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

Abstract

BackgroundIn 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.MethodsTo 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.ResultsMaxam–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.ConclusionsData 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0066-8) contains supplementary material, which is avail...

Humanized anti-DEspR IgG4S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis ratnu/nu model

“…Immunofluorescence staining was performed as previously described [ 11 , 18 ]. Panc1 TCs and CSCs were fixed with 2% paraformaldehyde (Thermo Scientific, AAJ19943K2) for 15-min at 4 °C.…”

“…Double immunostaining was done using 10 μg/ml hu--AF568, αSMA (Sigma, A5228), and collagen 1/3 alpha-1 chain-AF488 (Santa Cruz, sc-293,182, human/rat reactive), incubated overnight in 4 °C. Imaging was performed with a Zeiss Axioskop fluorescence microscope, as previously described [ 11 , 18 ].…”

“…Cell surface expression of Panc1 TCs and CSCs was performed as previously described [ 18 ]. Cells were labeled at 4 °C in 2% Fetal Bovine Serum (FBS) (Thermo Scientific, 10,438,026) in Hank’s Buffered Salt Solution (HBSS) (ThermoFisher, 14,170,112) for 30-min.…”

“…Immunofluorescence staining was performed as previously described [ 11 , 18 ]. Binding was performed at 4 °C with 10 μg/ml 7c5-AF568.…”

“…DEspR is indeed a ‘missing puzzle piece’ as it is annotated as a non-coding gene, due to a stop codon [T-G-A] in the NCBI DNA database [ 16 ], in lieu of tryptophan-codon [T-G-G] at amino acid position-#14 (tryp#14). Given multiple experimental evidence detecting DEspR protein, functionality, and RNA-sequences with [T-G-A/G]-tryp#14 in placenta RNA-seq dataset [ 11 , 17 , 18 ], analysis for potential ADAR1 RNA-editing would clarify this DNA-protein discordance.…”