Heparin prevents the binding of phospholipase A2 to phospholipid micelles: importance of the amino-terminus

Abstract

The activity of the major isoform of porcine pancreatic phospholipase A2 (PLA2), designated B-PLA2, against micellar substrates is inhibited by heparin. Inhibition is a consequence of binding of the enzyme to heparin, documented by a heparin-induced alteration in the intrinsic fluorescence of B-PLA2 and in the 8-anilino-1-naphthalene sulfonate fluorescence and by the enhanced rate of chemical modification of the active site residue His-48. As a consequence of heparin binding, the conformation of B-PLA2 at the active site and at the amino-terminus is altered, and the enzyme does not bind to phospholipid micelles. In spite of the heparin-induced conformational changes, B-PLA2 retains its ability to catalyze the hydrolysis of monomeric phospholipid. Other glycosaminoglycans can bind to and inhibit the activity of B-PLA2 toward organized phospholipids, but none tested is as effective as heparin. An isoform of the pancreatic enzyme, designated UB-PLA2 and which corresponds to iso-pig PLA2, does not bind to nor is its catalytic activity influenced by heparin. A peptide corresponding to the amino-terminal 26 residues of B-PLA2 can rescue PLA2 from heparin inhibition. A similar peptide corresponding to the amino-terminus of UB-PLA2 has no effect on heparin inhibition. A model for the inhibition of B-PLA2 by heparin is proposed in which the catalytically significant effect of heparin is to interact directly with the amino-terminus of B-PLA2, the interfacial recognition site, to prevent the enzyme from binding to micellar substrates.

The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme

“…The spectroscopic results indicate that heparins initially bind to the BthTx-I dimer interface region, and this hypothesis is confirmed by the decrease in the ITFE, which supports the results of UVCD experiments and is reinforced by FTIR results, which show a little secondary structure rearrangement in relation to heparin size. However, previous reports showed that heparins with well-known structural variety, can bind to PLA 2 with different binding capacities and affinities [6,[18][19][20][21][22]. Even though these results were consistent with our spectroscopic studies for heparin binding to BthTx-I, it is still clear that both interactions, whether heparin or PLA 2 , share the characteristics of the Coulomb interactions, along with that of the distribution of the electrostatic potential on the surface.…”

“…It is well-recognized that PLA 2 action towards organized lipid interfaces is much higher than against isotropically dispersed phospholipids. The mechanism by which heparin inhibits PLA 2 [6] through a direct interaction with the enzyme is at the interfacial recognition site (IRS) [7], blocking its access to micellar substrates and inducing conformational changes at the enzyme active site and N-terminal and also by binding to a strongly cationic site located in the Cterminal [8]. However, the active site remains functional and can catalyze the hydrolysis of water-soluble phospholipids.…”

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

“…PLA # s have been shown to bind to heparin-like cell-surface molecules [19][20][21][22][23]. EF also binds heparin, and heparin inhibits EF activity by blocking its binding site on the cell membrane [14].…”

“…This suggests that the ability of the mutant EF to bind to the cell surface and bring about an increase in the binding of EGF was not affected. 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.…”

Heparin-antivenom association: differential neutralization effectiveness in Bothrops atrox and Bothrops erythromelas envenoming

“…Among the substances capable of interacting with heparin, are included proteins from the extracellular matrix (fibronectin, laminae, vitronectin), proteins involved in lipid metabolism, components of the complement system, serine protease inhibitors, viral proteins, and enzymes 21,22,33 . Within the enzymes, heparin can interact with phospholipase A 2 , present in many snake venoms, affecting 8,9,12 , or not 7,19 the enzymatic activity of these proteins.…”