The rs35217482 (T755I) Single-Nucleotide Polymorphism in Aldehyde Oxidase-1 Attenuates Protein Dimer Formation and Reduces the Rates of Phthalazine Metabolism

Abstract

Aldehyde oxidase 1 (AOX1) is a molybdenum-containing enzyme that catalyzes the oxidation of a range of aldehyde compounds and clinical drugs, including azathioprine and methotrexate.The purpose of this study was to elucidate the effects of single-nucleotide polymorphisms (SNPs) in the coding regions of the human AOX1 gene on protein dimer formation and metabolic activity. Six variants (Q314R and H1297R [rs3731722]), with allele frequencies greater than 0.01 in 1 or more population, were obtained from the genome aggregation and 1000 Genomes project databases. Protein expression and dimer formation were evaluated using HEK293T cells expressing the wild-type (WT) or different SNP variants of AOX1. Kinetic analyses of phthalazine oxidation were performed using S9 fractions of HEK293T cells expressing WT or each the different mutant AOX1. Although we detected no significant differences among WT AOX1 and the different variants with respect to total protein expression, native PAGE analysis indicated that one of the SNP variants, T755I, found in East Asian populations, dimerizes less efficiently than the WT AOX1. Kinetic analysis, using phthalazine as a typical substrate, revealed that this mutation contributes to a reduction in the maximal rates of reaction without affecting enzyme affinity for phthalazine. Our observation thus indicates that the T755I variant has significantly negative effects on both the dimer

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“…The variability between lots has been attributed to DMD-AR-2023-001379 differences in method of preparation, handling, and decline in activity during storage and in postmortem tissues (Barr et al, 2013). Alterations in inter-individual AO activity have also been reported due to the presence of allelic variants of AOX1 gene, resulting in proteins with increased or decreased activity (Hartmann et al, 2012;Ueda et al, 2022). The in vitro activity of AO for most of its oxidative substrates is nonlinear with time due to concurrent inactivation (Abbasi et al, 2019) and the enzyme is also liable to substrate inhibition (Barr et al, 2013).…”

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“…However, enzyme activity did not appear to vary with ethnicity 23 . Studies investigating AOX genetic polymorphism have identified two SNPs in the hAOX1 gene that affect in vitro activity 25–27 . One SNP, rs35217482 (T755I, found in East Asian populations), appears to suppress protein dimerization in vitro in S9 fractions of HEK293T cells expressing mutant AOX, resulting in a > 50% reduction in phthalazine oxidation 27 .…”

“…Although in vitro analysis of AOX enzyme activity has identified two SNPs that impact activity, there are no data confirming their effect in vivo , and most SNPs identified to date appear to have no effect on AOX activity. Whereas the two SNPs were identified in an East Asian population 27 and an Italian population, 26 the wider ethnic distribution of these SNPs has yet to be determined. Taken together, the in vitro expression data and clinical PK data from other drugs metabolized by AOX indicate that there is no relevant ethnic sensitivity in AOX metabolism, and that ethnic differences in PK exposure due to variations in AOX metabolism are not expected.…”

“…23 Studies investigating AOX genetic polymorphism have identified two SNPs in the hAOX1 gene that affect in vitro activity. [25][26][27] One SNP, rs35217482 (T755I, found in East Asian populations), appears to suppress protein dimerization in vitro in S9 fractions of HEK293T cells expressing mutant AOX, resulting in a > 50% reduction in phthalazine oxidation. 27 A second SNP, rs56199635 (R921H), was identified in an Italian population resulting in an hAOX1 variant with poor enzyme activity in vitro with four AOX substrates tested.…”

“…[25][26][27] One SNP, rs35217482 (T755I, found in East Asian populations), appears to suppress protein dimerization in vitro in S9 fractions of HEK293T cells expressing mutant AOX, resulting in a > 50% reduction in phthalazine oxidation. 27 A second SNP, rs56199635 (R921H), was identified in an Italian population resulting in an hAOX1 variant with poor enzyme activity in vitro with four AOX substrates tested. 26 However, the functional impact of either mutation has yet to be evaluated in vivo.…”