P.35 - Translational biochemistry for dystroglycanopathies

Abstract

ISPD (isoprenoid synthase domain containing) mutations cause a wide clinical spectrum of muscular dystrophies (dystroglycanopathies). Human ISPD is a cytidyltransferase, catalyses the reaction from CTP and Ribitol-5-phosphate to CDP-Ribitol, a novel nucleotide sugar for functional alpha-dystroglycan. hISPD has one conserved cytidyltransferase domain and a second functional unknown non-conserved domain. We aim to correlate clinical phenotypes to structural impacts of ISPD mutations to decipher the underlying molecular mechanisms. First we established a high throughput in vitro protein thermostablity screening assay (Thermofluor assay, TF), by which we screened for the ligands of recombinant hISPD (purified from E.coli) and found out that Ribitol-5-Phosphate can only alter the protein thermostability in the presence of CTP. The thermostablities of missense mutants are also assessed by TF assay, together with protein half-life cycloheximide assay in HEK cells transfected with mutants revealing that some mutants have significant lower stabilities than wild type (p.Thr238Ile). Surprisingly, few mutants have a higher stability than wild type. Interestingly, we found that C-terminal truncating mutations of the second domain showed very low protein levels in HEK cells (p.Arg395, p.Arg396*), consistent with the fact that secondary domain is essential for protein stability and dimerization and with the associated severe form of CMD. Enzyme activates were measured by Pyrophosphate Fluormetric assay, revealed that most missense mutants have 30%-50% reduced activity. However, few mutations have a high enzymatic activity, those are currently being investigated for their ligand binding affinity (Km). In summary, we assessed the effects of the single amino acid changes on ISPD protein stability, protein structure, enzyme activities, and correlated this with the patient phenotype. Ultimately, using the high throughput Thermofluor assay, we screened a small chemical chaperones library as a proof-of-principle for personalized treatment of dystroglycanopathy patients.