Allele-specific Characterization of Alanine: Glyoxylate Aminotransferase Variants Associated with Primary Hyperoxaluria

Abstract

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.

Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1

“…Thus, DECA seems to be promising for further studies in animal models. Recently, an additional 75 missense mutations in AGT have been identified, and almost half are uncharacterized (7,29). DECA or similar small molecules may be beneficial for rescuing PH1 caused by these new AGT mutants.…”

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview

“…On the other hand, the purification and characterization of the variants expressed in E. coli are suitable to dissect and quantify the effect of a mutation on the functional properties of AGT as well as on its secondary, tertiary and quaternary structures [11][12][13]17,18,35,36,[51][52][53][54][55][56][57][58]. Yeast and cell-free systems allow a rapid but semi-quantitative determination of the impact of each mutation on the intracellular activity, stability and assembly of the protein [37,53,54,59]. Thus, only the combination of more than one approach can provide a detailed picture of the molecular defect of a pathogenic variant.…”

“…It has been demonstrated that PH1-causing mutations can affect both the thermodynamic and kinetic stability of the protein and decrease the activation free energy for denaturation [17]. Moreover, many studies show that the thermal destabilization of variants associated with the minor allele is exacerbated by the presence of the P11L polymorphism [59]. Additionally, an increased susceptibility to proteolytic cleavage, often observed in the variants, gives important information about conformational changes leading to the exposure of flexible regions [18,59].…”

“…Moreover, many studies show that the thermal destabilization of variants associated with the minor allele is exacerbated by the presence of the P11L polymorphism [59]. Additionally, an increased susceptibility to proteolytic cleavage, often observed in the variants, gives important information about conformational changes leading to the exposure of flexible regions [18,59]. In some cases, the structural changes caused by pathogenic mutations in AGT lead to a reduced stability of the dimeric structure and/or to an increased aggregation propensity.…”

“…The first case is much less frequent than the second one. In fact, the vast majority of pathogenic mutations impact the AGT folding, leading to a variety of downstream effects including a reduced protein level (as in the case of M195R and M195L variants) [59], a reduced half-life (as in the case of the A112D variant) [54], or an increased intracellular aggregation (as in the case of the I244T and G41R variants) [46,50]. A mutation can also cause more than one effect, as in the case of Gly161 variants, which show a strongly reduced expression level due to both the reduced half-life and the cytosolic aggregation of the protein [58].…”