Fargue, S; (2012) From folding to function through compartmentalisation : influence of aminoacid changes and pyridoxal-5'-phosphate on the cell biology of alanine:glyoxylate aminotransferase. Doctoral thesis , UCL (University College London).

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Abstract

From folding to function through compartmentalisation - influence of aminoacid changes and roles of pyridoxal-5'-phosphate on alanine:glyoxylate aminotransferase. Primary hyperoxaluria type 1 (PH1), is a rare inherited disease caused by a deficiency in the liver-specific, pyridoxal-5’-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT). AGT is normally localized to the peroxisomes where it catalyses the conversion of the intermediary metabolite glyoxylate to glycine. In a subset of patients with PH1, linked with the Gly170Arg mutation, AGT is mistargeted to mitochondria and patients may respond to pharmacological doses of pyridoxine, a precursor of PLP. Using a model system of stably transformed CHO cells expressing different AGT variants, we have studied four mutations – Gly170Arg, Phe152Ile, Ile244Thr, Gly41Arg - on the background of a common polymorphism, Pro11Leu, characteristic of a minor AGXT allele. All four mutations unexpectedly result in the peroxisome-to-mitochondrion mistargeting of AGT. However, significant differences were found on AGT quaternary status and catalytic activity. These results emphasize the synergistic effect of the Pro11Leu polymorphism and disease-causing mutations and its potential to act as a mitochondrial targeting signal. Varying the concentration of pyridoxine in cells had a biphasic effect on AGT expression, activity and compartmentalization that differed between constructs. Where some mutants had improved function with additional pyridoxine, normal AGT was inhibited at higher levels. The results provide an explanation for the therapeutic effect of pyridoxine in some patients. Overall, PLP was shown to have both chaperone and prosthetic group effects on mutant AGTs. Using a specially developed cell-based assay of indirect glycolate toxicity, we have shown the lower ability of mutant AGTs to detoxify glyoxylate. This is the basis for a functional assay of AGT in mammalian cells which could be used to screen drugs targeted at AGT.

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