The importance of glycine-30 for enzymatic activity of phospholipase A2.

The nearly conserved glycine-30 in porcine pancreatic phospholipase A2 has been replaced by serine. The resulting mutant G30S was expressed in Escherichia coli, purified and characterized. The mutation caused a significant drop in enzymatic activity towards monomeric and aggregated substrates, but had a limited effect on substrate binding. In contrast the affinity for calcium ions, the essential cofactor, was reduced 10-fold. The reduced enzymatic activity is attributed to a reduced stabilization of the transition state. The results are discussed in view of naturally occurring inactive phospholipase A2 homologues from snake venom.

Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2.

The ability of platelet secretory phospholipase A2 (sPLA2) to induce platelet activation was investigated. sPLA2 (group II) contained in an activated platelet supernatant, as well as high concentrations of purified recombinant platelet sPLA2, failed to induce platelet activation. Furthermore, sPLA2 did not modify platelet activation induced by various agonists. The possible relationship between the failure of this enzyme to induce platelet activation and its origin (mammalian) or its structural group (group II) was then investigated, using pancreatic PLA2s (group I) and venom PLA2s from groups I, II and III. All venom PLA2s induced platelet activation that was accompanied by the liberation of arachidonic acid and was abolished by aspirin. In contrast, as observed for platelet sPLA2, enzymes from hog or bovine pancreas were unable to induce platelet activation even when used at high concentrations. Interestingly, PLA2 able to induce platelet activation efficiently hydrolyse phosphatidylcholine, while those inactive on platelets did not. Taken together, these results suggest that the catalytic activity of added PLA2 is necessary but not sufficient to induce platelet activation. Moreover, the ability of PLA2 to induce platelet activation is not related to its structural group (I, II, III) but rather to its origin (venom vs. mammalian) and capacity to hydrolyse phosphatidylcholine, the major phospholipid of the outer leaflet of the plasma membrane.

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae.

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.

Crystal structure of a porcine pancreatic phospholipase A2 mutant. A large conformational change caused by the F63V point mutation.

The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.

An extended binding pocket determines the polar head group specificity of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69-->Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62-66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate.

The anticoagulant effect of the human secretory phospholipase A2 on blood plasma and on a cell-free system is due to a phospholipid-independent mechanism of action involving the inhibition of factor Va.

Blood platelets play a central role in haemostasis by leading to plug formation and by increasing the efficiency of blood coagulation. We have previously shown that blood platelets contain a group II secretory phospholipase A2 (sPLA2 grII) which is released into the extracellular medium upon activation but is unable to stimulate blood platelets. We presently reported an investigation of the putative involvement of the human sPLA2 grII (hsPLA2 grII) in the coagulation process, both in the absence and in the presence of activated platelets. We show that this enzyme prolongs the recalcification time of blood plasma even in the presence of activated platelets. The positive action of blood platelets on coagulation is correlated, at least in part, with the appearance at the cellular surface of procoagulant phospholipids which constitute a potential target for hsPLA2 grII. We therefore investigated the involvement of its enzymatic activity in the anticoagulant effect of this enzyme. We observed that the replacement of CaCl2 by SrCl2 to initiate the coagulation cascade did not suppress, but rather increased, the inhibitory action of hsPLA2 grII. Moreover, hsPLA2 grII hydrolyzed only a minor proportion of platelet phospholipids, and it did not affect plasma phospholipids. Taken together, these observations strongly suggest that the major action of hsPLA2 grII on blood coagulation does not involve the hydrolysis of phospholipids, in contrast with the strong anticoagulant effect of the group II venom phospholipase A2 from Crotalus durrissus terrificus. We next studied which step of the coagulation cascade was affected by hsPLA2 grII. Using purified coagulation factors, we demonstrated that hsPLA2 grII strongly inhibited the prothrombinase activity. This inhibitory effect was independent of the presence of phospholipids but required factor Va, leading to the hypothesis that hsPLA2 grII inhibited this factor. Further, the anticoagulant effect of hsPLA2 grII was observed on normal and factor-X-deficient plasma, but not on factor-V-deficient plasma. In conclusion, the anticoagulant action of hsPLA2 grII is based on a nonenzymatic mechanism of action involving the inhibition of factor Va.

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2.

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide.

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F.

Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A2. They are part of an extended hydrogen bonding system that links the N-terminal alpha-NH3(+)-group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine pancreatic phospholipase A2 mutant, in which residues 62-66 had been deleted (delta 62-66PLA2). The mutations did not affect the catalytic properties of the enzyme, nor the folding kinetics. The stability against denaturation by guanidine hydrochloride was decreased, however. To analyse how the enzyme compensates for the loss of the tyrosine hydroxyl group, the X-ray structures of the delta Y52F and delta Y73F mutants were determined. After crystallographic refinement the final crystallographic R-factors were 18.1% for the delta Y52F mutant (data between 7 and 2.3 A resolution) and 19.1% for the delta Y73F mutant (data between 7 and 2.4 A resolution). No conformational changes occurred in the mutants compared with the delta 62-66PLA2, but an empty cavity formed at the site of the hydroxyl group of the former tyrosine. In both mutants the Asp99 side chain loses one of its hydrogen bonds and this might explain the observed destabilization.