expression caused a fivefold reduction of cell proliferation and was associated with the development of cells with sub-G1 DNA content, nuclear condensation, and nuclear budding. In AP-positive cultures, there was a greatly increased number of floating cells; nick-labeling of DNA by terminal transferase and biotinylated dUTP showed a 15-fold increase of stained cells. These data indicate that low-level TN-AP expression enhances collagen phagocytosis, presumably through the TN-AP collagen-binding domain. High-level AP expression promotes cell deletion by apoptosis. We suggest that the expression of AP by fibroblasts indicates a novel role for this enzyme in collagen degradation by phagocytosis.
 ...
PMID:Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibroblasts. 928 52

We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses ( approximately 80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218-->Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162-->Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphosphatidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162-->Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation.
 ...
PMID:Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia. 956 33

A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.
 ...
PMID:Tissue-nonspecific alkaline phosphatase with an Asp(289)-->Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation. 1294 72

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.
 ...
PMID:Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation. 1664 97

Alkaline phosphatases are ubiquitous enzymes involved in many important biological processes. Mammalian tissue-nonspecific alkaline phosphatase (TNAP) has long been thought to play an important role in bone mineralization. In this study, we identified a full-length cDNA encoding a potential alkaline phosphatse from pearl oyster Pinctada fucata by RT-PCR and RACE and designated the encoded protein as PFAP. The sequence of PFAP shares an overall similarity of 67% with that of human TNAP. Prediction and analysis of its secondary and tertiary structure revealed that the PFAP contains two mammalian-specific regions, the crown domain, involved in collagen binding, and the calcium binding domain, which hint its potential ability to participate in biomineralization. RT-PCR and in situ hybridization showed that the PFAP mRNA distributes specifically in the hepatic duct of the digestive diverticula. These findings implied its possible role in calcium absorption and transportation. In vivo, PFAP could be specifically released by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting it is glycophosphatidylinositol-anchored to the plasma membrane. Therefore, a human growth hormone-PFAP fusion was constructed to locate the cleavage/attachment site. Immunofluorescent labeling and immunoblotting showed that Asn-477 is the cleavage/attachment site and the 25-residue peptide COOH-terminal to Asn-477 is removed during glycophosphatidylinositol anchoring. This research will hopefully pave the way to illustrate the role PFAP plays in calcium transportation related to pearl biomineralization.
 ...
PMID:A novel glycosylphosphatidylinositol-anchored alkaline phosphatase dwells in the hepatic duct of the pearl oyster, Pinctada fucata. 1762 76

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT(2B)R) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT(2B)R and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT(2B)R and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT(2B)R intrinsic activity or shRNA-mediated 5-HT(2B)R knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT(2B)R couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT(2B)R controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT(2B)R-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT(2B)R-null mice exhibit defects in TNAP activity.
 ...
PMID:Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C. 2057 58