Different primary ADNP antibodies mentioned in literature were tested for their ability to detect wild-type ADNP by means of immunoblot analysis. We started a preliminary evaluation with the following antibodies: goat polyclonal ADNP (E-20) antibody (Santa Cruz Biotechnology), mouse monoclonal ADNP (F-9) antibody (Santa Cruz Biotechnology), and rabbit polyclonal ADNP antibody (Bethyl Laboratories). Wild-type human ADNP has a predicted molecular weight of 124 kDa based on its amino acid sequence 14. However, a clear signal at the predicted molecular weight could not be detected as multiple bands were visible ranging from 50–250 kDa in total protein lysates from HEK293T, HeLa, SHSY-5Y and a control lymphoblastoid cell line (LCL) (Fig. 1A-C). Besides, we also selected a more recently developed ADNP antibody from the St. John’s Laboratory (STJ91502) to assess its western blot’s potency. Previous publications of Furman et al., (2014) and Gennet et al., (2008) reported that antibodies for ADNP detection present multiple bands after western blotting 19,21. This observation also applied for the St. John’s Laboratory ADNP antibody (STJ91502) tested here. The antibody only recognizes multiple bands ranging from 75–175 kDa in total protein lysates from HEK293T, HeLa, SHSY-5Y and LCL, only when using the SuperSignal™ West Femto Maximum Sensitivity Substrate (Fig. 1D). No visible bands were observed after incubation with a normal range Pierce™ ECL Western Blotting Substrate. Although the specific ADNP signal might be present alongside non-specific signals, use of the St. John’s Laboratory antibody failed to unambiguously detect ADNP expression levels. In summary, we were not able to unambiguously detect ADNP with the antibodies from Santa Cruz, Bethyl Laboratories and St. John’s Laboratory. The signals obtained after incubation with these antibodies resulted in a non-specific signal and none of them correspond to protein’s predicted molecular weight of 124 kDa.
Improved in vivo ADNP-specific protein detection by a new polyclonal N-terminal ADNP antibody (Aviva Systems)
Since we observed a decrease in the supposed ADNP signal after blocking with the immunization peptide in cell lines with the N-terminal antibody of Aviva Systems, we challenged the antibody’s capacity to detect a specific ADNP signal in different brain tissue regions of mouse, rat, and human origin. In line with the previous experiments, we performed the blocking peptide competition assay as assessment for the antibody specificity (Fig. 3). In the different mouse and rat brain regions a very clear ADNP signal can be observed at a molecular weight of 145 kDa, which totally disappears after supplementation with the blocking peptide (Fig. 3A-D). Expression was the lowest in the cerebellum compared to frontal cortex, hippocampus, and whole brain. These findings are indicative for reliable ADNP detection with the N-terminal antibody of Aviva Systems in different rodent brain regions. However, in post-mortem human brain lysates the situation seems less straightforward. Here, a clear ADNP signal appears in whole brain and frontal lobe lysates with a molecular weight of 145 kDa. Surprisingly, an additional signal of 85 kDa was observed in human brain only, which disappeared in the blocking peptide condition. This suggests that the 85 kDa band signal contains an epitope recognized by the N-terminal antibody, indicative for a proteolytic cleavage product of ADNP or an unknown protein isoform. Comparison of a post-mortem age-matched cerebellum of a healthy subject to a seven-year-old deceased boy carrying the heterozygous ADNP mutation p.His559Glnfs*3 showed no evidence for wild-type ADNP detection, expected at 150 kDa, possibly attributed to the long post-mortem interval. Besides, a clear degradation signal was present at the molecular weight of 30 kDa. In addition, we were unable to detect a mutant ADNP protein with a predicted molecular mass of 63 kDa (Fig. 3E-F).
Figure 3. A polyclonal N-terminal ADNP antibody from Aviva Systems detects ADNP specifically in murine and rat tissues and suggests proteolytic processing of the protein in the human brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats and humans were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems. (A-C) The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP in a range of 145 kDa with (E) additional lower mass signal of 85 kDa in all human brain regions. (B-D-F) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5x excess to antibody concentration reduced the signal observed at 145 kDa in all tested cell lines. Importantly, the 85 kDa band suggestive for proteolytic cleavage as well as degraded ADNP signal disappeared completely after immunization peptide supplementation. GAPDH was used as a loading control.
Different C-terminal ADNP antibodies show discrete specific immunoblot signals in a broad range of 35–145 kDa, significantly overlapping with the immunoblot pattern of the N-terminal antibody (Aviva Systems), suggestive of ADNP degradation mechanisms.
ADNP was initially discovered in the neurotrophic environment of glial cells, and hence can therefore potentially be secreted in the extracellular matrix 6. Many secreted proteins often undergo proteolytic processing by proteases, which can cause inconclusive western blot results 29. For this reason, we additionally tested several C-terminal antibodies raised against different amino acid residues of the ADNP protein, because N-terminal antibody detection can often be difficult because of epitope retention in the hydrophobic core of the protein which is not always fully exposed 6,30. The polyclonal C-terminal antibody of Protein Technology was raised against amino acids 757–1102 of ADNP and shows reactivity with mouse, rat, and humans. The antibody recognized a specific ADNP signal at 150 kDa in total protein lysates from HEK293T, HeLa, SHSY-5Y and a control LCL. However, additional signals were observed at 50 kDa and 37 kDa, possibly due to proteolytic cleavage, instability of the protein, or non-specific binding (Fig. 4A). Next, a polyclonal C-terminal antibody of Abcam was raised against amino acids 850–1050 of ADNP which shows immunoreactivity with mouse, rat, and humans. The antibody recognized a specific ADNP signal at 150 kDa in total protein lysates from HEK293T, HeLa, SHSY-5Y and a control LCL, contributing to the most intense signal of the three different C-terminal antibodies. However, clear signals were observed ranging from 50–125 kDa, suggestive of instability, degradation, or non-specific binding of the protein (Fig. 4B). Lastly, we implemented a polyclonal C-terminal antibody of the Sarma Laboratory, raised against amino acids 953–1102 with murine and human reactivity 24. The antibody also showed a clear ADNP signal at 150 kDa in total protein lysates from HEK293T, HeLa, SHSY-5Y and a control LCL. A smear of bands ranging from 65–150 kDa was detected and suggests decay of the protein (Fig. 4C). These results indicate that C-terminal antibodies raise reliable western blot results and uniformly conclude a molecular weight of 150 kDa of the non-processed ADNP protein in different in vitro sample materials.
Figure 4. Three independent commercially available C-terminal polyclonal ADNP antibodies detect ADNP specifically in different in vitro sample materials and show clear instability of the protein. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for three different C-terminal ADNP antibodies. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. All the tested antibodies recognized ADNP with a molecular weight of 150 kDa. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3.
Since ADNP protein could clearly be detected in different cell line lysates, antibodies were next evaluated for a reliable ADNP detection in in vivo sample materials. For this reason, we challenged the C-terminal antibody capacities to different brain regions of mouse, rat, and human origin, in parallel with the previously tested N-terminal antibody of Aviva Systems. Unfortunately, none of the tested commercial C-terminal antibodies provides peptide used for immunization and therefore no blocking strategy could be performed for verification. First, we performed a western blot analysis with these distinct C-terminal ADNP antibodies on different murine brain regions. Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124–250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A). The C-terminal antibody of Abcam showed a clear ADNP signal of 150 kDa with a suggested proteolytic cleavage band of 50 kDa (Fig. 5B). The C-terminal ADNP antibody of the Sarma Laboratory also indicated a clear band signal of 150 kDa (Fig. 5C). Next, we performed a western blot analysis with the same C-terminal antibodies on different rat brain regions. The C-terminal ADNP antibody of Protein Technology detected multiple faint bands ranging from 124–180 kDa in the tested brain regions (Fig. 5D). The Abcam antibody showed a specific ADNP signal at 150 kDa together with a possible proteolytic cleavage band of 82 kDa (Fig. 5E). The Sarma Laboratory antibody which is predicted to only react with mouse and human species, also showed a specific ADNP band in the rat cerebellum at 150 kDa with a fading signal in the other brain regions. In addition, visibility of multiple degraded band signals also suggests significant involvement of protein decay mechanisms (Fig. 5F). Ultimately, we subjected our selection of C-terminal antibodies to different adult and juvenile brain regions. The antibody of protein technology only detected wild type ADNP at 124–150 kDa in the adult frontal lobe and hippocampus, but not in the other adult or juvenile brain regions. Additionally, a clear proteolytic cleavage band signal of 50 kDa was observed in all tested brain lysates (Fig. 5G). Contrasting to the protein technology antibody, the Abcam antibody showed a clear ADNP signal at 150 kDa in the adult frontal lobe, but not in the other regions. However, two very strong band signals were observed in all the tested conditions at 65 kDa, respectively at 50 kDa, indicative for proteolytic processing of the protein (Fig. 5H). Like the observed ADNP signal at 150 kDa after incubation with the Abcam antibody, the Sarma Laboratory antibody was also able to capture a specific signal at the same molecular weight in the adult frontal lobe. A strong ADNP degradation signal can be visualized at the molecular weight of 37–75 kDa (Fig. 5I). These results highlight the complexity of ADNP detection and indicate that these results are antibody dependent. However, a combination of the N-terminal antibody together with C-terminal antibodies confirms a reliable molecular weight of ADNP at 145–150 kDa. We strongly recommend the implementation of these N-terminal and C-terminal antibodies in future research to monitor ADNP protein detection.
Figure 5. Different C-terminal ADNP antibodies detect ADNP in the range of 150 kDa and suggest proteolytic processing of the protein in the brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats, and humans were lysed in RIPA buffer and used as protein samples for the assessment with three C-terminal antibodies with the optimized dilutions listed in Table 3. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A-C) Murine samples indicate detection of ADNP in the range of 150 kDa with bands suggesting proteolytic processing at 50 kDa. (D-F) Rat samples indicate detection of ADNP in the range of 150 kDa with bands indicating proteolytic processing at 82 kDa after incubation with the C-terminal Abcam antibody. (G-I) Human brain samples indicate detection of ADNP at different molecular weights of 124–150 kDa in the adult frontal lobe and hippocampus and highlight the antibody differences in detection of ADNP. The three tested antibodies showed strong band signals at lower molecular weights, which could indicate proteolytic cleavage or degradation of the protein.
Verification of ADNP specific immunoblot detection strategies in a CRISPR/Cas9 Adnp knockout mESC cell model.
In mice, Adnp knockout homozygosity has detrimental effects on neural tube closure, leading to early embryonic death at day E8.5-E9 31. However, complete knockout of Adnp in mouse embryonic stem cells (mESCs) affects morphology, but not viability with cells expressing pluripotency markers. To study the molecular function of ADNP, deficient mESCs were created with CRISPR/Cas9 technology by (1) insertion a 3 kDa C-terminal Flag-AviTag (3x-DYKDDDDK) at the endogenous Adnp gene, (2) introduction of the homozygous mutations, p.Tyr718* and p.Lys407Valfs*31, fused to a C-terminal Flag-AviTag (3x-DYKDDDDK), and (3) complete Adnp knockout homozygosity, by targeting eight base pairs downstream of the ATG start codon in exon 3 and right downstream of the stop codon in exon 5 to cut out the entire coding region 2. Immunoblotting of total protein lysates of a parental control cell line and Adnp homozygous cell lines with the N-terminal antibody (Aviva Systems) resulted in detection of a band above the molecular weight marker of 150 kDa. Moreover, none of the two ADNP mutant proteins p.Tyr718* and p.Lys407Valfs*31 were detected at a lower expected molecular weight of 80 kDa and 48 kDa respectively. In contrast, strong bands were observed at lower molecular weights of 65 kDa and 37 kDa, which are also observed in the complete homozygous Adnp knockout condition, indicating non-specific binding (Fig. 6A). Administration of the blocking peptide caused complete loss of all observed signals after incubation with the N-terminal Aviva Systems antibody and may indicate remaining cross-reactive epitopes in homozygous Adnp knockout mESC cells, for example of ADNP2 32, and non-specific binding (Fig. 6B). To alternatively demonstrate stable translation of 3x-DYKDDDDK (Flag) wild-type ADNP and mutants, we performed the same western blot with incubation of a DYKDDDDK (Flag)-tag antibody. Here, we could confirm presence of wild-type ADNP protein around 150 kDa amongst a strong background signal together with the introduced mutants at the predicted lower molecular weights (Fig. 6C). To assess the molecular weight of ADNP, we tested three different C-terminal antibodies on the parental and CRISPR/Cas9 engineered mESC lines. Although none of the C-terminal antibodies contains the epitope for mutant ADNP detection, we questioned whether the wild-type band signal would disappear after antibody incubation in homozygous mutant cells. In fact, all C-terminal ADNP antibodies were able to detect wild-type ADNP with a strong signal of 150 kDa in the parental lines, respectively a faint signal in the CRISPR/Cas9-mediated cell line with wild-type ADNP 3x-DYKDDDDK. Although ADNP mutants could not be visualized with our C-terminal antibody panel, we observed complete disappearance of the wild-type ADNP signal in the Adnp homozygous knock-out condition, again confirming an observed molecular weight for endogenous ADNP of 150 kDa (Fig. 6D-F). In this section, we created Adnp mESCs containing either wild-type, homozygous mutants, or complete Adnp knockout to confirm its molecular weight and to verify the specificity of different N-terminal and C-terminal antibodies (described above). A combination of both antibodies raised against different ADNP epitopes as well as a complete disappearance of the ADNP signal in an Adnp homozygous knockout cell line provide definitive proof for an observed molecular weight of 150 kDa for wild-type ADNP. The presence of band signals at lower molecular weights in the knock-out conditions clearly indicates non-specific protein band as detected with the N-terminal ADNP antibody in mESCs.
Figure 6. Unambiguous detection of ADNP using homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines. mESCs containing either wild-type, homozygous mutants, or complete Adnp knockout were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal ADNP, 3x-DYKDDDDK, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight with additional lower mass signal of 37–65 kDa in Adnp homozygous and parental control mESCs. (B) Supplementation of the immunization peptide in a 5x excess to antibody concentration reduced all signals observed mESC lines, indicating that the N-terminal antibody does not bind ADNP specifically in mESCs. (C) Detection of wild-type and homozygous Adnp mutants by means of a C-terminal 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 150 kDa in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line using a DYKDDDDK antibody. Truncated ADNP mutants, p.Tyr718* and p.Lys407Valfs*31, were detected at a lower molecular weight of 80 kDa, respectively 48 kDa. (D-F) Wild-type ADNP detection by means of three different C-terminal antibodies in mESC lines. Wild-type ADNP was detected with a strong signal at 150 kDa in the parental control line with a rather decreased signal in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line. Disappearance of the 150 kDa band was observed in the mESC line with complete Adnp homozygosity, indicating a reliable molecular weight of 150 kDa for ADNP.
Unambiguous detection of ADNP with an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector: a dual antibody approach for reliable ADNP detection.
In addition to commercial ADNP antibodies, we next confirmed the molecular weight of 150 kDa by studying two expression clones of human recombinant ADNP with patient mutations in both an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector fused to human ADNP transfected in HEK293T cells. In both cases, the GFPSpark and N-DYKDDDDK (Flag) tag ensure direct specific detection of ADNP. To validate whether mutant forms of ADNP are stably synthesized, site-directed mutagenesis of the human ADNP expression vectors was applied to evaluate effects of nonsense and frameshift stop mutations similar to Helsmoortel-Van der Aa syndrome patient mutations. Protein lysates of HEK293T cells transfected with either wild-type or mutated ADNP constructs were analyzed by immunoblotting for anti-GFP, anti-DYKDDDDK (Flag), and the N-terminal antibody of Aviva Systems, since Helsmoortel-Van der Aa mutations are characterized by a premature stop codon truncating the C-terminus of ADNP 14. A GFP-positive signal was observed at 175 kDa in HEK293T cells transfected with the wild-type GFPSpark construct, while a DYKDDDDK-signal for ADNP was seen at 150 kDa (Fig. 7A/C). This dual approach confirms the previously observed 150 kDa signal for ADNP (see above), because GFP itself accounts for 25 kDa of the total molecular weight. The mutant forms of ADNP were observed at a lower molecular weight in the lysates of p.Tyr719*, p.Leu823Hisfs*6 and p.Asn832Lysfs*81 compared to a wild-type overexpression lysate, confirming the formation of a shorter mutant protein as an effect of the genetic mutation. The p.Arg173* nonsense mutation only appeared after immunoblotting of the GFP construct, but not in the DYKDDDDK-condition, perhaps due to stabilization of mutant ADNP by the larger GFP fusion protein, in line with our previous study which showed that N-terminal ADNP mutants are rapidly degraded by the proteasome 7. The DYKDDDDK-antibody showed a lot more non-specific binding after immunoblotting and for this reason we repeated the experiment under the same conditions with the selected N-terminal ADNP antibody (Aviva Systems) to compare the results of the Western blot for anti-GFP and anti-DYKDDDDK. Since immunoblotting using an anti-fusion tag antibody allows unambiguous detection of ADNP, the observed signal is a reliable reference to validate the signals of the anti-ADNP antibody, which showed inconclusive results in in vitro sample materials (Fig. 7B/D). Both immunoblots show similar results, detecting wild-type ADNP at a molecular weight of 175 kDa (GFPSpark) and 150 kDa (DYKDDDDK Flag). In line, the mutant constructs are detected at a lower molecular weight of 45 kDa (p.Arg173*), 105 kDa (p.Tyr719*), 118 kDa (p.Leu823Hisfs*6), and 127 kDa (p.Asn832Lysfs*81). Respectively, the flag-tagged ADNP mutants are detected at 20 kDa (p.Arg173*), 80 kDa (p.Tyr719*), 93 kDa (p.Leu823Hisfs*6), and 103 kDa (p.Asn832Lysfs*81), accounting for a molecular weight difference of 25 kDa for GFP. In this section, we expressed N-GFPSpark and N-DYKDDDDK tagged human ADNP clones for immunoblot assessment, which further corroborated a molecular weight of 150 kDa for wild-type ADNP. Additionally, we were also able to detect mutant ADNP forms of both expression vector constructs with a lower molecular weight with antibodies recognizing the epitope tag as well as the N-terminal antibody (Aviva Systems), indicating mutant ADNP protein at cellular levels.
Figure 7. Unambiguous detection of ADNP using an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector. (A) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-GFPSpark and mutated constructs using an anti-GFP antibody. (B) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-GFPSpark and mutated constructs using the N-terminal ADNP antibody (Aviva Systems). (C) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-DYKDDDDK (Flag) and mutated constructs using an anti-DYKDDDDK antibody. (D) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-DYKDDDDK and mutant constructs using the N-terminal ADNP antibody (Aviva Systems). The observed molecular weight of wild-type ADNP-GFPSpark is 175 kDa (including 25 kDa GFPSpark tag), respectively ADNP-DYKDDDDK 150 kDa, with each of their mutants showing a lower molecular weight as a consequence of the truncating mutations. Detection with antibodies for GFP, DYKDDDDK (Flag), and ADNP gave comparable results. GAPDH was used as a loading control in all experiments.
Detection of heterozygous truncated ADNP mutants in a CRISPR/Cas9-engineerd HCT116 colon cancer cell line.
Previously, we have demonstrated stable translation of ADNP mutants in homozygous CRISPR/Cas9 endonuclease-mediated mESCs and an in an overexpression cellular model. However, these artificial systems do not approach the actual human disease condition e.g., Adnp homozygosity is embryonically lethal 1,31 and ADNP patients are all unified by heterozygous ADNP mutations 13,14, with endogenous ADNP protein expressed at nearly femtomolar concentrations 1,17. For this reason, we further implemented a HCT116 colon cancer cell line, expressing an intact ADNP allele fused to 3xFlag-V5-loxP-neonGreen-STOP[-loxP], increasing the molecular weight by an additional 32 kDa together with a mutant allele coupled to 3xHA-loxP-mCherry-STOP[-loxP], attributing an additional 25 kDa. The mutant allele carried the most prevalent ADNP nonsense mutation, p.Tyr719* 14. Expression of one intact wild-type allele and one ADNP mutant allele models the human condition in closer proximity. Western blotting of total protein lysates of an unmodified HCT116 control and the ADNP heterozygous lines resulted in detection of a non-specific band above the molecular weight marker of 150 kDa. Additionally, expression of the ADNP mutant allele was not observed at a lower molecular weight of 105 kDa. Absence of the expected mutant went hand in hand with a non-specific band at 45 kDa (Fig. 8A). Administration of the blocking peptide caused complete loss of all observed signals after incubation with the N-terminal Aviva Systems antibody, proving this antibody is not suitable for reliable ADNP detection in HCT116 cells (Fig. 8B). To detect stable translation of wild-type and p.Tyr719* mutant ADNP, we implemented a western blot with incubation of a DYKDDDDK (Flag)-tag antibody, indicating the wild-type ADNP allele, and a HA-tag antibody for detection of the mutant protein. Using the DYKDDDDK (Flag)-tag antibody, we could show presence of wild-type ADNP approximately at 182 kDa amongst lower background signals (Fig. 8C). Detection of the p.Tyr719* mutation was mediated by incubation with a HA-tag antibody, showing presence of a truncated ADNP protein of 105 kDa with a lower degrading smear, indicating instability of the mutant protein (Fig. 8D). Since C-terminal antibodies do not detect the epitope for p.Tyr719* mutant recognition, we mainly detect endogenous and CRISPR-engineered wild-type ADNP in HCT116 mutant and control cell lines. Here, we could detect endogenous ADNP at a molecular weight of 150 kDa in the control cell line. Moreover, the modified Adnp wild-type allele shows expression of an intact ADNP protein, 32 kDa above its observed molecular weight of 150 kDa as detected by each of the three different C-terminal antibodies. Degrading signals were observed at lower molecular weights (Fig. 8E-G). Altogether, we show evidence for detection of a truncated mutant ADNP protein at lower molecular weight in an Adnp heterozygous colon cancer cell line. In line with previous observations, we detected endogenous ADNP levels at a molecular weight of 150 kDa, confirming an immunoblot signal above its theoretical mass.
Figure 8. Western blotting of ADNP in a HCT116 colon cancer cell line, carrying the prevalent heterozygous p.Tyr719* mutation. HCT116 cells containing a wild-type and p.Tyr719* mutant allele were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal antibody, 3x-DYKDDDDK, HA-tag, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control in all experiment. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight an additional signal of 45 kDa, indicating proteolytic cleavage or non-specific binding. (B) Administration of the immunization peptide in a 5x excess to antibody concentration reduced all signals, indicating that the N-terminal antibody does not bind ADNP specifically in HCT116 cells. (C) Detection of wild-type ADNP by means of the 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 182 kDa in the 3xFlag-V5-loxP-neonGreen/3xHA-loxP-mCherry engineered line using a DYKDDDDK antibody, 32 kDa by tag insertion. (D) Detection of mutant ADNP by means of the HA-epitope tag. A truncated mutant p.Tyr719 ADNP protein was detected in at 105 kDa in the 3xFlag-V5-loxP-neonGreen/3xHA-loxP-mCherry engineered line using a HA-antibody, 25 kDa above its predicted molecular weight by tag insertion. Instability of the truncated protein was observed by a degrading smear. (E-G) Wild-type ADNP detection by means of three different C-terminal antibodies. Non-processed ADNP was detected with a strong signal at 150 kDa in the control line and at a molecular weight of 182 kDa in the genome-edited cell line. In both cases, a degrading smear was observed, indicating instability of the wild-type protein.
Lack of detectable mutant ADNP protein after immunoblot analysis in patient-derived sample materials.
Our artificial expression systems allowed assessment of the validity of the blocking peptide strategy, and it also provided evidence on the effect of ADNP mutations at the protein level. To determine whether truncated ADNP is retained in patients carrying the heterozygous N-terminal lys408Valfs*31 and C-terminal Asn832Lysfs*81 mutations, we examined ADNP protein levels in their corresponding mutant hiPSCs compared to an unrelated control. We used ADNP antibodies that specifically recognize a region within the N-terminus (Aviva Systems) and C-terminus (Protein Technology, Abcam, and Sarma Laboratory) of ADNP. Western blot analysis showed that a truncated fragment with a theoretical molecular weight of 48 kDa for the lys408Valfs*31 mutation, respectively 127 kDa for the Asn832Lysfs*81 mutation together with a wild-type ADNP signal could not be detected after N-terminal antibody incubation. The N-terminal ADNP antibody only recognized an epitope at the molecular weight of 25 kDa, suggestive for degradation (Fig. 9A). The signal of 25 kDa disappeared after administration of our blocking peptide competition assay, suggesting recognition of an ADNP epitope in the degraded signal (Fig. 9B). All C-terminal antibodies recognize wild-type ADNP at a molecular weight of 150 kDa with a degrading smear, confirming previous observations in our artificial overexpression model as well as in cell lines and tissue samples (Fig. 9C-E). Interestingly, the C-terminal antibody of Protein technology was raised against aa757-1102 and can therefore detect a possible Asn832Lysfs*81 mutant protein with an expected molecular weight of 127 kDa. Again, no mutant ADNP protein could be detected (Fig. 9C). Signal quantification of ADNP in mutant to control cell lines normalized by GAPDH resulted in a decreased relative expression for the C-terminal Asn832Lysfs*81 mutation for all three antibodies, but not for the lys408Valfs*31 mutation where the mutant-to-wild-type expression ratio showed a higher signal with the antibodies of Protein Technology and Abcam (Fig. 9E).
Figure 9. Western blotting of ADNP in human induced pluripotent stem cells (hiPSCs), carrying distinct heterozygous ADNP mutations mediated by CRIPSR/Cas9. (A-B) hiPSCs were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems) with application of our blocking peptide competition assay. Here, no reliable ADNP signal was detected. The molecular weight of the ADNP mutant lines is expected to decrease to 127 kDa for the Asn832Lysfs*81, respectively to 48 kDa for the lys408Valfs*31 line. However, no signal is observed at the predicted weight for the mutations. (C-E) The C-terminal antibodies of Protein Technology, Abcam, and the Sarma Laboratory were able to visualize wild-type ADNP at 150 kDa. Possessing the desired epitope for mutant ADNP detection, the C-terminal antibody of Protein technology was not able to capture the predicted truncated protein. GAPDH was used as a loading control. The ADNP signal was quantified determining the ratio of the wild-type protein in mutant to control cell lines. Here, the relative ADNP expression decreased in the Asn832Lysfs*81 cell line compared to the control, whereas mutant-to-wild-type expression ratio showed a higher signal with the antibodies of Protein Technology and Abcam in the lys408Valfs*31 cell line.
In order to further investigate the presence of endogenous mutant ADNP, we performed western blotting experiments with the same antibody panel in LCLs of four healthy subjects and six ADNP patients carrying distinct mutations spread all over the protein, e.g., Q40*, Ser404*, Leu831Ilefs*82, and Asn832Lysfs*81. First, all three C-terminal antibodies were able to detect wild-type ADNP at a molecular weight of 150 kDa. The antibody of Protein Technology, raised against aa757-1050 and thus in part able to recognize the mutant proteins formed of the p.Leu831Ilefs*82 and p.Asn832Lysfs*81 ADNP mutations, did not show a truncated ADNP protein with a predicted molecular weight of 127 kDa, and solely detected expression of the wild-type protein with no difference (ns, p = 0.32; student T-test) in ADNP levels in LCLs of patients compared to healthy subjects. Moreover, two strong non-specific bands were observed at 75 kDa and 50 kDa, as previously shown in mESCs (Fig. 10C/F). The other C-terminal antibodies do not contain the necessary epitope for mutant ADNP recognition and showed a significant decrease in the expression of ADNP (*p = 0.04; student T-test) with the C-terminal antibody of Abcam and (*p = 0.02; student T-test) with the C-terminal antibody of the Sarma Laboratory in LCLs of patients compared to healthy controls (Fig. 10C-F). On the other hand, the N-terminal antibody recognized a wild-type ADNP signal at 150 kDa alongside other protein bands at lower molecular weights, showing no difference in the expression (ns, p = 0.42; student T-test) in the patient LCLs compared to the control lines (Fig. 10A). The wild-type ADNP signal at 150 kDa together with the non-specific lower band signals disappeared after administration of the blocking peptide (Fig. 10B). Surprisingly, no mutant proteins could be detected in the ADNP heterozygous LCLs from different patients after N-terminal antibody incubation. Moreover, non-specific band signals at 50 kDa and 75 kDa were observed in all LCLs with a stronger intensity in patient lines. In contrast to the specific ADNP signal in murine, rat and human brain sections, we report that the N-terminal antibody recognizes a specific ADNP signal alongside non-specific band signals in lymphoblastoid cell lines, which can be attributed to the higher signal-to-noise ratio in brain versus lymphoblastoid model systems. Taken together, western assessment of different patient-derived sample materials showed no detectable mutant ADNP protein levels irrespective of the selected antibody, suggestive for degradation and/or absence of a mutant ADNP protein in the Helsmoortel-Van der Aa syndrome.
Figure 10. Absence of a mutant ADNP protein after immunoblotting of different lymphoblastoid cell lines from Helsmoortel-Van der Aa syndrome patients. (A) LCLs of four control subjects and six patients were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems). The expected wild-type ADNP signal presented at 150 kDa together with two non-specific bands at 50 kDa and 75 kDa with no difference in expression (p = 0.42; ns) of the wild-type protein. However, the ADNP mutants at a lower molecular weight of 127 kDa for the Asn832Lysfs*81 and Leu831Ilefs*82 mutations, respectively to 45 kDa for the Ser404* mutation, and to 10 kDa for the cell line carrying the Gln40* mutation could not be visualized. (B) Administration of the immunization peptide in a 5x excess to antibody concentration reduced all signals, indicating that the N-terminal antibody recognized ADNP specifically in LCLs alongside non-specific band signals. (C-E) C-terminal antibodies detected wild-type ADNP at a molecular weight of 150 kDa. No mutant ADNP was observed with the antibody of Protein Technology which is capable to recognize a part of the truncated Asn832Lysfs*81 and Leu831Ilefs*82 mutations. (F) All C-terminal antibodies visualized wild-type ADNP at 150 kDa, with only the Abcam (p = 0.04; *) and Sarma Laboratory (p = 0.02; *) antibodies showing the expected reduction of ADNP in LCLs of Helsmoortel-Van der Aa syndrome patients. GAPDH was used as a loading control.