Inhibition of human secretory class II phospholipase A₂ by heparin

Abstract

By means of kinetic analyses using Triton X-100/deoxycholic acid/dilauroylglycerophosphoethanolamine (4 : 2 : 1, molar ratio) mixed micelles we examined the effects of heparin on the activity of several phospholipases A, (PLA,). Heparin avidly bound cationic PLA,s including human secretory class I1 PLA, and thereby inhibited their hydrolysis of phospholipids in the mixed micelles. Initial velocity measurements indicated that heparin behaved as a competitive inhibitor for human secretory class I1 PLA, and closely related A.h. blomhoffii PLA, and A.p. piscivorus PLA,. In particular, heparin showed the highest specificity for human secretory class I1 PLA,. In the absence of deoxycholic acid in mixed micelles, A.h. blomhoffii PLA, was also strongly inhibited by heparin. The observed inhibition was not due to the interaction of heparin with the active site of PLA, because heparin did not inhibit the hydrolysis of monomeric substrates by PLA,s. Both kinetic measurements and fluorescence measurements of PLA,-bound 8-anilino-1-naphthalene sulfonate in the presence of varying amounts of heparin showed that a heparin molecule bound about seven molecules of PLA,. When positive charges of four lysines in the amino-terminal region of A.h. blomhoffii PLA, were neutralized by limited carbamoylation, heparin neither bound the carbamoylated A.h. blomhofii PLA, nor inhibited the hydrolysis of Triton X-l OO/dilauroylglycerophosphocholine mixed micelles by the carbamoylated A.h. blornhoffii PLA, that retained 50% activity of native A.h. blornhofii PLA,. Also, heparin did not inhibit the hydrolysis of mixed micelles by 7,lObis(octanoy1)ated A.p. piscivorus PLA, in which two lysines in the amino-terminal a-helix are acylated. These results indicate that the inhibition of human secretory class I1 PLA, and related cationic PLA,s by heparin originates from the interaction of heparin with cationic residues in the aminoterminal region that forms a part of interfacial binding site. In addition, unique structural features of human secretory class I1 PLA,, together with its unique mode of interaction with heparin, suggest that this PLA, might have an additional heparin-binding site. Although the heparin-PLA, binding diminished as the ionic strength of reaction medium increased, the inhibition of human secretory class T I PLA, by heparin remained significant at the physiological ionic strength. An estimated value of inhibition constant (K,) was 0.1 pM under physiological conditions, which suggests that a normal pharmaceutical dose of heparin might inhibit human secretory class I1 PLA, and regulate its biological effects.Phospholipase A, (PLA,) catalyzes the hydrolysis of the fatty acid ester in the 2-position of 3-sn-phospholipids [ 1, 21. This reaction is generally believed to produce arachidonic acid which is a precursor of eicosanoids [3, 41. In certain inflammatory cells, the activation of PLA, leads to the formation of platelet activating factor [51. Because these lipids are potent mediators of inflammatory reactions, PLA, has been impl...

Mechanism of Human Group V Phospholipase A2(PLA2)-induced Leukotriene Biosynthesis in Human Neutrophils

“…3) suggests the presence of cationic patches on both sides of molecular surface. Membrane and heparin binding measurements indicate that cationic residues on the surface containing Trp 31 , which was shown to be critical for binding to zwitterionic membranes (18), are involved in binding to anionic membranes, whereas those on the opposite face are involved in heparin binding. The effects of the K6E, K11E, and R34E mutations on the energetics of binding to anionic vesicles, which can be estimated using an equation, ⌬⌬G 0 ϭ ϪRT ln[relative affinity] under the standard conditions with the concentration of free phospholipid set at 1 M, are 0.54 kcal/mol or less at 25°C.…”

“…Intuitively, it is more likely that the K6E mutation induces local structural changes of hVPLA 2 , which in turn interfere with the interactions of other critical residues, such as Trp 31 (18), with zwitterionic membranes. Heparin Affinities of hVPLA 2 and Mutants-Binding of hV-PLA 2 with heparin and its derivatives is a complex process that involves multiple interaction sites, which makes it difficult to quantitatively determine the binding affinity (31,32). We previously reported the use of immobilized heparin and heparan sulfate columns to semiquantitatively assess the binding affinities of hIIaPLA 2 and mutants for heparinoids (16).…”

Ultrastructural Localization of Secretory Type II Phospholipase A 2 in Atherosclerotic and Nonatherosclerotic Regions of Human Arteries

“…50 One consequence of its net positive charges is that snpPLA 2 binds to sulfated GAGs. 33,51 For its activity, snpPLA 2 requires millimolar concentrations of calcium. Some of these characteristics indicate that snpPLA 2 can be active extracellularly.…”

Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule

“…Diccianni et al [23] have shown that a peptide corresponding to 26 residues at the N-terminal end of porcine pancreatic PLA # can rescue PLA # from heparin inhibition. Dua and Cho [24] have also reported that the cationic residues at the N-terminal region of the human sPLA # are responsible for interaction with the heparin. Based on the present data and our earlier studies [14], we propose that the N-terminal basic residues, Arg"!, Lys"#, Lys"& and Arg"', could be involved in heparin binding.…”