Native Peptide Inhibition

Abstract

The binding of low molecular weight type II phospholipase A 2 (EC 3.1.1.4) to membrane surfaces and hydrolysis of phospholipid are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. The floor and right side of the channel are provided by hydrophobic residues 2, 5, and 9 of an amphipathic amino-terminal helix. The channel is postulated to form via a conformational change in this helix and inward movement of a hydrophobic flap (residue 69 side chain). We show that the amino-terminal tryptic peptide of human type II phospholipase A 2 forms a noncovalent complex with the tryptic peptide from residues 70 -74 of the enzyme. Further, the 70 -74-peptide sequence (FLSYK) dose-dependently inhibits phospholipid hydrolysis in a mixed micelle assay. This native peptide inhibition also occurred with type II enzymes from Crotalus durissus and Crotalus atrox, which have different amino acid sequences at the amino terminus as well as different 70 -74 regions of the molecules. Despite significant conservation of tertiary structure among the enzymes, inhibition by each peptide is specific to the enzyme from which the peptide sequence is derived. We propose that these native peptides inhibit enzyme activity via a sequencespecific, noncovalent interaction with the amino-terminal residues of the enzyme, thereby preventing the conformational change on binding to the micelle interface. These experiments demonstrate a new method for specific inhibition of phospholipase A 2 which, in principle, would be applicable to other biologically active polypeptides and proteins.Secretory phospholipases A 2 (PLA 2 ) 1 are a family of calciumdependent 14-kDa enzymes that catalyze the hydrolysis of the S n 2 fatty acyl ester bond of phospholipids (1). These enzymes, which were first described as components of snake venoms and later in mammals, are classified into two major classes, type I and type II, based on their primary structures. Type I PLA 2 of mammalian origin is mainly found in the pancreas (2), while the type II enzyme is stored in secretory granules in blood platelets, macrophages, and neutrophils (3, 4) and in tissues is localized in mast cells, Paneth cells, and chondrocytes (5, 6). It is also found in fluids derived from patients with inflammatory conditions (7,8) and is induced in several cell types in response to inflammatory stimuli (5). Despite differences in their primary sequences (ϳ30% homology only), crystal structures of PLA 2 from bovine pancreas (type I) and human synovial fluid (type II) are almost superimposable and, not surprisingly, the active sites from both types of enzymes are virtually identical (9). Asp-99 and His-48 form an essential catalytic dyad in the fashion of serine proteases (10). Tyr-52 and Tyr-73 appear to be associated with these two residues via a hydrogen bonding network, but there is evidence (11) that Tyr-52 is not essential for the catalytic reaction.From structure-function studies of type I and II enzymes, the first eight residu...

A Novel Approach to the Design of Inhibitors of Human Secreted Phospholipase A2 Based on Native Peptide Inhibition

“…Materials-sPLA 2 -IIA was purified from conditioned medium from a Chinese hamster ovary cell line (5A2) stably transfected with human sPLA 2 -IIA cDNA (30). Cells were grown in 10-liter fermentation chambers for 72 h, conditioned medium was harvested, and cell pellets were extracted with 1.0 M NaCl containing 10 mM Tris-HCl, pH 8.0.…”

“…The enzyme was quantified by a specific enzyme-linked immunosorbent assay as described previously (31) and filter-sterilized through Acrodisc 0.2 m syringe filters (Gelman Sciences, Ann Arbor, MI). Recombinant sPLA 2 -IIA was visualized as a single 14-kDa band on silver-stained 4 -20% gradient Tris-glycine polyacrylamide gels (Novex Novel Experimental Technology, San Diego; Silver Stain Kit, Bio-Rad) and its identity confirmed by NH 2 -terminal amino acid sequence analysis (30). The preparations contained less than 0.1 ng of endotoxin/g sPLA 2 -IIA protein in the assay (Limulus Amebocyte Lysate Pyrochrome Associates of Cape Cod, Inc., Falmouth, MA).…”

“…Surface residues surrounding the active site of sPLA 2 -IIA, including exposed residues of the NH 2 -terminal helix, are important for binding of the enzyme to aggregated lipid surfaces prior to lipid hydrolysis (26). We have previously described enzyme-specific-inhibitory pentapeptides with amino acid sequences corresponding to residues 70 -74 of the sPLA 2 native protein (30). The peptide 70 FLSYK 74 of human sPLA 2 -IIA was originally identified as a result of copurification with the NH 2 -terminal peptide 1 NLVNFHR 7 on HPLC analysis following tryptic digestion of human sPLA 2 -IIA.…”

“…Because the N-terminal helix of sPLA 2 is known to be important for enzyme activity, we postulated and subsequently showed that exogenous addition of the 70 -74 pentapeptide sequence inhibits enzyme activity. We propose that unlike active site inhibitors, these peptides inhibit by binding to the NH 2 -terminal helix of sPLA 2 -IIA, thereby interfering with a conformational change that occurs in the NH 2 -terminal helix of the enzyme during interfacial binding to the substrate (30).…”

Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera

“…Additionally, heparin and heparan sulphate [16,17], lipocortin [18][19][20], lung surfactant protein A [21] and, uniquely, peptides of PLA # enzymes themselves [22] have been shown to inhibit PLA # enzymic activity.…”