Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone, but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. While studies suggest the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of yeast expressing minor allele mutants reflects reduced protein levels, indicating these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (SUPREX), and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5'-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized, and that pyridoxal 5'-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. As previous studies suggest stabilizing compounds (i.e., pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high-throughput screens for compounds that stabilize AGT mutants.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
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