Molecular and cellular insights into defects of human alanine:glyoxylate aminotransferase variants associated with Primary Hyperoxaluria Type I

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in the absence of appropriate treatments, in the whole body. PH1 is caused by the deficiency of human liver specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). AGT is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and calcium oxalate formation. Only two curative therapeutic approaches are currently available for PH1: the administration of pyridoxine, a precursor of PLP, which is only effective in a minority of patients (10-30%), and liver transplantation, a very invasive procedure. AGT is encoded by the gene AGXT for which two main polymorphisms can be found: the major allele (AGT-Ma) and the minor allele (AGT-Mi). Up to now, more than 150 mutations have been identified that lead to PH1 and several studies have tried to clarify the genotype/phenotype correlations. However, the mechanisms by which each mutation causes AGT deficiency at the protein level are still poorly understood. Therefore, we performed a side-by-side comparison between normal AGT and nine purified pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization and thermal stability of both the holo- and apo-form. Moreover a detailed analysis of the structural properties of the S187F-Ma variant and of the molecular and cellular properties of Gly161 variants has been undertaken. Altogether, the data obtained has allowed us (i) to provide evidence for the structural and/or functional effects caused by each mutation on the protein, (ii) to reassess previous data obtained with crude cellular extracts, and (iii) to indicate a suitable therapy among those already available, and to suggest new treatments strategies for patients bearing the mutations analysed.