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

Abstract

Background: Secretory 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.

Results: Chicken 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.

Conclusion: ChPLA2-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.

Figure 1

Thin layer chromatography of PC and PE before or after incubation with ChPLA₂-IIA or ChPLA₂-IB. After solvent migration the silica plate was exposed to iodine vapor to reveal the various spots. Lane (1), PC. Lane (2), PC incubated with PLA₂ from chicken pancreas (PLA₂-IB) and showing a complete hydrolysis of the PC. Lane (3), PC incubated with PLA₂ from chicken intestine (PLA₂-IIA). Lane (4), PE. Lane (5), PE incubated with PLA₂-IB and showing a complete hydrolysis of the PE. Lane (6), PE incubated with PLA₂-IIA and showing a complete hydrolysis of the PE. Lane (7), free fatty acid C 18:1.

Figure 2

Purification of ChPLA2-IIA. (A) Gel filtration chromatography of intestinal ChPLA₂-IIA on Sephadex G-50. The column (1.5 cm × 34 cm) equilibrated in 20 mM Tris-HCl buffer pH 8.0 containing 20 mM CaCl₂ and 2 mM benzamidine. Elution was performed with the same buffer at a flow rate of 40 ml.h^-1 and 3 ml samples were collected. ChPLA₂-IIA activity was measured as described in Material and methods section using egg yolk emulsion as substrate. The pooled fractions containing the PLA₂ activity were indicated by horizontal line. (B) Mono-S Sepharose chromatography. The column (5 cm × 2 cm) was equilibrated with 20 mM Tris HCl buffer pH 8.0 containing 20 mM CaCl₂ and 2 mM benzamidine; and then washed with the same buffer containing 0.3 M NaCl. Linear salt gradient (0.3 to 1 M NaCl, dotted line) was applied to the column; gradient chamber 75 ml; 2 ml fraction; flow rate, 40 ml/h. The pooled fractions containing the PLA₂ activity were indicated by horizontal line. SDS-PAGE (15%) analysis of pure ChPLA₂-IIA was inserted in Figure 2B. Lane 1, molecular mass markers (MM); Lane 2, 15 μg of proteins obtained after Sephadex G-50 chromatography; Lane 3, 15 μg of purified ChPLA₂-IIA, obtained after Mono-S chromatography.

Figure 3

Effect of Ca²⁺ and NaTDC on ChPLA2-IB and ChPLA2-IIA activities. (A) Effect of Ca²⁺ concentration on ChPLA₂-IIA (black circle) and ChPLA₂-IB (white circle) activities. Enzyme activity was measured at various concentrations of Ca²⁺ using egg yolk emulsion as substrate at pH 9.0 and at 40°C in the presence of 10 mM NaTDC. The star indicates the phospholipase activity measured in the absence of CaCl₂ and in the presence of 10 mM EDTA or EGTA. (B) Effect of increasing concentration of bile salts (NaTDC) on ChPLA₂-IIA (black circle) and ChPLA₂-IB (white circle) activities. PLA₂ activity was measured using egg yolk emulsion as substrate at pH 9.0 and at 40°C in the presence of 10 mM Ca²⁺.

Figure 4

Effect of temperature and pH on ChPLA2-IB and ChPLA2-IIA. Effects of temperature (A, B) and pH (C, D) on ChPLA₂-IIA (black circle) and ChPLA₂-IB (white circle) activities (A, C) and stability (B, D). PLA₂ were tested for activity at various temperatures (A) and pH (C) as described in material and methods. To study the stability of PLA₂, 1 mg.ml^-1 of each enzyme was incubated during 30 min at various temperatures (B) and pH (D). Residual PLA₂ activity was measured using egg yolk as substrate in the presence of 10 mM Ca²⁺ and 10 mM NaTDC. For temperature stability studies, enzymes were incubated in 10 mM Tris (pH 8.0) and 10 mM CaCl₂. For pH stability studies, Tris buffer was replaced by the appropriate buffer for the pH range.

Figure 5

Nucleotide sequence of the cDNA of ChPLA₂-IIA and the deduced amino acid sequence. Sequencing was performed in triplicate with three independent PCRs. The amino acid sequence obtained by N-terminal amino-acid sequence of the pure ChPLA₂-IIA is shown in italic. M1-C18 in bold, signal peptide; Y41-G49 in square, Ca²⁺ loop; and H64-D65 with stars, active site.

Figure 6

3D modelling structure of ChPLA2-IIA. (A) Cartoon representation the 3D model of the mature ChPLA₂-IIA. Secondary structure elements labels are indicated. α-helices and β-strands are colored in red and yellow, respectively. The catalytic network and the Ca²⁺-binding residues are indicated and shown as sticks. The disulfide-bridges are shown in yellow. (B) Cartoon representation of the ChPLA₂-IIA structure showing amino acids (represented by sticks) interacting with the substrate (PG). The substrate is indicated as a stick representation. The Ca²⁺ ions are represented by a blue spheres. This figure was generated using the PYMOL software.