Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.
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.



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.



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.



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.
Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.|Department of Medicine, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.
Victoria L M
VL
Herrera
Department of Pathology and Biomedical Engineering, Boston University, Boston, USA.
Martin
M
Steffen
Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.|Department of Medicine, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.
Ann Marie
AM
Moran
Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.|Department of Medicine, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.
Glaiza A
GA
Tan
Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.|Department of Medicine, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA.
Khristine A
KA
Pasion
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724, USA.
Keith
K
Rivera
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724, USA.
Darryl J
DJ
Pappin
Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA. nruizo@bu.edu.|Department of Medicine, Boston University School of Medicine, 700 Albany Street, Boston, MA, 02118, USA. nruizo@bu.edu.
Nelson
N
Ruiz-Opazo
D000818:Animals; D023102:Anoikis; D045744:Cell Line, Tumor; D003062:Codon; D037483:Galectin 1 / Q000032:analysis / Q000378:metabolism; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D060066:Protein Interaction Maps; D011544:Pseudogenes / Q000235:genetics*; D051381:Rats; D014364:Tryptophan / Q000235:genetics
D003062:Codon; C571813:FBXW7-AS1 non-coding RNA, human; D037483:Galectin 1; D014364:Tryptophan
DEspR; DEspR protein–protein interactions; Pseudogene