Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells

Abstract

Failure to detoxify the intermediary metabolite glyoxylate in human hepatocytes underlies the metabolic pathology of two potentially lethal hereditary calcium oxalate kidney stone diseases, PH (primary hyperoxaluria) types 1 and 2. In order to define more clearly the roles of enzymes involved in the metabolism of glyoxylate, we have established singly, doubly and triply transformed CHO (Chinese-hamster ovary) cell lines, expressing all combinations of normal human AGT (alanine:glyoxylate aminotransferase; the enzyme deficient in PH1), GR/HPR (glyoxylate/hydroxypyruvate reductase; the enzyme deficient in PH2), and GO (glycolate oxidase). We have embarked on the preliminary metabolic analysis of these transformants by studying the indirect toxicity of glycolate as a simple measure of the net intracellular production of glyoxylate. Our results show that glycolate is toxic only to those cells expressing GO and that this toxicity is diminished when AGT and/or GR/HPR are expressed in addition to GO. This finding indicates that we have been able to reconstruct the glycolate-->glyoxylate, glyoxylate-->glycine, and glyoxylate-->glycolate metabolic pathways, catalysed by GO, AGT, and GR/HPR respectively, in cells that do not normally express them. These results are compatible with the findings in PH1 and PH2, in which AGT and GR/HPR deficiencies lead to increased oxalate synthesis, due to the failure to detoxify its immediate precursor glyoxylate. These CHO cell transformants have a potential use as a cell-based bioassay for screening small molecules that stabilize AGT or GR/HPR and might have use in the treatment of PH1 or PH2.

Four of the Most Common Mutations in Primary Hyperoxaluria Type 1 Unmask the Cryptic Mitochondrial Targeting Sequence of Alanine:glyoxylate Aminotransferase Encoded by the Polymorphic Minor Allele

“…The different AGT constructs used in this study are described in Table 1. AGT-MA, AGT-mi, AGT-anc, AGT-170, AGT-152, and AGT-41 were prepared as described previously (10,18,26,27). All of the AGT constructs except AGT-MA and AGT-244 already existed and were recloned from a pHYK vector into pcDNA3.1.…”

“…CHO cell lines expressing AGT variants were established by retransforming a CHO cell line previously transformed with GO and created for other purposes not described (27). Three cell lines expressed normal AGT variants: AGT-MA expressing AGT encoded by the major allele of AGXT, with AGT-mi expressing AGT encoded by the minor allele of AGXT, and AGT-anc expressing a variant of AGT-MA to which a more usual cleavable N-terminal MTS was added, similar to marmoset AGT (28).…”

Influence of nutrition on feline calcium oxalate urolithiasis with emphasis on endogenous oxalate synthesis

“…3). Any glyoxylate that is not reduced to glycolate or transaminated to glycine is oxidised to oxalate, a reaction catalysed by cytosolic L-lactate dehydrogenase (61) (Fig. 3(a), step I).…”

“…Species such as man and rabbits expressing AGT1 almost exclusively in peroxisomes have lost the first translation start site during evolution resulting in an MTS-lacking protein. In cats and (61,69) ; II, conversion of glyoxylate into L-glycine catalysed by alanine:glyoxylate aminotransferase 1 (AGT1) (64,74,75,82,83,92,100,101,105) ; III, conversion of glyoxylate into glycolate catalysed by glyoxylate reductase/hydroxypyruvate reductase (GR/HPR) (67,68,69,72,155,156) ; IV, conversion of hydroxypyruvate into D-glycerate catalysed by glyoxylate reductase/hydroxypyruvate reductase (67,68,69,72,155,156) . Essential metabolic conversions in situation B are indicated with Ia -d, II, III and IV, meaning: Ia, conversion of cytosolic D-fructose, D-glucose and D-galactose into D-glycerate (86,87,89) ; Ib, conversion of D-glycerate into hydroxypyruvate; Ic, conversion of hydroxypyruvate into glycolaldehyde (80) ; Id, conversion of glycolaldehyde into glycolate (80) ; II, conversion of peroxisomal glycolate into oxalate catalysed by glycolate dehydrogenase (GD) (97) ; III, conversion of peroxisomal glycolate into glyoxylate catalysed by glycolate oxidase (GO) (97) ; IV, conversion of glyoxylate into L-glycine catalysed by alanine:glyoxylate aminotransferase 1 (64,74,75,101,157) .…”

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

“…To confirm that DECA treatment restored AGT function to cells, we took advantage of a CHO-K1 cell model in which glycolate oxidase (GO) with AGT or AGT P11LG170R was stably expressed (25). This cell line was chosen because this glyoxylate pathway is not active in this cell type.…”