Herrera, V. L., Steffen, M., Moran, A. M., Tan, G. A., Pasion, K. A., Rivera, K., Pappin, D. J., Ruiz-Opazo, N. (June 2016) Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein. BMC Mol Biol, 17 (1). p. 15. ISSN 1471-2199 (Electronic)1471-2199 (Linking)
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Abstract
BACKGROUND: In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. METHODS: To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. RESULTS: Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. CONCLUSIONS: Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | DEspR DEspR protein-protein interactions Pseudogene |
| Subjects: | bioinformatics > genomics and proteomics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification |
| CSHL Authors: | |
| Communities: | CSHL Cancer Center Program > Signal Transduction CSHL labs > Pappin lab CSHL Cancer Center Program > Cellular Communication in Cancer Program |
| Depositing User: | Matt Covey |
| Date: | 14 June 2016 |
| Date Deposited: | 23 Jun 2016 17:01 |
| Last Modified: | 26 Oct 2020 16:57 |
| PMCID: | PMC4906906 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/32874 |
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