Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature

Vaccaro, Anna Maria; Tatti, Massimo; Ciaffoni, Fiorella; Salvioli, Rosa; Barca, Alessandra; Roncaioli, Paola

doi:10.1016/0005-2736(93)90024-t

Cited by 23 publications

(33 citation statements)

References 29 publications

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“…The pH optimum of glucosylceramidase in the Sap C-PS LUV system is in the range 5.s5.4, slightly lower than that previously found when PS SUV were used as activators (pH 5.8, Ref. [16]). …”

Section: Effect Of Sap C On Glucosylceramidase Activation By Ps Luvmentioning

confidence: 73%

“…PS vesicles were prepared as previously described [16]. In short, small unilamellar vesicles (SUV) were obtained by submitting the lipid suspension to sonication under nitrogen in a Branson B 15 Sonifier, while large unilamellar vesicles (LUV) were prepared by filter exclusion through two stacked 0.1 pm pore size polycarbonate filters (Nucleopore Corp., Pleasanton, CA) using a high pressure extrusion apparatus (Lipex Biomembranes Inc., Vancouver, BC).…”

Section: Vesicle Preparatronmentioning

confidence: 99%

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Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes

et al. 1993

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The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.

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“…The pH optimum of glucosylceramidase in the Sap C-PS LUV system is in the range 5.s5.4, slightly lower than that previously found when PS SUV were used as activators (pH 5.8, Ref. [16]). …”

Section: Effect Of Sap C On Glucosylceramidase Activation By Ps Luvmentioning

confidence: 73%

Section: Vesicle Preparatronmentioning

confidence: 99%

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes

et al. 1993

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“…Vesicle Preparation-LUV were prepared by filter exclusion using a high pressure extrusion apparatus (Lipex Biomembranes, Vancouver, Canada) as described previously (21,30). In short, the dry lipids were dispersed by Vortex mixing in 2 mM L-histidine, 2 mM TES, 150 mM NaCl, 1 mM EDTA, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

“…Liposomal Glucosylceramide-We have previously shown that PS LUV, as opposed to PS SUV, are unable to fully stimulate glucosylceramidase activity toward the artificial substrate MUGlc (21). Here, we have investigated whether the liposome size has a similar effect on the enzyme activity toward glucosylceramide, the physiological enzyme substrate, inserted in vesicles containing, besides glucosylceramide (5%), cholesterol, PC, and an acidic phospholipid such as PS.…”

Section: Influence Of Liposome Size On the Enzymatic Hydrolysis Ofmentioning

confidence: 99%

“…In vitro, the Sap C-induced stimulation of glucosylceramidase requires the presence of a negatively charged phospholipid such as phosphatidylserine (PS) (5,19). In earlier studies, we provided evidence that Sap C was able to favor the association of glucosylceramidase with PS membranes and that the physical characteristics of PS vesicles markedly influenced their interaction with glucosylceramidase (20,21).…”

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Effect of Saposins A and C on the Enzymatic Hydrolysis of Liposomal Glucosylceramide

Vaccaro

Tatti

Ciaffoni

et al. 1997

Journal of Biological Chemistry

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The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV). The separate addition of either Sap A or Sap C to the LUV system partially stimulated the sphingolipid degradation while saposins B and D had no effect. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV.The characteristics of glucosylceramidase activation by Sap C were different from those of Sap A. Sap C increased the rate of hydrolysis of both the artificial water soluble substrate, 4-methylumbelliferyl-␤-D-glucopyranoside, and the lipid substrate, glucosylceramide, while Sap A only stimulated degradation of the sphingolipid. Also the binding properties of Saps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidase with the membrane. In contrast, Sap A had poor affinity for the membrane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase binding.In conclusion, our results show that both Sap A and Sap C are required for maximal hydrolysis of glucosylceramide inserted in PS-containing LUV, that their effects are synergistic, and that their mode of action is different. Sap C is responsible for the membrane binding of glucosylceramidase, while Sap A stimulation is possibly related to its effect on the conformation of the enzyme. It can be envisaged that Sap A in conjunction with Sap C might have a physiological role in glucosylceramide degradation.

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Proteomics study on the protective mechanism of soybean isoflavone against inflammation injury of bovine mammary epithelial cells induced by Streptococcus agalactiae

Niu

Zhang

et al. 2021

Cell Stress and Chaperones

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This study aimed to verify the anti-inflammatory effect of soybean isoflavones (SI) on the inflammatory response induced by Streptococcus agalactiae (S. agalactiae) of bovine mammary epithelial cells (bMECs) and to elucidate its possible mechanism. BMECs were pretreated with SI of different concentrations (20, 40, 60, 80, 100 μg/mL) for 0.5, 3, 6, 9, 12, 15, 18, 24 h. And then, S. agalactiae was used to infect bMECs for 6 h (MOI = 50:1) to establish the inflammation model. Cell viability, growth curves of S. agalactiae, cytotoxicity, and S. agalactiae invasion rate were determined. A proteomics technique was used to further detect differential proteins and enrichment pathways. SI (40 μg/mL) improved the viability of bMECs at 12 h (p < 0.05) and 60 and 80 μg/mL of SI greater (p < 0.01). Moreover, 60 μg/mL of SI protects cells from bacterial damage (p < 0.05). SI could inhibit S. agalactiae growth and internalization into bMECs in a time-and dose-dependent manner. In addition, proteomics results showed that 133 proteins were up-regulated and 89 proteins were down-regulated significantly. The differentially significantly expressed proteins (DSEPs) were mainly related to cell proliferation, differentiation, apoptosis, and migration. GO annotation showed that 222 DSEPs were divided into 23 biological processes (BP) terms, 14 cell components (CC) terms, and 12 molecular functions (MF) terms. DSEPs were significantly enriched in 10 pathways, of which the immune pathway was the main enrichment pathway.

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Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature

Cited by 23 publications

References 29 publications

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes

Effect of Saposins A and C on the Enzymatic Hydrolysis of Liposomal Glucosylceramide

Proteomics study on the protective mechanism of soybean isoflavone against inflammation injury of bovine mammary epithelial cells induced by Streptococcus agalactiae

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