Cloning and Recombinant Expression of a Structurally Novel Human Secreted Phospholipase A2

Abstract

Mammals contain a diverse set of secreted phospholipases A 2 (sPLA 2 s) that liberate arachidonic acid from phospholipids for the production of eicosanoids and exert a variety of physiological and pathological effects. We report the cloning, recombinant expression, and kinetic properties of a novel human sPLA 2 that defines a new structural class of sPLA 2 s called group XII. The human group XII (hGXII) cDNA contains a putative signal peptide of 22 residues followed by a mature protein of 167 amino acids that displays homology to all known sPLA 2 s only over a short stretch of amino acids in the active site region. Northern blot and reverse transcription-polymerase chain reaction analyses show that the tissue distribution of hGXII is distinct from the other human sPLA 2 s with strong expression in heart, skeletal muscle, kidney, and pancreas and weaker expression in brain, liver, small intestine, lung, placenta, ovaries, testis, and prostate. Catalytically active hGXII was produced in Escherichia coli and shown to be Ca 2؉ -dependent despite the fact that it is predicted to have an unusual Ca 2؉ -binding loop. Similar to the previously characterized mouse group IIE sPLA 2 s, the specific activity of hGXII is low in comparison to that of other mammalian sPLA 2 , suggesting that hGXII could have novel functions that are independent of its phospholipase A 2 activity.

Potentiation of Tumor Necrosis Factor α-induced Secreted Phospholipase A2 (sPLA2)-IIA Expression in Mesangial Cells by an Autocrine Loop Involving sPLA2 and Peroxisome Proliferator-activated Receptor α Activation

“…Briefly, assay mixtures (1 ml) contained 100 mM TrisHCl (pH 7.0), 10 mM CaCl 2 , [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleate-labeled E. coli (Ϸ10,000 cpm), and 5 l of cell supernatants, which produces less than 5% of substrate hydrolysis. Reaction mixtures were incubated for 30 min at 37°C in a thermomixer.…”

“…When exogenously added to other cell types, sPLA 2 -IB was also found to activate the expression of a number of pro-inflammatory genes including cyclooxygenase-2 (20,21), sphingomyelinase, and ceramidase (22). On the other hand, exogenously added sPLA 2 -IIA can induce the activation of cPLA 2 and cyclooxygenase-2 (3,23,24), the release of elastase (25) and ␤-glucuronidase (26), and the expression of Mac-1 (27), , CD-69 (28), inducible nitricoxide synthase (29), and Fas ligand (23) on different cell types. The nature of the sPLA 2 -IIA cellular target involved in these biological effects remains, however, to be clearly identified.…”

Inhibitory Effects of Surfactant Protein A on Surfactant Phospholipid Hydrolysis by Secreted Phospholipases A2

“…The sPLA 2 s form a growing family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids, leading to the production of free fatty acids and lysophospholipids that serve as precursors for a variety of lipid-derived mediators involved in numerous biological activities (5,6). To date, 10 different sPLA 2 s, referred to as groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII, have been characterized in mammals (7)(8)(9)(10)(11)(12). Of particular interest, levels of sPLA 2 activity in bronchoalveolar lavage fluids (BALF) of patients with ARDS often correlate positively with the severity of the disease (13).…”

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

“…Generation of Recombinant sPLA 2 Proteins-Recombinant human GIIA, GIIF, GX, and GXII sPLA 2 were produced by refolding the inclusion body protein obtained from expression in E. coli, as described previously (17,26,30,54). The preparation of recombinant human GIB, GIIE, and GV sPLA 2 will be reported elsewhere.…”

“…One study identified GX sPLA 2 mRNA in peripheral blood leukocytes (25), whereas two others did not (17,29). Transcripts for GXII sPLA 2 were identified in multiple tissues, including myocardium, skeletal muscle, kidney, pancreas, and type 2 helper T cells (30,31).…”