Abstract 1549 PLA2 and PLA1 dual activity of Pigment Epithelium-derived factor receptor (PEDF-R) and its stimulation by PEDF-derived peptides

Pigment epithelium-derived factor receptor (PEDF-R) (also called ATGL) is encoded by patatin-like phospholipase domain-containing 2 (PNPLA2) gene. In the retina, PEDF-R exhibits phospholipase A2 (PLA2) activity. Pigment epithelium-derived factor (PEDF), which is encoded by SERPINF1, binds PEDF-R, and stimulates its PLA2 activity to promote survival of retinal cells. Accumulation of lysophospholipid in the retina is found in mice that lack PNPLA2. A peptide derived from the PEDF neurotrophic region binds and promotes retina survival, but it remains to be determined if such a peptide activates PEDF-R and whether PEDF-R is a PLA1 enzyme. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human membrane protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. To overexpress and produce human recombinant PEDF-R, we have subcloned the cDNA of human PNPLA2 that codes for a C-terminal truncated version of PEDF-R (PEDF-R (1-288)) with peptide tags at its N-terminal and C-terminal ends in an expression vector. The recombinant protein was produced in Escherichia coli strain Rosetta (DE3) and purified from soluble fractions of cell lysates by a two-step of affinity column chromatography using an automated purification system. The highly purified and soluble bacterially derived recombinant PEDF-R (1-288) protein was assayed for PLA2 and PLA1 activities using commercial assay kits. The recombinant protein catalyzed the substrates for PLA1 and PLA2 efficiently. PLA2 activity of recombinant PEDF-R (1-288) depended on the pH of the buffer, but PLA1 activity was pH independent. CaCl2 inhibited PLA1 activity of recombinant PEDF-R (1-288), but not PLA2 activity. Shock freezing and storage of PEDF-R (1-288) did not affect its phospholipase activity. More importantly, we tested the effect of peptides modified from the neurotrophic PEDF region on the PLA activities of PEDF-R. We found that selected peptides that stimulated both, PLA1 and PLA2 of PEDF-R protected retinal cells against death in a mouse model of retinal degeneration. Thus, in addition to providing techniques to express, produce and purify large amounts of soluble, stable, and active recombinant PEDF-R (1-288) from Escherichia coli, our results point to the discovery of a new PLA1 activity of PEDF-R that is efficiently stimulated by peptides modified from the neurotrophic PEDF region. These technologies will prove useful for further biochemical, structural, and biological studies. This research was supported by NEI-NIH intramural research program ZIAEY000438 and ZIAEY000306.