Molecular aspects of glutathione synthetase deficiency

Sammanfattning: The tripeptide glutathione (GSH) is involved in several crucial pathways in the cell, for instance regulation of enzyme activity, DNA synthesis, and free radical scavenging. Synthesis of GSH takes place via two steps in the gamma-glutamyl cycle. In the first step, catalysed by gamma-glutamylcysteine synthetase, a peptide bond is formed between glutamate and cysteine. The enzyme glutathione synthetase (GS) subsequently adds glycine to the dipeptide forming GSH. Although inborn errors in GSH metabolism are rare, the most frequent disease is caused by mutations in the GS encoding gene (GSS) located on chromosome 20q11.2. Approximately 30 different mutations have been reported from a total of nearly 70 patients. Patients with GS deficiency present with a clinical picture ranging from compensated haemolytic anaemia to a complex disorder with additional symptoms including metabolic acidosis, 5-oxoprolinuria, and central nervous system impairment. The severity of the disease has been postulated to be related to the residual GS activity. Studies of wild-type recombinant GS revealed binding properties deviating from Michaelis-Menten kinetics for the binding of gamma-glutamyl substrates; two apparent Km. values were identified, indicating that human GS shows homotropic negative cooperativity in the binding of gamma-glutamyl substrate (Paper I). To elucidate the effect on the catalytic properties of GS, kinetic analyses were performed of 11 naturally occurring missense mutations from GS deficient patients by expressing recombinant mutated enzymes (Papers II and III). This identified mutations causing decreased enzyme stability, decreased substrate affinity, decreased catalytical velocity, and also showed that the mutations could affect the cooperative binding of the gamma-glutamyl dipeptide. In about 25 % of the patients, no mutations could be identified based on sequence analysis of the exons and exon/intron boundaries. Analysis of GS mRNA in cultured fibroblasts identified aberrant splicing patterns in all these patients (Paper IV). We conclude that to identify splice mutations and to avoid misclassification of splice mutations in coding exons as missense mutations, the mutation analysis should be complemented with RNA analyses. To elucidate the relationship between genotype and phenotype, data on genotype, enzyme activity, enzyme activity, metabolite levels, and clinical phenotype were analysed in 41 patients (Paper V). Ile patients were divided into three groups based on the severity of the disease. Correlation was found between GSH levels and GS activity in cultured fibroblast. The mild clinical phenotype could be distinguished from the moderate and severe phenotypes based on level of predicted GS activity. In addition, all mutations causing frameshift and premature stop codons were associated with the moderate and severe phenotypes. Moderate and severe phenotype could not be distinguished. We conclude that prediction of a mild versus a moderate or severe phenotype is possible based on genotype, GSH levels and predicted enzyme activity.