Alcohol dehydrogenase 5 Is a source of formate for de novo purine biosynthesis in HepG2 cells

Abstract

Background: Formate provides one-carbon units for de novo purine and thymidylate (dTMP) synthesis and is produced via both folate-dependent and folate-independent pathways. Folate-independent pathways are mediated by cytosolic alcohol dehydrogenase 5 (ADH5) and mitochondrial aldehyde dehydrogenase 2 (ALDH2), which generate formate by oxidizing formaldehyde. Formate is a potential biomarker of B-vitamin-dependent one-carbon metabolism. Objective: This study investigated the contributions of ADH5 and ALDH2 to formate production and folate-dependent de novo purine and dTMP synthesis in HepG2 cells. Methods: ADH5 knockout and ALDH2 knockdown HepG2 cells were cultured in folate-deficient [0 nM (6S) 5-formyltetrahydrofolate] or folate-sufficient [25 nM (6S) 5-formyltetrahydrofolate] medium. Purine biosynthesis was quantified as the ratio of [14C]-formate to [3H]-hypoxanthine incorporated into genomic DNA, which indicates the contribution of the de novo purine synthesis pathway relative to salvage synthesis. dTMP synthesis was quantified as the ratio of [14C]-deoxyuridine to [3H]- thymidine incorporation into genomic DNA, which indicates the capacity of de novo dTMP synthesis relative to salvage synthesis. Results: The [14C]-formate-to-[3H]-hypoxanthine ratio was greater in ADH5 knockout than in wild-type HepG2 cells, under conditions of both folate deficiency (+30%; P < 0.001) and folate sufficiency (+22%; P = 0.02). These data indicate that ADH5 deficiency increases the use of exogenous formate for de novo purine biosynthesis. The [14C]-deoxyuridine-to-[3H]-thymidine ratio did not differ between ADH5 knockout andwild-type cells, indicating that ADH5 deficiency does not affect de novo dTMP synthesis capacity relative to salvage synthesis. Under folate deficiency, ALDH2 knockdown cells exhibited a 37%lower ratio of [14C]-formate to [3H]-hypoxanthine (P < 0.001) comparedwith wild-type HepG2 cells, indicating decreased use of exogenous formate, or increased endogenous formate synthesis, for de novo purine biosynthesis. Conclusions: In HepG2 cells, ADH5 is a source of formate for de novo purine biosynthesis, especially during folate deficiency when folate-dependent formate production is limited. Formate is also shown to be limiting in the growth of HepG2 cells.