Effects of the propeptide of group X secreted phospholipase A2 on substrate specificity and interfacial activity on phospholipid monolayers (original) (raw)
Related papers
Biophysical Journal, 2001
Phospholipase A 2 (PLA 2) hydrolyzes phospholipids to free fatty acids and lysolipids and thus initiates the biosynthesis of eicosanoids and platelet-activating factor, potent mediators of inflammation, allergy, apoptosis, and tumorigenesis. The relative contributions of the physical properties of membranes and the structural changes in PLA 2 to the interfacial activation of PLA 2 , that is, a strong increase in the lipolytic activity upon binding to the surface of phospholipid membranes or micelles, are not well understood. The present results demonstrate that both binding of PLA 2 to phospholipid bilayers and its activity are facilitated by membrane surface electrostatics. Higher PLA 2 activity toward negatively charged membranes is shown to result from stronger membrane-enzyme electrostatic interactions rather than selective hydrolysis of the acidic lipid. Phospholipid hydrolysis by PLA 2 is followed by preferential removal of the liberated lysolipid and accumulation of the fatty acid in the membrane that may predominantly modulate PLA 2 activity by affecting membrane electrostatics and/or morphology. The previously described induction of a flexible helical structure in PLA 2 during interfacial activation was more pronounced at higher negative charge densities of membranes. These findings identify a reciprocal relationship between the membrane surface properties, strength of membrane binding of PLA 2 , membrane-induced structural changes in PLA 2 , and the enzyme activation.
Journal of Biological Chemistry, 2002
Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.
Mapping the Interfacial Binding Surface of Human Secretory Group IIa Phospholipase A2
Biochemistry, 1997
Human secretory group IIa phospholipase A 2 (hIIa-PLA 2) contains a large number of prominent cationic patches on its molecular surface and has exceptionally high affinity for anionic surfaces, including anionic membranes. To identify the cationic amino acid residues that support binding of hIIa-PLA 2 to anionic membranes, we have performed extensive site-directed mutagenesis of this protein and measured vesicle binding and interfacial kinetic properties of the mutants using polymerized liposomes and nonpolymerized anionic vesicles. Unlike other secretory PLA 2 s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA 2 is driven in part by electrostatic interactions involving a number of cationic residues forming patches on the putative interfacial binding surface. Among these residues, the amino-terminal patch composed of Arg-7, Lys-10, and Lys-16 makes the most significant contribution to interfacial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For these mutants, complete vesicle binding occurs in the presence of high vesicle concentrations, and under these conditions the mutants display specific activities comparable to that of wild-type enzyme. These studies indicate that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial binding of hIIa-PLA 2 to anionic vesicles and that cationic residues closest to the opening of the active-site slot make the most important interactions with the membrane. However, because the wild type binds extremely tightly to anionic vesicles, it was not possible to exactly determine what fraction of the total interfacial binding energy is due to electrostatics.
Surface pressure-dependent cross-modulation of sphingomyelinase and phospholipase A2 in monolayers
Lipids, 1998
We investigated the ways in which phospholipase A2 and sphingomyelinase are mutually modulated at lipid interfaces. The activity of one enzyme is affected by its own reaction products and by substrates and products of the other enzyme; all this depends differently on the lateral surface pressure. Ceramide inhibits both the sphingomyelinase activity rate and the extent of degradation, and decreases the lag time at all surface pressures. Dilauroyl- and dipalmitoylphosphatidylcholine, the substrates of phospholipase A2 (PLA2), do not affect sphingomyelinase activity. The products of PLA2, palmitic acid and lysopalmitoylphosphatidylcholine, strongly enhance and shift to high surface pressures the activity optimum and the cutoff point of sphingomyelinase. Palmitic acid also shifts to high surface pressures the cut-off point of PLA2 activity. Sphingomyelin strongly inhibits PLA2 at surface pressures above 5 mN/m, while ceramide shifts the cut-off point and the activity optimum to high surface pressures. The sphingolipids increase the lag time of PLA2 at low surface pressures. Both phosphohydrolytic pathways involve different levels of control on precatalytic steps and on the rate of activity that appear independent on specific alterations of molecular packing and surface potential. The mutual lipid-mediated interfacial modulation between both phosphohydrolytic pathways indicates that phospholipid degradation may be self-amplified or dampened depending on subtle changes of surface pressure and composition.
Journal of Molecular Biology, 1997
Activation of phospholipase A 2 (PLA 2 ) upon binding to phospholipid assemblies is poorly understood. X-ray crystallography revealed little structural change in the enzyme upon binding of monomeric substrate analogs, whereas small conformational changes in PLA 2 complexed with substrate micelles and an inhibitor were found by NMR. The structure of PLA 2 bound to phospholipid bilayers is not known. Here we uncover by FTIR spectroscopy a splitting in the a-helical region of the amide I absorbance band of PLA 2 upon binding to lipid bilayers. We provide evidence that a higher frequency component, which is only observed in the membrane-bound enzyme, is a property of more¯exible helices. Formation of exible helices upon interaction with the membrane is likely to contribute to PLA 2 activation.
Biochemistry, 2007
They are regulated by diverse factors, including the membrane charge, fluidity, mode of membrane binding (insertion, orientation), and allosteric conformational effects. Relative contributions of these factors to the complex kinetics of PLA 2 activation are not well understood. Here we examine the effects of thermal phase transitions and the surface charge of phospholipid membranes on the activation of human pancreatic PLA 2. The temperature dependence of the initial catalytic rate of PLA 2 peaks around the lipid phase transition temperature (T m) when T m is not too far from physiological temperatures (30-40°C), and the peak is higher in the presence of anionic membranes. High PLA 2 activity can be induced by thermal perturbations of the membrane. Temperature-dependent fluorescence quenching experiments show that despite dramatic effects of the lipid phase transition on PLA 2 activity, the membrane insertion depth of PLA 2 increases only modestly above T m. The data show that membrane structural disorder, and not the depth of membrane insertion, plays a major role in PLA 2 activity.
Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers
The Biochemical journal, 1989
The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
Surface pressure Isochoric method Bothrops diporus sPLA 2 Bothrops asper myotoxin We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Π opt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Π opt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA 2 s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA 2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA 2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Π opt , validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN•m −1 and 10 mN•m −1 , termed here as LR 20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA 2 s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A 2 .
Interaction of phospholipase A2 and phospholipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
Binding of phospholipase A 2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PCt4) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC 14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of diaikyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of the pig-pancreatic enzyme to vesicles of the diether-PC t4 could not be detected even after long incubation (up to 24 h) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.