Pigment epithelium-derived factor-receptor (PEDF-R/ATGL) exhibits phospholipase B activity

Authors

• S Debnath
• SP Becerra

Abstract

Pigment epithelium-derived factor-receptor (PEDF-R), encoded by the PNPLA2 gene, exhibits phospholipase A2 (PLA2) activity and is stimulated by the neurotrophic domain of PEDF (17-mer) to promote cell survival. This study aims to overexpress, produce, and characterize recombinant human PEDF-R for biochemical studies. Human PNPLA2 cDNA encoding a truncated PEDF-R version (PEDF-R[1-288]) was subcloned into an expression vector with His-, SUMO and Strep-peptide tags for efficient overexpression in Escherichia coli. Recombinant PEDF-R[1-288] was purified from soluble cell lysates using ammonium sulfate precipitation, and a two-step affinity column chromatography that included His-affinity and Strep-affinity columns. The highly purified recombinant protein was soluble in zwitterionic detergent containing buffers and formed protein complexes larger than 1236 MW. The soluble PEDF-R[1-288] protein catalyzed both PLA1 and PLA2 substrates. The enzyme remained stable and active under shock-freezing and storage conditions. The PLA1 enzyme was active over a broad pH range, while PLA2 had an optimum at basic pH. Both activities were calcium-independent, but CaCl2 inhibited PLA1 activity. ATP and MgCl2 slightly inhibited PLA2 activity, but only MgCl2 inhibited PLA1 activity up to 2.5 mM. Bromoenol lactone selectively inhibited PLA2 activity, while atglistatin and NG-497 had no effect on PLA1 or PLA2 activities. The 17-mer peptide stimulated both PLA1 by more than 5-fold and PLA2 by more than 3-fold. The study identifies a novel phospholipase B (PLB) activity in PEDF-R, and established a robust method for producing large quantities of active recombinant PEDF-R.

Scientific Focus Area: Molecular Biology and Biochemistry

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