Analyses of Patatin‐like Phospholipase Domain‐containing 2 (PNPLA2) Transcripts: Discrepancies between Expressed Sequence Tag (EST) and Experimental Data

Abstract

Pigment epithelium‐derived factor‐receptor (PEDF‐R), a PNPLA2 protein found in the retina, has phospholipase A2 (PLA2) activity that is enhanced upon binding PEDF, a retina survival factor. Amino acid D166 forms part of the PLA2 catalytic dyad required for activity and is encoded on PEDF‐R exon 4a (E4a). Interestingly, EST databases predict that PEDF‐R has multiple isoforms: one with an E4a deletion in mouse sequence, and another with an exon 6 (E6) deletion in mouse and rat sequences. It is unknown whether alternatively spliced PEDF‐R transcripts exist in the retina. Here, we explored the proposed PEDF‐R isoforms in cell lines of retinal origin (R28, 661W) and in various mouse tissues. We designed PCR primer pairs that encompassed the predicted E4a deletion and/or E6 deletion to amplify the full length and predicted splice variants. The PCR products resolved by gel electrophoresis showed a single band consistent with full length PEDF‐R rather than splice variants. Additionally, we performed western blots of R28 and 661W cell extracts and found that multiple PEDF‐R antibodies detected only a single band migrating as expected for full length PEDF‐R. The data point to a single transcript and no PEDF‐R alternative spliced variants. Considering ESTs offer partial gene coverage and possible sequence errors, our results underlie the importance of validating conclusions drawn from EST databases. Supported by NIH‐NEI‐IRP.