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X. Xu, S. Aparicio, A.R. Tink, N. Lara, S. Sawant, S.S. M. Zhang, C.J. Barnstable, J. Tombran–Tink; PEDF and the SERPINS: Phylogeny, Sequence Conservation, and Functional Domains . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3106.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: PEDF is an important neuroprotective and antiangiogenic factor. The purpose of this study was to use an array of bioinformatics tools to assemble and compare PEDF sequences from a broad range of species to identify common structural features that can shed light on the function of this serpin. Methods: All sequence data were downloaded from NCBI databases. ClustalW, RasMol, and Protein Explorer were used to assemble and analyze the sequence data. The pyhlogenetic trees were constructed using "drawgram" from PHYLIP package. Standard immunocytochemistry and western blotting methods were used to examine PEDF localization in nuclear and cytoplasmic compartments of four human cell lines, RPE, Y79, neuroblastoma, and HepG2. Results: We assembled PEDF sequences for 9 additional species and performed a cross species alignment for 14 PEDF sequences to identify conserved structural domains in the gene. We found evolutionary conservation of a leader sequence, the RCL, a single C–terminal glycosylation site, collagen binding residues, and four specific conserved PEDF peptides. The C–terminus peptide 384–415 and an N–terminal peptide 78–95, are highly homologous with 97 and 39 inhibitory serpins, respectively. There is strong conservation of 39 of 51 consensus key residues involved in serpin structure and function and much less homology between PEDF and non–inhibitory serpins. Two peptides, 40–67 and 277–301, are unique to PEDF. Conserved residues at the N–terminus, helix d (hD), and helix A (hA) of PEDF form a structure similar to the heparin binding groove of other serpins. We identified a motif in PEDF that is homologous to the nuclear localization signals of other proteins. Immunocytochemistry and western blots show a bitopographical localization of PEDF in several cell types. Conclusions: PEDF is highly conserved throughout the vertebrate kingdom. Our results suggest that secretion is required for PEDFs activity, that PEDF is active in the nuclear compartment, and argue that PEDF has structural and functional features in common with inhibitory serpins as well as a number of unique structural motifs.
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