Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C.
Ontology highlight
ABSTRACT:
SUBMITTER: Low MG
PROVIDER: S-EPMC1183647 | biostudies-other | 1977 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC2924006 | biostudies-literature
Project description:Alkaline phosphatase was solubilized from plasma membrane of rat liver with butanol-ol, bile acids or sodium deoxycholate, and electrophoretically compared with a soluble form in serum which was derived from the liver. The three enzyme preparations from the plasma membrane migrated at the same position on polyacrylamide-gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulphate. The mobility of them, however, was distinctly different from that of the serum-soluble form of the liver-derived alkaline phosphatase. On the other hand, phosphatidylinositol-specific phospholipase C isolated from Bacillus cereus was used to release alkaline phosphatase from plasma membrane. The released alkaline phosphatase was demonstrated to have the same mobility as the serum-soluble form on polyacrylamide-gel electrophoresis in the presence or absence of detergents. The phospholipase C also converted the butan-1-ol-extracted membrane form into the serum-soluble form. The results suggest that release of alkaline phosphatase from the liver into serum is not simply caused by a detergent effect of bile salts, but involves an enzymic hydrolysis of phosphatidylinositol, with which alkaline phosphatase may strongly interact in the membrane.
| S-EPMC1144824 | biostudies-other
Project description:Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis , often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids, such as phosphatidylcholine, which is enriched in the outer leaflet of eukaryotic cells, are not well understood. A 500 ns long molecular dynamics simulation of PI-PLC at the surface of a lipid bilayer revealed a strikingly high number of interactions between tyrosines at the interfacial binding site and lipid choline groups with structures characteristic of cation-? interactions. Membrane affinities of PI-PLC tyrosine variants mostly tracked the simulation results, falling into two classes: (i) those with minor losses in affinity, Kd(mutant)/Kd(wild-type) ? 5 and (ii) those where the apparent Kd was 50-200 times higher than wild-type. Estimating ??G for these Tyr/PC interactions from the apparent Kd values reveals that the free energy associated with class I is ~1 kcal/mol, comparable to the value predicted by the Wimley-White hydrophobicity scale. In contrast, removal of class II tyrosines has a higher energy cost: ~2.5 kcal/mol toward pure PC vesicles. These higher energies correlate well with the occupancy of the cation-? adducts throughout the MD simulation. Together, these results strongly indicate that PI-PLC interacts with PC headgroups via cation-? interactions with tyrosine residues and suggest that cation-? interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins.
| S-EPMC3797534 | biostudies-literature
Project description:A phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was purified by a three-step procedure. The specific activity of the purified enzyme was approx. 6000 times that of the culture supernatant, with an overall recovery of approx. 10%. Estimation of the molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by gel filtration gave values of 33000 and 20000 respectively. A thiol group appears to be necessary for the activity of the enzyme. The purified enzyme had no detectable delta-haemolytic activity and was unable to hydrolyse S. aureus phospholipids. Phosphatidyl-inositol in erythrocyte 'ghosts' was readily hydrolysed by the purified phospholipase C. However, in contrast with our previous preliminary observations, phosphatidylinositol in intact erythrocytes was not significantly hydrolysed. These results suggest that at least 75-80% of the phosphatidylinositol is located at the inner leaflet of the membrane.
| S-EPMC1164594 | biostudies-other
Project description:Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.
| S-EPMC1138605 | biostudies-other
Project description:Staphylococcus aureus (S. aureus) commonly colonizes the human skin and nostrils. However, it is also associated with a wide variety of diseases. S. aureus is frequently isolated from the skin of patients with atopic dermatitis (AD), and is linked to increased disease severity. S. aureus impairs the skin barrier and triggers inflammation through the secretion of various virulence factors. S. aureus secretes phosphatidylinositol-specific phospholipase C (PI-PLC), which hydrolyses phosphatidylinositol and cleaves glycosylphosphatidylinositol-anchored proteins. However, the role of S. aureus PI-PLC in the pathogenesis of skin diseases, including AD, remains unclear. In this study, we sought to determine the role of S. aureus PI-PLC in the pathogenesis of skin diseases. PI-PLC was observed to enhance the invasion and persistence of S. aureus in keratinocytes. Besides, PI-PLC promoted the penetration of S. aureus through the epidermal barrier in a mouse model of AD and the human organotypic epidermal equivalent. Furthermore, the loss of PI-PLC attenuated epidermal hyperplasia and the infiltration of Gr-1+ cells and CD4+ cells induced by S. aureus infection in the mouse model of AD. Collectively, these results indicate that PI-PLC eases the entry of S. aureus into the dermis and aggravates acanthosis and immune cell infiltration in infected skin.
| S-EPMC7575579 | biostudies-literature
Project description:Calcium-dependent phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus (saPLC1) catalyzes hydrolysis of phosphatidylinositol (PI) into inositol 1-phosphate by a unique mechanism involving formation of inositol 1,6-cyclic phosphate (1,6-IcP) as an intermediate. This work examines the rates and products of cleavage of phosphorothioate and phosphorodithioate analogues of PI in which sulfur was introduced into the phosphate moiety at a nonbridging position (pro-R or pro-S), a bridging position, or both. The replacement of the pro-S oxygen in the phosphoryl moiety of PI by sulfur results in a 3 x 10(7)-fold decrease of the catalytic rate constant, whereas alteration of the pro-R oxygen results in only a modest rate reduction. The addition of the second sulfur atom into the bridging position of the S(p) isomer of the phosphorothioate analogue causes a dramatic (2 x 10(5)-fold) increase of the rate of cleavage but has a negligible effect on the R(p) isomer. These differences are consistent with a change in the mechanism for the S(p) isomer of the phosphorodithioate analogue into a more dissociative type, where the leaving group carries a large amount of negative charge. In addition, hydrolysis of the diastereomers of the phosphorothioate analogues of 1,6-IcP, inositol cis-1,6-IcPs and inositol trans-1,6-IcPs, affords two distinct products, inositol 1-phosphorothioate and inositol 6-phosphorothioate, respectively. Formation of inositol 6-phosphorothioate is explained by the binding of trans-1,6-IcPs in the active site in a rotated orientation that interchanges the oxygen atoms at the 1- and 6-positions, thereby allowing the hydroxyl group at the 1-position to act as a leaving group. The reorientation of the intermediate is driven by formation of favorable interactions of the enzyme active site with the nonbridging oxygen in the trans intermediate.
| S-EPMC3733342 | biostudies-literature
Project description:Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-? as a partner of the Spry1 and Spry2 proteins. Spry-PLC? interaction was dependent on the Src homology 2 domain of PLC? and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLC? phosphorylation and decreased PLC? activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLC?1 or PLC?2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLC?. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLC? activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.
| S-EPMC2947483 | biostudies-literature
Project description:Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.
| S-EPMC1136395 | biostudies-other
Project description:Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.
| S-EPMC1137374 | biostudies-other