Expression of porcine pancreatic phospholipase A<sub>2</sub>. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from<i>Escherichia coli</i>

Geus, Pieter de; Bergh, C.J. van den; Kuipers, Oscar P.; Verheij, Hubertus M.; Hoekstra, W. P. M.; Haas, G.H. de

doi:10.1093/nar/15.9.3743

Cited by 63 publications

(34 citation statements)

References 38 publications

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“…Thus, the pancreatic enzymes are deslgned by nature to become activated by trypsin in the intestine. Our previously developed expression systems of PP-PLA2 as fusion protein in bakers yeast or in E. coli De Geus et al, 1987) has shown that activation peptides as long as 400 amino acids are efficiently removed by trypsin, indicating that also in these constructs the cleavable arginine bond is readily accessible to trypsin. In the present study we have shown that the same approach, refolding of a fusion protein followed by cleavage at an engineered tryptic site, was ineffective for HP-PLA2.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a mutant human platelet phospholipase A₂ expressed in Escherichia coli

Franken

Berg

Huang

et al. 1992

European Journal of Biochemistry

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Both methionine residues in phospholipase A, (€'LA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity.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-PLA,) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA, sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA, 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 > phosphatidylethanolamine $ 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-PLA,, porcine pancreatic PLA2 and PLA2 from Nuju melanoleuca venom to lipidlwater 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 < phosphoethanolamine < phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-l -phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA, active site.Phospholipase A, (PLA,) activity is found in almost every mammalian cell (Van den Bosch, 1980). In concert with other esterases and acyl transferases, PLA2 plays a role in the maintenance of the phospholipid composition of cellular membranes. Furthermore, since PLA2 hydrolyses the sn-2 ester bond, its activity gives rise to the production of arachidonic acid and lysophospholipids which are precursors of biologically active lipids such as prostaglandins, leukotrienes,

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of a mutant human platelet phospholipase A₂ expressed in Escherichia coli

Franken

Berg

Huang

et al. 1992

European Journal of Biochemistry

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“…Refolding was initiated by removal of denaturant in the presence of free cysteine to remove proteinbound sulfonates. This method was used previously by us and others to generate native sPLA 2 s (43,44,(55)(56)(57)(58), but the yields are low (typically less than a few percent) because of protein precipitation in refolding buffer. By evaluating two different denaturant removal procedures (slow dialysis or rapid dilution) and systematically exploring the use of protein-solubilizing agents including water-miscible organic solvents and nonionic detergents (59), we have been able to improve dramatically the refolding yields for most of the mouse and human sPLA 2 s.…”

Section: Production Of Recombinant Mouse and Human Spla 2 S-mentioning

confidence: 99%

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

Singer¹,

Ghomashchi²,

Calvez³

et al. 2002

Journal of Biological Chemistry

314

506

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Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A 2 (sPLA 2 s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA 2 s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 M range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA 2 s, with Me-Indoxam being the most generally potent sPLA 2 inhibitor. A dramatic correlation exists between the ability of the sPLA 2 s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA 2 s are uniquely efficient in this regard.

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“…The mutant protein was obtained by tryptic cleavage of reoxidized fusion protein (De Geus et al, 1987) and purified by CM-cellulose chromatography at pH 4.8 and subsequently at pH 6.0. Final purification to homogeneity was achieved on DEAE-cellulose at pH 8.0 and was checked on FPLC.…”

Section: Expression and Purificationmentioning

confidence: 99%

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

Lugtigheid

Otten-Kuipers

Verheij

et al. 1993

European Journal of Biochemistry

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The X-ray structure of a mutant porcine pancreatic phospholipase A, inhibitor complex [Thunnissen et al. (1990) Nature 347, 689-6911 has been determined. This structure shows several interactions between the sn-2-acyl chain and the phosphate moiety of the inhibitor at sn-3 and the protein. The interactions of the remaining part of the polar head group are less clear. Because Arg53 is in close proximity to the head group, we tested the importance of charge at position 53 on enzymatic activity and specificity. Arg53 has been replaced by a glutamine and a glutamic acid in mutants R53Q and R53E, respectively. The effects of the mutations were tested with both zwitterionic and anionic substrates. With monomeric, zwitterionic, (R,S)-1,2-dihexanoyldithiopropyl-3-phosphocholine as substrate, the mutants R53Q and R53E display twofold and sevenfold, respectively, increased kJKm values, composed of increased k,, and decreased K , values. Tested on micelles of zwitterionic (R)-l,2-dioctanoylglycero-3-phosphocholine the mutants R53Q and R53E are more active than the native enzyme, whereas these mutations have an opposite effect on the activity on anionic (R)-l,2-dioctanoylglycero-3-phosphoglycol. Thus, whereas the native enzyme is 0.3 times as active on zwitterionic as on the anionic substrate, these ratios are 1.0 (R53Q) and 1.7 (R53E) for the mutants. No changes in activity were observed with the anionic substrate (R)-1,2-dioctanoylglycero-3-sulfate. Binding studies with substrate-derived inhibitors confirmed the increased affinity for zwitterionic phospholipids and the reduced affinity for anionic phospholipids. The kinetic and binding data indicate the involvement of the charge of residue 53 in head-group specificity and suggest a position of residue 53 closer to the choline or glycol than to the phosphate.The lipolytic enzyme phospholipase A, (PLA,) hydrolyzes the sn-2 ester bond of phosphoglycerides in a calcium-dependent and stereospecific reaction. Phospholipases occur both extracellularly and intracellularly. The extracellular enzymes are found abundantly in mammalian pancreas and in snake or bee venoms serving a digestive function. In addition the venom phospholipases show a wide range of pharmacological actions. The intracellular phospholipases are present in low concentrations in almost every mammalian cell and have an important role in inflammation processes by Abbreviations. PLA,, phospholipase A,; R53Q and R53E, mutants of PLA, with Arg53 changed to glutamine and glutamic acid, respectively ; Oco,GroSO,H, (R)-l,2-dioctanoylglycero-3-sulfate ; Oco,GroPGlo, (R)-l,2-dioctanoylglycero-3-phosphoglycol; Oc0,GroPCho, (R)-l,2-dioctanoylglycero-3-phosphocholine; Lau,GroPCho, (R)-l,2-didodecanoylglycero-3-phosphocholine; HxoS,GroPCho, (R,S')-1,2dihexanoyldithiopropyl-3-phosphocholine; DodPCho, n-dodecylphosphocholine; HxdPCho, n-hexadecylphosphocholine; MyrAholPCho, (R)-2-tetradecanoylaminohexanol-l-phosphocholine ; MyrAholPGlo, (R)-2-tetradecanoylaminohexanol-1 -phosphoglycol; KL, two-dimensional Michaelis Menten const...

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Expression of porcine pancreatic phospholipase A₂. Generation of active enzyme by sequence-specific cleavage of a hybrid protein fromEscherichia coli

Cited by 63 publications

References 38 publications

Purification and characterization of a mutant human platelet phospholipase A₂ expressed in Escherichia coli

Purification and characterization of a mutant human platelet phospholipase A₂ expressed in Escherichia coli

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂

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Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein fromEscherichia coli

Cited by 63 publications

References 38 publications

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A2

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Expression of porcine pancreatic phospholipase A₂. Generation of active enzyme by sequence-specific cleavage of a hybrid protein fromEscherichia coli

Purification and characterization of a mutant human platelet phospholipase A₂ expressed in Escherichia coli

Purification and characterization of a mutant human platelet phospholipase A₂ expressed in Escherichia coli

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A₂