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

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Abstract Heterozygousde novomutations in theActivity-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 mutantADNPmRNA. 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 inE. coliexpression 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.

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