Purification, Analysis, and Enzymatic Activity of Recombinant Human Synovial Fluid Phospholipase A2 and N-Terminal Variants

Marco, Stefania Di; Märki, F; Hofstetter, H.; Schmitz, A.; Oostrum, Jan van; Grütter, Markus G.

doi:10.1093/oxfordjournals.jbchem.a123904

Cited by 20 publications

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“…Crystallographic experiments on porcine pancreatic type I PLA 2 showed that a free ␣-amino group is an essential requirement for enzyme activity (15,17,32,33). In type II PLA 2 from C. atrox, strong hydrogen bonding occurs between the NH 2 -terminal ␣-amino group and the side chain of residue 4 with the backbone carbonyls and amides of residues 71 and 73 (25).…”

Section: Discussionmentioning

confidence: 99%

“…Both chemical modification (14) and site-directed mutagenesis (15)(16)(17) studies have shown that the NH 2 -terminal residue is crucial for activity on micellar substrates. Further studies with type I PLA 2 (17,18) have shown that significant alterations of the invariant hydrophobic face of the NH 2 -terminal amphipathic helix (residues 2, 5, or 9) are detrimental to enzyme activity.…”

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confidence: 99%

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Native Peptide Inhibition

Tseng

Inglis

Scott

1996

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Native Peptide Inhibition

Tseng

Inglis

Scott

1996

Journal of Biological Chemistry

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“…The membranes were hybridized and washed as instructed by the manufacturer. The probe used was a 0.45-kb cDNA covering the whole protein coding area of human group II PLA2 cloned into the HindIII-BamHI site of pUC18 plasmid [13]. The insert was labeled with 32 P-dCTP (Amersham, Buckinghamshire, UK) by random oligonucleotide primer extension [14] to a specific activity of approximately 10 9 cpm/g.…”

Section: Northern Hybridizationmentioning

confidence: 99%

Group II phospholipase A2 in human male reproductive organs and genital tumors

et al. 1998

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“…Briefly, a 0.45-kb cDNA sequence covering the protein coding area of group II PLA2 (Di Marco et al 1992) was inserted into the HindIII-BamH1 site of the pGEM-3Z transcription vector (Promega; Madison, WI). RNA transcripts were generated by using the T7 (anti-sense probe) or SP6 (sense probe) polymerase (Boehringer Mannheim; Mannheim, Germany) after linearization of the vector with the appropriate restriction enzymes (HindIII and BamHI).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Expression of Human Group II Phospholipase A₂in Transgenic Mice

Nevalainen

Laine

Grass

1997

J Histochem Cytochem.

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SUMMARY Group II phospholipase A 2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group lI PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group ll PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group ll PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A 2 .

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Purification, Analysis, and Enzymatic Activity of Recombinant Human Synovial Fluid Phospholipase A2 and N-Terminal Variants

Cited by 20 publications

References 0 publications

Native Peptide Inhibition

Native Peptide Inhibition

Group II phospholipase A2 in human male reproductive organs and genital tumors

Expression of Human Group II Phospholipase A₂in Transgenic Mice

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Purification, Analysis, and Enzymatic Activity of Recombinant Human Synovial Fluid Phospholipase A2 and N-Terminal Variants

Cited by 20 publications

References 0 publications

Native Peptide Inhibition

Native Peptide Inhibition

Group II phospholipase A2 in human male reproductive organs and genital tumors

Expression of Human Group II Phospholipase A2in Transgenic Mice

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Product

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Expression of Human Group II Phospholipase A₂in Transgenic Mice