<scp>C</scp>
            ‐terminal
            <scp>Lys</scp>
            or
            <scp>Arg</scp>
            Clipping in Proteins

doi:10.1002/9781119053354.ch3

Cited by 2 publications

(1 citation statement)

References 9 publications

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“…Many secreted proteins often undergo proteolytic processing by proteases, which can cause inconclusive western blot results 30 . 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 di cult because of epitope retention in the hydrophobic core of the protein which is not always fully exposed 16,31 . 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.…”

Section: Resultsmentioning

confidence: 99%

In Search of the Hidden Protein: Optimization of Detection Strategies for autism-associated Activity-Dependent Neuroprotective Protein (ADNP) mutants.

D’Incal

Cappuyns

Choukri

et al. 2022

Preprint

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Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) protein were found to be the common cause underlying the Helsmoortel-Van der Aa syndrome (HVDAS). With most of the patient mutations situated in the last exon, we previously demonstrated the predicted escape from nonsense-mediated decay by detecting mutant ADNP mRNA. In this study wild-type and mutant forms of ADNP are investigated at the protein level and therefore optimal detection of the protein is required. We postulate that detection of ADNP by means of western blotting has been ambiguous and address different strategies to optimize the ADNP signal. Validation of a new N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. By means of both a GFPSpark® and and Flag®-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. However, western blot assessment of immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect mutant ADNP protein, a scientific paradox up to today not yet resolved. This study aims to shape awareness for critical western blot assessment of ADNP variants and stimulates further research regarding ADNP expression by means of a validated multi-antibody approach.

show abstract

Section: Resultsmentioning

confidence: 99%

In Search of the Hidden Protein: Optimization of Detection Strategies for autism-associated Activity-Dependent Neuroprotective Protein (ADNP) mutants.

D’Incal

Cappuyns

Choukri

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

Tracing the Invisible Mutant ADNP protein in Helsmoortel- Van der Aa Syndrome patients

D'Incal,

Cappuyns,

Choukri

et al. 2023

Preprint

View full text Add to dashboard Cite

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines, showing disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detectnative mutant ADNP protein,whereas alternative experimental protein enrichment strategies such as co-immunoprecipitation and mass spectrometry also depend on appropriately characterized antibodies that are not currently available. This study aims to shape awareness for critical assessment of ADNP mutant protein analysis in Helsmoortel-Van der Aa syndrome.

show abstract

C ‐terminal Lys or Arg Clipping in Proteins

Cited by 2 publications

References 9 publications

In Search of the Hidden Protein: Optimization of Detection Strategies for autism-associated Activity-Dependent Neuroprotective Protein (ADNP) mutants.

In Search of the Hidden Protein: Optimization of Detection Strategies for autism-associated Activity-Dependent Neuroprotective Protein (ADNP) mutants.

Tracing the Invisible Mutant ADNP protein in Helsmoortel- Van der Aa Syndrome patients

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