Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2

Abstract

BackgroundSecretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds.ResultsChicken intestinal group IIA phospholipase A2 (ChPLA2-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didn't hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl2, a specific activity of 160 U.mg-1 for purified ChPLA2-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA2-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A2. The gene encoding the mature ChPLA2-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA2-IIA was built using the human intestinal phospholipase A2 structure as template.ConclusionChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA2-IIA.

The Finding of a Group IIE Phospholipase A2 Gene in a Specified Segment of Protobothrops flavoviridis Genome and Its Possible Evolutionary Relationship to Group IIA Phospholipase A2 Genes

“…Abbreviations: Bt , Bos taurus ; Cf , Canis lupus familiaris ; Gg , Gallus gallus ; Mc , Macaca mulatta ; Oa , Ornithorhynchus anatinus ; Oc , Oryctolagus cuniculus ; and Pn, Pan troglodytes . References: Bt IIEPLA 2 (NP_001179015) [ 33 ], Cf IIEPLA 2 (XP_544525) (automated computational prediction by GNOMON); Dt Dis-2 (AFH66959) [ 17 ]; Gg IBPLA 2 (NP_001138961) [ 34 ]; Gg IIEPLA 2 (NP_001171878) [ 35 ]; Hs IBPLA 2 (NP_000919) [ 36 ]; Hs IIAPLA 2 (NP_001155199) [ 37 ]; Hs IICPLA 2 (NP_001099042) [ 38 ]; Hs IIDPLA 2 (NP_036532) [ 39 ]; Hs IIFPLA 2 (NP_073730) [ 40 ]; Hs VPLA 2 (NP_000920) [ 38 ]; Lm Lei-2 (AFH66961) [ 17 ]; Lm Lei-3 (AFH66962) [ 17 ]; Mc IIEPLA 2 (XP_001094364) (automated computational prediction by GNOMON); Mm IBPLA 2 (NP_035237) [ 41 ]; Mm IIAPLA 2 (NP_001076000) [ 42 ]; Mm IICPLA 2 (NP_032894) [ 41 ]; Mm IIDPLA 2 (NP_035239) [ 39 ]; Mm IIFPLA 2 (NP_036175) [ 20 ]; Mm VPLA 2 (NP_001116426) [ 41 ]; Oa IIEPLA 2 (XP_001505559) (automated computational prediction by GNOMON); Oc IIEPLA 2 (XP_002716050) (automated computational prediction by GNOMON); Oo IIEPLA 2 (this work); Pe IIEPLA 2 (this work); Pf IIEPLA 2 (this work); Pn IIEPLA 2 (XP_001163677) (automated computational prediction by GNOMON); and Pt IIEPLA 2 (this work).…”

DEPTOR at the Nexus of Cancer, Metabolism, and Immunity

“…It is interesting to point out that DEPTOR is not the only mTOR inhibitor whose expression is induced by GCs. The expression of regulated in DNA damage and development 1 (REDD1), a protein that inhibits mTORC1 (161) (FIGURE 2), is also induced in response to GCs (129,232,270,283). These results show that GCs act on several molecules to negatively regulate mTOR signaling.…”

Drastic changes in the tissue-specific expression of secreted phospholipases A2 in chicken pulmonary disease

“…Karray et al have biochemically characterized an active sPLA2-IB from chicken pancreas (ChPLA2-IB) [12]. Another biochemical and structural study of avian non-pancreatic PLA2 was performed from chicken intestine (ChPLA2-IIA) [13]. We also demonstrated in a recent study that pure ChPLA2-IIA, but not ChPLA2-IB, displays potent bactericidal activity against grampositive bacteria but lacks bactericidal activity against gram negative ones [14].…”

“…Group IB and group IIA sPLA2 were found to be the most abundant sPLA2, at least in some chicken tissues including pancreas and small intestine (43.15 and 9.45 relative expression level, respectively, in healthy chickens). This is probably the reason why ChPLA2-IB and ChPLA2-IIA could be purified and characterized from pancreas and intestine respectively [12,13]. A similar distribution of sPLA2-IB and -IIA has been also observed in human and mouse tissues at the mRNA level [26] and at the protein level [27e30].…”