Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein
© The Author(s) 2016
Received: 28 January 2016
Accepted: 20 May 2016
Published: 14 June 2016
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.
KeywordsDEspR Pseudogene DEspR protein–protein interactions
In contrast to rat and mouse databases listing Dear as a gene, DNA and RNA sequence databases list the human Dear gene or the dual-endothelin1/VEGFsp receptor (DEspR) as a pseudogene  (Additional file 1: Figure S1). Automated DNA sequence databases report a stop codon [TGA] instead of tryptophan [TGG] at codon#14 reported in single gene study . The current NCBI pseudogene annotation updated in May 2016 and referenced in other sites is discrepant with the single research group single-gene studies of human DEspR showing expression in human kidney via immunohistochemistry using a polyclonal anti-DEspR antibody, and functional studies of human DEspR expressed in permanent Cos1 cell transfectants detecting the predicted protein size by Western blot analysis as well as binding to DEspR-ligands (endothelin-1 and VEGFsp) . The NCBI pseudogene annotation is also discrepant with the single gene study demonstrating DEspR-specific functional roles in cancer and putative regulation at the splicing level with detection of both unspliced and spliced DEspR RNA in human tumor cells by allele-specific amplification-refractory mutation system (ARMS) methodology .
Experimental clarification is warranted since the basis for the NCBI pseudogene annotation, automated DNA/RNA-sequencing, is known to have reproducible systematic sequencing errors, regardless of technology . Occurrences of, hence risks for, systematic errors eliminate the a priori assumption that multiple occurrences negate errors. More specifically, systematic errors in high throughput DNA sequencing has been observed to occur “even in overlapping paired reads from high-coverage data, approximately one in 1000 bp, and are highly replicable across experiments” . Moreover, given discrepancies among methodologies, the Sanger sequencing is the final determinant of sequence discrepancy since “any difference from the Sanger sequence is defined as a sequencing error” .
Further support for the need for scientific clarification is found in the GENCODE Pseudogene Resource which states that “the definition of a pseudogene is based on the presence of specific characteristics such as premature stop codon, coding sequence frame shift, truncation, or disabling insertion/deletion—unless evidence (transcriptional, functional, publication) shows that the locus represents a protein-coding gene” . Concordant with Pei et al. , Kageyama et al.  explicitly states that “before a particular transcript can be determined to be a long non-coding RNA (or ‘transcribed pseudogene’), there must be somewhat convincing evidence for its lack of translatability.” Hence, deductions from automated sequence databases need to be evaluated in the context of experimental evidence for translatability and functionality.
To address the need for scientific clarification of DEspR (Dear) as a gene or pseudogene, we therefore upheld established standards that (1) “the translatability of the candidate can be validated with specific antibodies against amino acid sequences predicted from the ORF,”  and that (2) “an assessment of … a protein’s molecular activity by biochemical methods should be the final certification of an active gene product” . These perspectives are codified in the Central Protein Resource UNIPROT criteria for translatability: “evidence for existence of a protein at the protein level, such as via antibody detection, is the highest level of evidence” .
Here we confirm the existence of the DEspR gene product at the nucleotide and protein level. We show that both Sanger and Maxam–Gilbert sequencing detect two G’s for a tryptophan [TGG]-codon#14. We also determine that some RNA-seq entries also contain two G’s but with an extra A or TA, as the region in question spans a canonical Yamakawa compression motif . Monoclonal anti-DEspR antibodies to different DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein from membrane-bound proteins from human tumor cells and the Cos1-DEspR + permanent transfectants cells. More importantly, DEspR molecular activity, protein–protein interactions, protein-specific properties and functionality are shown, and found to play key roles in cancer stem cell anoikis resistance and growth.
Confirmation of two G’s and compression site
Importantly, queries against subsets of the NCBI sequence read archive (SRA) database reveals DEspR-specific exon sequences which would not be expected for the FBXW7 transcript in the opposite strand (Fig. 1b). Notably, five entries show the two G’s out of 124 data entries in said file (Additional file 3: Figure S3), and that these two G’s are associated with an insertion of a “T–A’ or an “A” (Additional file 3: Figure S3). These observations show that the two G’s are indeed detected and are located within a problematic sequencing region, concordant with observations in manual sequencing gels of nucleotide compression spanning a known sequence motif for compression . These data show the questioned two G’s in support of tryptophan [TGG] codon in 5/124 sequences similar to manual sequencing runs. Given that Sanger-sequencing is the accepted standard final determinant of nucleotide discrepancies , these experimental data support the need for clarification and demonstration of DEspR protein expression.
Analysis of translatability and protein-specific properties
Proteins pulled-down with anti-DEspR mAb 5g12e8 from U87 CSC membranes
Actin, cytoplasmic 1 (ACTB)
Tubulin beta-4B chain (TUBB4B)
Tubulin beta chain (TUBB)
Tubulin beta-2A chain (TUBB2A)
Galectin-1 (LGALS1) a
Tubulin beta-3 chain (TUBB3)
Tubulin beta-6 chain (TUBB6)
Ras-related protein Rab-8A (RAB8A)
Ras-related protein Rab-7a (RAB7A)
Ras-related protein Rab-10 (RAB10)
Ras-related protein Rab-13 (RAB13)
Ras-related protein Rab-8B (Fragment) (RAB8B)
Transmembrane emp24 domain-containing protein 10 (TMED10) a
Transmembrane emp24 domain-containing protein 7 (TMED7)
Transmembrane emp24 domain-containing protein 9 (TMED9)
Ras-related protein Rap-1b (Fragment) (RAP1B)
However, because of the abundance of Galectin-1 in the pull-down products, we performed more Western blot analyses to eliminate the possibility that anti-DEspR antibody cross reacts with Galectin-1 and vice versa. As shown in Fig. 2c, the anti-human DEspR 5g12e8 mAb detects the 17.5 kDa glycosylated DEspR in the pull-down products from U87-CSC cell membrane proteins and from the membrane proteins isolated from DEspR-positive Cos1-permanent transfectant cells. These DEspR + Cos1-permanent transfectants cells were previously shown to express human DEspR by immunostaining, and to competitively bind ligands and anti-DEspR (7c5b2) mAb . After PNGase F digestion, 5g12e8 also detected the smaller, hence deglycosylated DEspR (Fig. 2c, middle panel). In contrast, anti-Galectin-1 mAb detected a 14 kDa protein band with markedly different size and expression levels to DEspR in all three lanes (Fig. 2c, right panel).
To further confirm this distinction, we performed double Western blot analyses of Galectin-1 recombinant protein. As shown in Fig. 2d, anti-Galectin-1 mAb detected human Galectin-1, in contrast to the anti-human DEspR 5g12e8 mAb which did not cross react to both 100 and 500 ng of purified recombinant human Galectin-1 on Western blot analysis, thus confirming that DEspR in the pull-down product is distinct from other pull-down products (Fig. 2b) especially Galectin-1 (Fig. 2c). Additionally, since Galectin-1 is known not to be glycosylated  as confirmed in Fig. 2c, the PNGase F-treated sample showing a decrease in size cannot, therefore, be Galectin-1.
To further corroborate the detection of human DEspR protein in human tumor cells, we performed sequential probing of the identical Western blot with two different anti-human DEspR mAbs targeting different epitopes. As shown in Fig. 2e, the 6g8g7 mAb targeting the epitope spanning the disputed tryptophan [TGG]-codon#14 (Fig. 2a) detects both the glycosylated 17.5 kDa and the less glycosylated 12.5 kDa DEspR bands. After removal of the 6g8g7 antibody and demonstration of no residual signals, the Western blot was re-probed with the 5g12e8 mAb used in the pull-down experiment and which targets a human DEspR-specific epitope spanning a peptide present only if correctly spliced (Fig. 2a). This 5g12e8 probed western blot detected the identical protein bands (Fig. 2e), thus showing that two different anti-hDEspR mAbs 6g8g7 and 5g12e8, which target different epitopes, detect the same protein bands representing spliced and glycosylated DEspR.
Protein–protein interactions: analysis of co-localization of DEspR and Galectin-1
Analysis of functionality at multiple levels certifies DEspR protein
As shown in Fig. 4, we studied DEspR-inhibition effects on anoikis resistant cancer stem-like cells (CSCs) isolated from human U87 glioblastoma tumor cell line , human MDA-MB-231 triple negative breast cancer cell line, human H460 non-small cell lung cancer cell line and human Panc1 pancreatic ductal adenocarcinoma cell line . To first demonstrate that DEspR protein is expressed on the different CSCs, we performed FACS analysis using AF568-fluorescently labeled 7c5b2 anti-DEspR mAb (Fig. 2a). Figure 4a shows that, compared to isotype control, fluorescently labeled 7c5b2-mAb specifically detected DEspR on the cell membrane in ~50 % of U87-CSCs, ~60 % of MB-231-CSCs, ~71 % of H460-CSCs and ≥55 % of Panc1-CSCs in the suspension culture conditions used.
To demonstrate the functionality of DEspR proteins present on the cell membrane, in vitro inhibition studies were performed using the 7c5b2, 5g12e8 and 6g8g7 anti-human DEspR mAbs which target different DEspR epitopes (Fig. 2a). As shown in Fig. 4b, all three mAbs significantly inhibited CSC growth in the four cancer tissue type-CSCs tested (P < 0.0001, One Way ANOVA with Tukey’s multiple comparisons test), in contrast to non-treated contemporaneous controls respectively. We note that CSC growth is representative of survival in suspension culture, proliferation and ability to form stem-cell like spheroids in suspension culture conditions. We also note some variations in the inhibition of CSC growth in vitro by the different mAbs, with 7c5b2 an 6g8g7 performing better than 5g12e8 (Fig. 4), but that all mAbs significantly inhibit survival, proliferation, and spheroid formation (CSC-growth) of U87-CSCs, MDA-MB-231-CSCs, H460-CSCs and Panc1-CSCs.
Stop [TGA] codon vs tryptophan [TGG] codon within a sequence-compression region
The detection of the two G’s to make tryptophan [TGG]-codon#14 in manual sequencing Sanger-dideoxy sequencing and Maxam and Gilbert sequencing, along with confirmation of sequence compression at the site within a Yamakawa compression-motif whether sequencing the sense strand (Maxam–Gilbert) or the antisense strand (Sanger dideoxy sequence), strongly suggests that the NCBI designation of human Dear/DEspR as a pseudogene based on stop codon#14 is erroneous. Following the accepted standard that manual Sanger sequencing defines the correct sequence rather than the automated sequence, the single gene analysis data indicates that the TGG codon is present rather than the TGA stop codon reported in automated DNA sequencing databases. This interpretation is supported by the fact that systematic sequencing errors are documented to occur with both position- and sequence-specificity even in “next generation sequencing technologies at rates greater than prior technologies” . Existence of the tryptophan[TGG]-codon#14 is independently supported by detection of the DEspR protein by mAbs 5g12e8 and 7c5b2 whose peptide epitopes exist only if the DEspR RNA is spliced, and in the case of 6g8g7, only if the contested tryptophan[TGG]-codon#14 is present (Fig. 2a). Notably, 5g12e8 and 6g8g7 detect DEspR-protein as a glycosylated 12.5–17.5 kDa protein band, much greater than the 1.5 kDa predicted from a 13 amino acid peptide should there be a stop codon at codon#14. Furthermore, functional validation of the glycosylation and internalization recognition sequences beyond codon#14 (Figs. 2, 5) experimentally refute a stop codon at codon#14.
Insights regarding other high throughput database reports
The non-detection of DEspR sequences in normal human tissue RNA-Seq databases is unsurprising since DEspR is not expressed or minimally expressed in normal tissues. Moreover, detection of unspliced DEspR-specific RNA as the dominant species  does not indicate non-translation, since regulation at the splicing level has recently been described for granulocyte differentiation whereby the dominant RNA species in the cell is the unspliced form [11, 12], similar to observations for detection of both spliced and unspliced DEspR-transcripts by ARMS-PCR .
Moreover, since DEspR overlaps with a larger transcript, FBXW7, on the opposite strand , RNA-seq databases that specifically exclude double stranded DNA  will also exclude RNA–RNA sequences such as DEspR-FBXW7 hybrids. The process-exclusion of RNA–RNA hybrids in RNA-Seq entries in current methodologies is due to the standard of excellence requiring elimination of all double stranded DNA in RNA-Seq libraries , which inadvertently will also exclude RNA–RNA hybrids. More specifically, since RNA–RNA hybrids can form in standard RNA isolation methods with phenol extraction , and can form during RNA-seq library preparation using primer annealing reactions at temperatures lower than the melting temperatures (Tm) of RNA–RNA hybrids, and given process-exclusion of any dsDNA or RNA–RNA or dsDNA-RNA hybrids, there is an inadvertent bias introduced against the detection of DEspR-specific transcripts due to RNA–RNA hybrids formed between DEspR transcript and exon#5 (or #6 depending on dataset numbering) of FBXW7 transcript on the opposite strand.
In parallel, the non-detection in proteomic databases is concordant with difficulties in detecting membrane proteins with single transmembrane domains that are less than 150 kDa , especially if glycosylated, since PNGase F treatments do not necessarily ensure detection by MS . The current non-detection of DEspR transcripts in transcriptomics could also be due to the exclusion of RNA–RNA hybrids, and in primer pair-specific amplification due to the absence of DEspR-specific primers given the non-recognition of the DEspR gene by NCBI.
Confirmation of translatability
Demonstration of translatability through different experimental designs and using anti-human-DEspR mAbs specific for different epitopes substantially fulfill UNIPROT criteria for determining a protein’s existence at the highest level such as detection of protein by antibody, demonstration of protein–protein interactions, demonstration of post-translational modifications, and demonstration of protein functionality .
Pull-down protein analysis experiments confirm the existence of DEspR via direct detection of the protein in human tumor cells by Western blot analysis of DEspR using multi-epitope anti-DEspR mAbs. The detection of the identical protein band by Western blot ‘walking’ with distinct anti-DEspR mAbs targeting two different epitopes, 5g12e8 mAb binding to DEspR epitope-1 that spans a splice junction, and 6g8g7 binding to DEspR epitope 2 that spans tryptophan[TGG]-codon#14, demonstrate the existence of the DEspR protein. If the stop codon were indeed present at codon#14 instead of tryptophan, there would simply be a 13 amino acid peptide with 1.5 kDa MW rather than the glycosylated 17.5 and 12.5 kDa protein bands, or the non-glycosylated DEspR-sequence predicted ~10 kDa protein.
Moreover, if there were indeed a stop codon at codon#14, DEspR would not be glycosylated nor internalized as the consensus sequence motifs for these sites are beyond the reported stop codon#14. The detection of the identical-sized protein on Western blot analysis of DEspR-positive Cos1 cell permanent transfectants and U87 tumor cells corroborates the protein product of the transfected plasmid DEspR mini-gene construct previously reported, and corroborates splicing of the unspliced DEspR cDNA mini-gene construct .
Detection of DEspR by Western blotting but not by mass spectrometry analysis
We note that although DEspR was detected consistently in denatured conditions via Western blot analysis of membrane protein pull-down products using 5g12e8 and 6g8g7 anti-DEspR mAbs, MS did not detect DEspR-specific peptides. However, non-detection by MS does not negate DEspR protein existence for several reasons. First, based on deduced amino acid sequence predicting a single transmembrane integral membrane ~10 kDa protein that is glycosylated to 17.5/12.5 kDa, non-detection by peptide mass fingerprinting methods using techniques such as MALDI-TOF MS analysis is not surprising given that only 204 integral membrane proteins were detected on mass spectrometry in a study of rat endothelial cells at the National Center for Proteomics Research, and that no VEGF receptor-2 (151 kDa) was detected in this study . Using 6718 as total number of membrane proteins , Mirza et al.  detected 3 % integral membrane proteins on MS, while Peng et al.  detected 301 integral membrane proteins via SDS-PAGE shotgun proteomics or 4.5 % of 6718 total membrane proteins, and highest count using SCX-RPLC-MS/MS (MudPIT) strategy detected 876 integral membrane proteins or 13 % of 6718 integral membrane proteins in murine NK cells . Notably, of the ones detected by MS using an improved method that detects more membrane proteins using a centrifugal proteomic reactor, all the proteins detected were >150 kDa and ≥2 transmembrane domains .
Other factors could also account for non-detection by MS with current methodologies. As stated by Bensalem et al. , peptide mass fingerprinting of membrane proteins, using techniques such as MALDI-TOF MS, remains a significant challenge for at least three reasons: (1) membrane proteins are naturally present at low levels, (2) many detergents strongly inhibit proteases and have deleterious effects on MALDI spectra, and (3) despite the presence of detergent, membrane proteins are unstable and often aggregate . Additionally, glycosylation of DEspR also impedes current proteomic detection as reported by Cao et al.  stating that “deglycosylation of plasma membrane proteins by treatment with PNGase-F did not yield detection of additional hydrophobic proteins”. Therefore, peptide mass fingerprinting MS analysis of PNGase-treated pull-down proteins which did not identify DEspR does not negate the existence of DEspR protein. These observations by others account for the non-detection by MS of the single transmembrane receptor <150 kDa DEspR protein, thus excluding MS non-detection as counter evidence for the existence of DEspR protein, especially given multiple other experimental evidence that fulfill UNIPROT criteria for determining a protein .
Demonstration of protein interactions, protein-specific properties and functionality
Aside from ligand-specific activated DEspR signaling , detection of DEspR-protein interactions in the DEspR-Galectin-1 complex, along with other pull-down protein partners, complies with a UNIPROT criterion for ascertaining existence of a protein as a gene product through the demonstration of protein-interactions . The distinct functionality of DEspR vs Galectin-1 in terms of anoikis resistance indicates DEspR protein-specific functions, rather than the potential counter-argument that DEspR functionality is merely due to the known Galectin-1 functions.
More specifically, DEspR-specific inhibition by blocking antibodies induces anoikis in four cancer tissue type CSCs, corroborating earlier observations for Panc1 and U87 CSCs . In contrast, Galectin-1 is pro-anoikis . Given these two observations, we hypothesize that glycosylated DEspR plays a role in anoikis resistance by binding to Galectin-1 and inhibiting its pro-anoikis function, much like glycosylation of p16-INK4a de-induces Galectin-1 pro-anoikis functions through upregulation of Galectin-3 . Confirmation of the DEspR-Galectin-1 complex is demonstrated by co-localization on double-immunostaining experiments, thus further strengthening evidence for translatability and functionality by the detection of protein–protein interactions following UNIPROT criteria . Distinguishing the DEspR protein band by size and quantity from its pull-down partners identified from MS by their corresponding antibodies in serial sequential probing of the identical western blot, corroborates their detection by MS, as well as eliminates the potential of erroneous cross-reactivity pull-down results.
More importantly, given that demonstration of protein functionality certifies a protein’s existence [7, 8], the demonstration of DEspR expression in >50 % of CSCs in four different cancer tissue types by FACS analysis and DEspR functionality via blocking antibody inhibition of CSC growth in all four different cancer tissue type CSCs—glioblastoma, triple negative breast cancer, non-small cell lung cancer and pancreatic ductal adenocarcinoma, provide compelling evidence of the existence of DEspR protein based on UNI-PROT criteria and standards in the field [7, 8].
Furthermore, the demonstration of internalization of DEspR protein via live cell imaging is consistent with a consensus internalization recognition sequence downstream to amino acid#14. This not only confirms translatability of the DEspR protein beyond 13 amino acids, thus refuting the stop codon presence, but also demonstrates a protein-specific property, i.e. receptor internalization and expands DEspR’s multi-functionality. Concordantly, these new data on functionality are supported by previous findings of DEspR roles in tumor angiogenesis, invasiveness, growth, as well as CSC survival in adverse conditions .
Altogether, in the context of an error prone site at codon#14, multiple protein assays independently and collectively demonstrate the existence of the DEspR protein, and analyses that show DEspR protein properties (protein–protein interactions, glycosylation, internalization) and functional roles in multiple cancer tissue type CSCs, collectively certify the DEspR protein based on multiple UNIPROT criteria and demonstrate that the human DEspR gene is not a pseudogene. Given that fulfillment of a single UNIPROT criterion for a protein’s existence is sufficient, fulfillment of multiple criteria present compelling experimental evidence, at the highest protein level, that functional human DEspR protein exists.
We performed Maxam–Gilbert sequencing essentially as described . The hDEspR cDNA was 5′-end labelled with polynucleotide kinase using gamma-32P ATP. Chemical treatments for Guanine (G), Guanine + Adenine (G + A), Cytosine + Thymine (C + T) and Cytosine (C) cleavages were done as described . Cleavage products from the four reactions were size separated by 8 % denaturing polyacrylamide gel electrophoresis and fragments visualized by autoradiography. Electrophoresis was performed at three different fixed wattages: 25 watts (#1), 35 watts (#2) and 50 watts (#3).
Cell lines and antibody development and characterization by ELISA
Verified glioblastoma U87 MG (cat# ATCC HTB-14), triple negative breast cancer MDA-MB-231 (cat# ATCC HTB-26), non-small cell lung cancer NCI-H460 (cat# ATCC HTB-177) and pancreatic cancer Panc-1 (cat# ATCC CRL-1469) cell lines were obtained from ATCC. Isolation and propagation of U87, MB-231, H460 and Panc1 cancer stem cell-like cells (CSCs) was done as described . CSCs were maintained and expanded through passage-5 in complete MammoCult® medium (Stem Cell Technologies, BC, Canada) containing 0.5 % Methylcellulose (Stem Cell Technologies, BC, Canada) in 100 mm ultra-low attachment plates in 5 % CO2 humidified incubator at 37 °C. Testing for increased tumorigenicity was performed in vivo at passage-5. Monoclonal antibody development was custom performed by ProMab Biotechnologies (Richmond, CA). For 7c5b2  and 5g12e8 mAbs we used a nine amino-acid peptide, M1TMFKGSN E9 at the amino-terminal end of hDEspR  and a ten amino-acid peptide, E9MKSRWNWGS18, for 6g8g7 mAb as antigens respectively. Screening of hybridoma supernatants and characterization of monoclonal antibodies were performed by ELISA using corresponding antigenic peptides. Serial dilutions of primary antibodies were incubated at 37 °C for 1 h. The wells were then incubated with horse radish peroxidase (HRP)-labeled anti-IgG (Sigma) at 37 °C for 1 h. Reactions were analyzed at 450 nm after addition of 3,3′5,5′-tetramethylbenzidine substrate at 37 °C for 10 min.
Magnetic bead immunoprecipitation (pull-down) of DEspR protein-complex
U87 CSC membranes, Cos1-hDEspR permanent cell transfectant membranes and U87 xenograft tumor (in nude rats) membranes were isolated by differential centrifugation as described . Antibody coupling to magnetic Dynabeads M-450 Epoxy (Invitrogen) was performed as per manufacturer’s instructions using 200 μg of anti-hDEspR 5g12e8 mAb per 1 mL of beads (4 × 108 beads). For target binding 100 μL of 5g12e8-coupled beads (4 × 107 beads) and 1.3 mg membrane protein were incubated for 2 h at 4 °C in 1 ml ice-cold PBS containing 2 mM EDTA. To capture mAb-bound protein, the tube was placed in a magnet for 2 min, the supernatant discarded and the beads washed five times with 1 mL PBS buffer containing 2 mM EDTA and 0.2 % Tween-20 at room temperature. This washed magnetic bead-‘pull-down’-protein complex was then analyzed using different methods. (a) For SDS-PAGE (18 % polyacrylamide) the beads were resuspended in 20 µL of 1X Laemmle sample buffer and proteins denatured at 65 °C × 30 min in order to size-separate and assess size(s) of protein(s) ‘pulled-down’. (b) For PNGase F (SIGMA) treatment of pull-down proteins to assess glycosylation states of proteins pulled-down, the beads were resuspended in 20 μL of a buffer containing 20 mM ammonium bicarbonate pH 8.0, 1 % SDS, 100 mM beta-mercaptoethanol and incubated for 30 min at 65 °C. The tube was then placed in a magnet for 2 min. The supernatant was collected to which 20 μL of 0.25 M KCl was added to eliminate SDS by precipitation. To lower KCl concentration the supernatant (40 μL) was collected, diluted 10X with 20 mM ammonium bicarbonate pH 8.0 and concentrated by using a Microcon YM-3 centrifugal filter (Millipore). The concentrated sample was treated with PNGase F (2.5 units) in a final volume of 20 μL for 3 h at 37 °C. Gels were stained either with QC Colloidal Coomassie Stain (Bio-Rad) or Silver (BioRad Silver Stain Plus Kit) following manufacturer’s instructions.
Western blot analysis
Western blot analysis was done essentially as described  using pull-down proteins extracted from 2 × 107 5g12e8-coupled Dynabeads. Serial Western blot analyses of proteins pulled down—hDEspR, hGalectin-1, hRab-1b and hTMED10—were done sequentially by stripping and re-probing the same blot in the following order: 1st, human-specific DEspR mAb (5g12e8 40 μg/ml, secondary anti-mouse IgG at 1:20,000), 2nd, Rab-1b polyclonal antibody (pAb) (Santa Cruz Biotechnology, cat # sc-599, at 1 μg/ml, secondary anti-rabbit IgG at 1:20,000), 3rd, human-specific Galectin-1 (Abcam, cat # ab25138 at 0.4 μg/ml, secondary anti-rabbit IgG 1:20,000) and 4th, pAb TMED10 (Abcam, cat # ab72666, at 10 μg/ml, secondary anti-rabbit IgG 1:20,000).
Serial Western blot analyses of U87 tumor cell membrane and Cos1-hDEspR cell membrane pull-downs were done sequentially by stripping and re-probing the same blot in order: 1st, hDEspR mAb (5g12e8 40 μg/ml, secondary anti-mouse IgG at 1:20,000) and then 2nd, hGalectin-1 (Abcam, cat#ab25138, at 0.4 μg/ml, secondary anti-rabbit IgG 1:20,000).
Serial western blot of U87 tumor membrane pull-down was also reacted sequentially by stripping and re-probing the same blot using first the following in order: 1st, hDEspR mAb (6g8g7 40 μg/ml, secondary anti-mouse IgG at 1:20,000), and 2nd, hDEspR mAb (5g12e8 40 ug/ml, secondary anti-mouse IgG at 1:20,000).
Immunoreactive proteins were detected by chemi-luminescence using the ECL Western Detection kit (GE Healthcare) as per manufacturer’s specifications.
Mass spectrometry analysis
Mass spectrometry of pull-down proteins was custom-performed by Creative Proteomics (Shirley, NY). Briefly, proteins were reduced for 40 min with 5 mM dithiothreitol in 25 mM NH4HCO3 at room temperature and alkylated for 40 min with 15 mM iodoacetamide in 25 mM NH4HCO3 in the dark. After washed and dehydrated, the alkylated samples were digested overnight at 37 °C with trypsin in a 1:50 enzyme-to-substrate ratio (Promega, V5113). Following digestion, the peptide mixtures were acidified with trifluoroacetic (TFA) to 1 %, and desalted by home-made C18 tips. Finally, the desalted peptide samples were dried and dissolved in 10 μL of 0.1 % formic acid in water and subjected to nanoLC-MS/MS analysis in a Q Exactive mass spectrometer. The raw MS files were analyzed and searched against Uniprot human protein sequence database using Proteome Discoverer 1.4 (Thermo Fisher Scientific, USA). The parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable), and myristyl on glycine (variable), Asn to Asp (variable); the enzyme specificity was set to trypsin; the maximum missed cleavages were set to 2; the precursor ion mass tolerance was set to 10 ppm, and MS/MS tolerance was 0.6 Da.
Immunofluorescence analysis of subQ xenograft U87-CSC tumors
Double immunofluorescence staining was done as described . Human-specific anti-DEspR mAb (5g12e8) and human-specific anti-Galectin-1 antibody (Abcam, cat#ab25138) were labeled with AlexaFluor(AF)-488 or AF568, and used at 1 μg/ml for anti-Galectin-1 and at 100 μg/ml for 5g12e8 mAbs on fixed, paraffin-embedded sections following antigen-retrieval. Digital photomicroscopy was done using a Zeiss Axioskop fluorescence microscope with auto-exposure settings.
FACS analysis of U87-CSCs, MB-231-CSCs, H460-CSCs and Panc1-CSCs
U87-CSCs, MB-231-CSCs, H460-CSCs and Panc1-CSCs were incubated in ice-cold Hank’s balanced salt solution (HBSS, Invitrogen, NY) plus 2 % FBS containing: (a) 10 μg/ml AF-568 labeled 7c5b2 mAb, or (b) 10 μg/ml AF-568 labeled IgG2b as isotype control. Duplicate samples were incubated for 20 min at 4 °C, washed, resuspended in 1 % FBS/HBSS, 1 % PFA, filtered and analyzed on an LSR-II-FACS instrument. Analysis was done using FloJo Flow Cytometry Analysis Software (http://www.FloJo.com).
DEspR-inhibition of CSC-growth
DEspR-inhibition studies were performed as described . CSCs (2000/well) were seeded in ultra-low attachment 96-well plate and treated with different blocking anti-hDEspR mAbs (5g12e8, 7c5b2 and 6g8g7) at 100 μg/ml, compared with control non-treated CSCs with six replicates for each. CSCs were cultured in optimal (5 % CO2, humidified incubator at 37 °C) non-adherent conditions. Anti-hDEspR (5g12e8, 7c5b2 and 6g8g7) mAbs were added at seeding, day-2 and day-4. Live and dead CSCs were counted using Trypan Blue on day-5.
RNA sequence analysis
The NCBI Sequence Read Archive was searched on 7/17/2013 with a query sequence provided by the Genbank accession for “Homo sapiens dual endothelin-1(VEGFsp)/angiotensin II receptor (DEAR) mRNA, complete cds,” gi|144,954,325|gb|EF212178.1|, against 727 sequencing runs (Additional file 5).
Live cell imaging
Fluorescence and transmitted light images were acquired with a Zeiss LSM 710 Duo confocal microscope (Carl Zeiss, Thornwood NY). Excitation was from a 561 nm DPSS laser. A 63X 1.4 NA planapochromat oil immersion objective was used. The cells were in 35 mm dishes with coverglass bottoms. Emission was collected from 575–725 nm. Cells were maintained under physiological conditions using a Pecon stage-top incubation system which maintained 37° centigrade and 5 % CO2. Analysis was done using Image J (Image J, Wayne Rasband, NIH) with identical settings for brightness and contrast adjustments for all images. Live cell imaging with AF-568 labeled isotype IgG2b control was performed in identical conditions. At 60 min, cells were fixed briefly and mounted with Vectashield mounting medium with DAPI (Vector Laboratories, CA, Cat.# H-1200). Photomicroscopy was done using a Zeiss Axioskop fluorescence microscope (Carl Zeiss, Thornwood NY) with differential interference contrast (DIC).
All data were analyzed for normality and descriptive statistics. The following statistical tests were performed using SigmaPlot 11.0 or PRISM 5: one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test (MCT) for CSC-growth inhibition experiments. A P < 0.05 was considered statistically significant.
dual endotheiln-1/VEGFsp receptor
vascular endothelial growth factor signal peptide
cancer stem-like cell
allele-specific amplification-refractory mutation system
open reading frame
F-box and WD repeat domain containing 7
transmembrane emp24 domain-containing protein 10
ras-related protein Rab-1B
- PNGase F:
VLMH wrote the manuscript, co-supervised experimental design and data analysis, performed immunofluorescence analysis, co-supervised FACS analyses; MS performed RNA-seq analysis and revised manuscript; AMM performed Maxam–Gilbert sequencing, pull-down experiments and western blot analysis; GT performed pull-down experiments, western blot analysis, FACS analysis and DEspR inhibition of CSC-growth; KAP performed FACS analysis; KR and DJP performed mass spectrometry analysis; DJP advised on pull-down experiments and analysis; NR-O designed the experiments, supervised the studies, performed all statistical analyses and contributed to writing the manuscript. All authors read and approved the final manuscript.
We thank Francis J. Carr, Ph.D. for helpful discussions and manuscript editing; and Michael T. Kirber, Ph.D. for Live Cell Imaging in the Evans Department of Medicine Cell Imaging Core.
Availability of data and material
The datasets supporting the conclusions of this article are included within the article and its additional files.
Boston University has filed patents on DEspR for diagnostic and therapeutic agents with NRO and VLMH as co-inventors.
This work was partially supported by Ignition Awards from BU Office of Technology Development, and CTSI Award from BU-Clinical and Translational Science Institute Grant NIH-U54TR001012. Some Mass Spectrometry analyses were performed at Cold Spring Harbor Laboratory Shared Resources, which are funded by the Cancer Center Support Grant 5P30CA045508. The funding body had no role in design, in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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