Detergent‐Mediated Reconstitution of a Glycosyl‐Phosphatidylinositol‐Protein into Liposomes

Angrand, Muriel; Briolay, Anne; Ronzon, Frédéric; Roux, B.

doi:10.1111/j.1432-1033.1997.t01-1-00168.x

Cited by 54 publications

(51 citation statements)

References 31 publications

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“…7, A, B, E, and F). Consistent with our specificity studies, detergent-assisted reconstitution of another GPI-anchored protein (bovine intestine alkaline phosphatase) into raft-like liposomes was shown to be GPI anchor-dependent (34). Admittedly, we cannot entirely rule out the possibility of another PI-PLC-released protein indirectly facilitating the SCRL binding of PI-PLC-released PrP-sen, although our experiments with purified PrP-sen would indicate that other accessory proteins are not required for this binding activity.…”

Section: S]gpisupporting

confidence: 85%

“…This alternative, GPI anchor-independent mode of membrane association was detected in control experiments with GPI Ϫ PrP-sen during our attempts to reconstitute GPI ϩ PrPsen in SCRLs. These reconstitution experiments included the mixing of PrP-sen with SCRLs in the presence of a low concentration of Sarkosyl as transient exposure to detergent is commonly required to permit insertion of GPI-anchored proteins into liposomes (15,16,34,35). Presumably, the detergent is required to disrupt the pseudomicelles formed by GPI-anchored proteins in the absence of detergent (36) and/or to transiently perturb the liposome membranes to facilitate insertion of the GPI anchor.…”

Section: Fig 8 Peg and Pi-plc Assist Cell-free Conversion Of Scrlasmentioning

confidence: 99%

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Effect of Glycosylphosphatidylinositol Anchor-dependent and -independent Prion Protein Association with Model Raft Membranes on Conversion to the Protease-resistant Isoform

Baron¹,

Caughey

2003

Journal of Biological Chemistry

110

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Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raftlike liposomes (SCRLs). Both full-length (GPI ؉ ) and GPI anchor-deficient (GPI ؊ ) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34 -94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI ؉ PrPsen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositolspecific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI ؉ PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI ؊ PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res.Prion protein (PrP) 1 is a glycoprotein usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor (1). Like other GPI-anchored proteins, PrP is enriched in sphingolipidand cholesterol-rich membrane microdomains known as detergent-resistant membranes (DRMs), or rafts (2). Several lines of evidence from biochemical and molecular biological approaches suggest raft association is required for conversion of the normal protease-sensitive isoform (PrP-sen) to the transmissible spongiform encephalopathy-associated protease-resistant isoform (PrP-res) in a cell culture model of infection (2-7). Although cell-free studies using purified PrP molecules have provided new insights into binding and conversion of PrP-sen by PrP-res (reviewed in Ref. 8), few studies have considered the membrane-associated nature of PrP (9 -13) and the influence of this association on PrP-sen/PrP-res interactions (14).Given the complex composition of cellular raft membranes, which contain molecules other than PrP-sen that might influence interactions with PrP-res, investigations into the effect of PrP-sen association with rafts on these interactions would benefit from the use of a defined system that replicated raft membranes in the absence of other raft-associated molecules. One candidate system involves the use of sphingolipid-cholesterolrich raft-like liposomes (SCRLs...

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Section: S]gpisupporting

confidence: 85%

Section: Fig 8 Peg and Pi-plc Assist Cell-free Conversion Of Scrlasmentioning

confidence: 99%

Effect of Glycosylphosphatidylinositol Anchor-dependent and -independent Prion Protein Association with Model Raft Membranes on Conversion to the Protease-resistant Isoform

Baron¹,

Caughey

2003

Journal of Biological Chemistry

110

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“…Released TNAP without GPI anchor, obtained by PI-PLC treatment, migrated more slowly as indicated by a band of apparent molecular mass of ϳ130 kDa (Fig. 8, lane 2), in accordance with the observed behavior of the mammalian alkaline phosphatase lacking its GPI anchor (57). 80 -95% TNAP were usually released from MVs after the PI-PLC treatment as determined by the PME activity at pH 10.4 (TABLE ONE), consistent with a previous report (52).…”

Section: Characteristic Of Atp-induced Retardation Of Mineralsupporting

confidence: 58%

Phosphodiesterase Activity of Alkaline Phosphatase in ATP-initiated Ca2+ and Phosphate Deposition in Isolated Chicken Matrix Vesicles

Zhang

Balcerzak

Radisson

et al. 2005

Journal of Biological Chemistry

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Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-dayold chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 M ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.

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“…Removal of detergent from purified PrP-res does not improve infection efficiency. The initial experiments described above were performed without removing the sulfobetaine from the purified PrP-res due to the potentially beneficial role of detergents in GPI painting (2,41,56). To address the potential inhibitory effects of this detergent, infection experiments were performed using purified PrP-res that had been prewashed with PBS to remove the detergent.…”

mentioning

confidence: 99%

Mouse-Adapted Scrapie Infection of SN56 Cells: Greater Efficiency with Microsome-Associated versus Purified PrP-res

Baron¹,

Magalhães

Prado

et al. 2006

J Virol

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The process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.

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Detergent‐Mediated Reconstitution of a Glycosyl‐Phosphatidylinositol‐Protein into Liposomes

Cited by 54 publications

References 31 publications

Effect of Glycosylphosphatidylinositol Anchor-dependent and -independent Prion Protein Association with Model Raft Membranes on Conversion to the Protease-resistant Isoform

Effect of Glycosylphosphatidylinositol Anchor-dependent and -independent Prion Protein Association with Model Raft Membranes on Conversion to the Protease-resistant Isoform

Phosphodiesterase Activity of Alkaline Phosphatase in ATP-initiated Ca2+ and Phosphate Deposition in Isolated Chicken Matrix Vesicles

Mouse-Adapted Scrapie Infection of SN56 Cells: Greater Efficiency with Microsome-Associated versus Purified PrP-res

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