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Paige Nicole Winokur, Preeti Subramanian, Veronique Arocas, Patricia Becerra; Protease Nexin-1, a Novel Serpin in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2328.
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© ARVO (1962-2015); The Authors (2016-present)
Protease Nexin-1 (PN-1), encoded by the SERPINE2 gene, is a member of the serpin superfamily with demonstrable neurotrophic effects in the brain. Recently it was shown that PN-1 inhibits angiogenesis in the retina, but its neurotrophic capacities in the retina have not yet been evaluated. Pigment Epithelium-derived Factor (PEDF), another serpin exhibiting neurotrophic and antiangiogenic activities, is present in the retina and its SERPINF1 gene predominantly expressed in the retinal pigment epithelium (RPE). The principal objective of this study is to compare PN-1 and PEDF as potential neuroprotectors for the retina.
Sequence alignments were performed using ClustalW2 Multiple Sequence Alignment EMBL-EBI. Human ARPE-19 cell lines were cultured. Protein extracts were obtained from RPE and retina dissected from eyes of C57BL/6N [Crb1rd8] mice. PN-1 protein and Serpine2 transcript levels were evaluated by Western blotting and RT-PCR, respectively. Recombinant human PN-1, PEDF, and PEDF-R were prepared and used. PEDF-R, a critical cytoprotective receptor for PEDF, was prepared by in vitro protein synthesis (IVPS) via the Expressway™ Cell-Free E. coli Expression System (Invitrogen™). Binding to PEDF-R was assessed by pull-down assays and peptide affinity chromatography.
PN-1 (PDB: 4DY0_A) and PEDF (PDB: 1IMV_A) are remarkably analogous at the level of tertiary structure according to crystal structure analyses. Primary sequence alignments revealed that they are comparable in length, and PN-1 contains a region that shares homology with the neurotrophic active region of PEDF. Alpha helix C within this region is similar in both serpins, and contains the structural determinant for neurotrophic activity in PEDF. The homologous neurotrophic segment in PN-1 is widely conserved across species. SERPINE2 mRNA was detected in human ARPE-19, as well as in native murine RPE and retinal cells. PN-1 protein, like PEDF, bound to PEDF-R.
The RPE express the SERPINE2 gene and the PN-1 protein binds to PEDF-R and shares several structural similarities with PEDF. The data imply that PN-1 may play a neuroprotective role in the retina.
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