Inhibition by Gangliosides of Bacillus cereus Phospholipase C Activity Against Monolayers, Micelles and Bilayer Vesicles

Abstract

The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.Keywords: Bacillus cereus; phospholipase C ; gangliosides ; interfacial catalysis.Phospholipase C (PLC) from Bacillus cereus has been shown to catalytically induce unilamellar vesicle aggregation, lipid bilayer merging and fusion [l, 21. The molecular mechanism appears to involve isotropic lipid structures during the fusion process 131. It is also known that complex glycosphingolipids such as gangliosides have inhibitory effects on PLC and phospholipase AZ [4-71. In addition, it has been reported that gangliosides markedly inhibit bilayer-bilayer interaction and fusion promoted by basic proteins [S] or by PLC phosphohydrolase activity [2]. These effects are probably mediated by the modulatory properties of low proportions of gangliosides in the formation of non-bilayer phases [7].It is therefore of interest to investigate the effects of gangliosides on the enzymatic activity of B. cereus PLC with the aim of explaining previous findings by which complex gangliosides arrest the fusion triggered by this enzyme [2]. In this work we have studied the action of glycosphingolipids of different complexity on the activity of B. cereus PLC against mixed monolayers, micelles and small unilamellar vesicles. MATERIALS AND METHODSGlycosphingolipids were purified from bovine brain as described previously [4]. Egg PtdCho, Lau,GroPCho and Pam2- Abbreviations. PLC, phospholipase C ; Dec,GroPCho, didecanoylglycerophosphocholine; Lau,GroPCho, dilauroylglycerophosphocholine; Pam2GroPCho, dipalmitoylglycerophosphocholine; PyDec,GroPcho, 1,2-bis( 1 -pyrenedecanoyl)-sn-glycero-3-phosphocholine ; Cer, N-acylsphingoid; G,,, Gal~1-3Gal-NAc~l-4Ga1(3~2uNeuAc)~1-+4Glc~l -+Cer ; G,,,,, NeuAca2-3Gal~l-3Gal-NAc/J'l-+4Ga1(3~2aNeu-Ac)~1-4Glc~l-Cer; G., I h , NeuAcu2-+3Gal~l~3Gal-Nac~1-4Gal-(3+2aNeuAc8+-2aNeuAC)/J'1-4Glc/J'l+Cer; G,,,,, Galpl+3GalNAc-~l+4Ga1(3~2nNeuAc8+2aNeuAc)~1-4Glc~l+Cer; G,,,, mixture of total brain gangliosides (GM, 21 8; G,,,, 42%; G,,,, 18%; Gr,, 19%); GSLs, glycosphingolipids; Sulfatide, Gal(3+sulfate)/ll-l Cer.Enzymes. Phospholipase C (EC 3.1.4.3); phospholipase A? (EC 3.1.1.4); lipase (EC 3.1.1.3).GroPCho were from Avanti Polar Lipids (Birmingham AL). PyDec,GroPCho and 1 -pyrene-decanoic acid were from Molecular Probes (Eugene...

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

“…Gangliosides modulate the enzymatic activity of pancreatic phospholipase A 2 without acting directly on the active center or the interfacial recognition region of the enzyme (55,60). In addition, gangliosides also inhibit the fusion of large unilamellar liposomes induced by the B. cereus PLC (61,62), which lack the C2-like domain present in Cp-PLC. This inhibition seems to occur at the level of the lipid-water interface, affecting both the maximum rate of catalysis of the enzyme and the availability of the substrate (61,62).…”

“…In addition, gangliosides also inhibit the fusion of large unilamellar liposomes induced by the B. cereus PLC (61,62), which lack the C2-like domain present in Cp-PLC. This inhibition seems to occur at the level of the lipid-water interface, affecting both the maximum rate of catalysis of the enzyme and the availability of the substrate (61,62). It is not known whether Cp-PLC could induce a similar effect in that type of liposomes, but it is clear that its cytotoxic effect is not associated with fusion of the cellular membranes of the target cells and syncytia formation (15).…”

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

“…Several details regarding the kinetics, morphology, and mechanisms of formation of segregated domains by some phospholipases, including sphingomyelinase, have been elucidated using different model membrane systems and mixtures of lipids. The dependence of the activities of phospholipase A 2 (PLA 2 ), PLC, and sphingomyelinase on the lipid physical state, showing that these enzymes preferably degrade the more fl uid phase state, was demonstrated in early studies using lipid monolayers and bilayer vesicles (27)(28)(29)(30)(31)(32). Our previous studies of PLC-promoted membrane fusion correlated enzyme activity and the generation of nonlamellar structures that would in turn be fusion intermediates ( 33 ).…”

“…Control vesicles, i.e., those containing PC:SM:PE:Chol (in equimolar proportions) become stained at a somewhat higher rate than those containing DAG and/or Cer in addition. Note that phase characterization in an identical system ( 22 ) and by the various important early ( 30,32,57 ) and contemporary ( 42,49,(58)(59)(60) studies of lipases and lipid domains that have been carried out using natural lipids. GUV are examined individually in most cases, without contact with other vesicles and under reduced mobility conditions; thus, vesicle aggregation or fusion cannot be observed under those conditions.…”

“…The present contribution is intended to provide a link between the bulk (in cuvette) studies of lipase activity on lipid bilayers in different physical states (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44) and the morphological data obtained largely with GUV of homogenous lipid compositions (45)(46)(47)(48)(49). This work is based on our previous studies of phase separation in GUVs ( 23 ) as well as our experience with PlcHR 2 ( 8,50 ).…”