Abstract:
Congenital disorder of glycosylation disease type Ic (CDG-Ic) is a human inherited disorder characterised by mis-glycosylation in cells for which the enzyme defect has been well characterised. The CDG-Ic disorder results in an accumulation of dolichylpyrophosphate-linked Man₉GlcNAc₂ within the cells of affected patients owing to lack of an active α-1, 3-glucosyltransferase (ALG6) and leading to cellular defects because of protein under-glycosylation.
The N-glycosylation pathway is conserved from yeast to humans, making Saccharomyces cerevisiae, a useful model to study the genetics and basic cell biology of the CDG-Ic disorder. To characterise the phenotypic signatures of cells associated with mis-glycosylation, we constructed a specific alg6 deletion mutant (alg6Δ) and the human CDG-Ic mutant with C998T point mutation in yeast. Four phenotypic assays namely growth inhibition phenotype, carboxypeptidase Y (CPY) glycosylation patterns, localisation of green fluorescent protein tagged proteins, and induction of the unfolded protein response were developed to measure the effects of lack of the α-1, 3-glucosyltransferase in the alg6Δ and the CDG-Ic mutants. Difference in these properties when compared to the laboratory wildtype yeast strain provided quantifiable phenotypes of the glycosylation disorder.
The CDG-Ic strain was treated with 3000 individual compounds in a primary drug screen from two commercial libraries comprising bioactive compounds to select for compounds able to revert growth phenotype to wildtype. A small panel of compounds was obtained and one compound, Se-(methyl) selenocysteine hydrochloride (SMSC), in a secondary screen caused significant improvement in growth, CPY glycosylation and caused a mislocalised glycoprotein reporter to revert to normal subcellular localisation. SMSC also substantially reduced the activation of the unfolded protein response in the CDG-Ic mutant. Based on such phenotypic rescue assays, we propose that SMSC may assist the misfolded proteins to fold correctly. This approach comprises a good basis for future drug screens to rescue the CDG-Ic disorder in animal models.
Synthetic genetic analysis (SGA) is a method for determining networks of genes modifying particular genes of interest. Results from the SGAs identified networks of modifying genes that were similar between the CDG-Ic mutation and the alg6 deletion mutant. Such SGA analyses revealed a limited set of back-up genes assisting the ALG6 function. It is proposed that the different clinical presentations observed between CDG patients could be a direct reflection of the combination of mutations in one or more genes of the interacting networks.
