Biochemical and cellular effects of a novel missense mutation of the AGXT gene associated with Primary Hyperoxaluria Type 1.

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal disease caused by mutations in AGXT that lead to the deficiency of alanine:glyoxylate aminotransferase (AGT). AGT is a liver pyridoxal 5'-phosphate (PLP)-dependent enzyme that detoxifies glyoxylate inside peroxisomes. The lack of AGT activity results in a build-up of glyoxylate that is oxidized to oxalate, then culminating in hyperoxaluria often leading to kidney failure. Most pathogenic mutations reduce AGT specific activity because of catalytic defects, improper folding, mistargeting to mitochondria, reduced intracellular stability, dimerization, and/or aggregation. Administration of pyridoxine (PN), a precursor of PLP, is a therapeutic option available for PH1 patients carrying responsive genotypes through the ability of the coenzyme to behave as a chaperone. Here, we report the clinical and biochemical characterization of the novel mutation c.1093G > T (p.Gly365Cys) identified in a Japanese patient. In silico studies predict that the p.Gly365Cys mutation causes a steric clash resulting in a local rearrangement of the region surrounding the active site, thus possibly affecting PLP binding and catalysis. Indeed, the purified p.Gly365Cys mutant displays proper folding but shows an extensive decrease of catalytic efficiency due to an altered PLP-binding. When expressed in AGXT1-KO HepG2 cells the variant shows reduced specific activity and protein levels in comparison with wild type AGT that cannot be rescued by PN treatment. Overall, our data indicate that the mutation of Gly365 induces a conformational change at the AGT active site translating into a functional and structural defect and allow to predict that the patients will not be responsive to vitamin B6, thus supporting the usefulness of preclinical studies to guide therapeutic decisions in the era of precision medicine.

Diet and Adaptive Evolution of Alanine-Glyoxylate Aminotransferase Mitochondrial Targeting in Birds.

Adaptations to different diets represent a hallmark of animal diversity. The diets of birds are highly variable, making them an excellent model system for studying adaptive evolution driven by dietary changes. To test whether molecular adaptations to diet have occurred during the evolution of birds, we examined a dietary enzyme alanine-glyoxylate aminotransferase (AGT), which tends to target mitochondria in carnivorous mammals, peroxisomes in herbivorous mammals, and both mitochondria and peroxisomes in omnivorous mammals. A total of 31 bird species were examined in this study, which included representatives of most major avian lineages. Of these, 29 have an intact mitochondrial targeting sequence (MTS) of AGT. This finding is in stark contrast to mammals, which showed a number of independent losses of the MTS. Our cell-based functional assays revealed that the efficiency of AGT mitochondrial targeting was greatly reduced in unrelated lineages of granivorous birds, yet it tended to be high in insectivorous and carnivorous lineages. Furthermore, we found that proportions of animal tissue in avian diets were positively correlated with mitochondrial targeting efficiencies that were experimentally determined, but not with those that were computationally predicted. Adaptive evolution of AGT mitochondrial targeting in birds was further supported by the detection of positive selection on MTS regions. Our study contributes to the understanding of how diet drives molecular adaptations in animals, and suggests that caution must be taken when computationally predicting protein subcellular targeting.

Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

Combined liver-kidney transplantation for primary hyperoxaluria type I in children: Single Center Experience.

Primary hyperoxalurias are rare inborn errors of metabolism with deficiency of hepatic enzymes that lead to excessive urinary oxalate excretion and overproduction of oxalate which is deposited in various organs. Hyperoxaluria results in serious morbid-ity, end stage kidney disease (ESKD), and mortality if left untreated. Combined liver kidney transplantation (CLKT) is recognized as a management of ESKD for children with hyperoxaluria type 1 (PH1). This study aimed to report outcome of CLKT in a pediatric cohort of PH1 patients, through retrospective analysis of data of 8 children (2 girls and 6 boys) who presented by PH1 to Wadi El Nil Pediatric Living Related Liver Transplant Unit during 2001-2017. Mean age at transplant was 8.2 ± 4 years. Only three of the children underwent confirmatory genotyping. Three patients died prior to surgery on waiting list. The first attempt at CLKT was consecutive, and despite initial successful liver transplant, the girl died of biliary peritonitis prior to scheduled renal transplant. Of the four who underwent simultaneous CLKT, only two survived and are well, one with insignificant complications, and other suffered from abdominal Burkitt lymphoma managed by excision and resection anastomosis, four cycles of rituximab, cyclophosphamide, vincristine, and prednisone. The other two died, one due to uncontrollable bleeding within 36 hours of procedure, while the other died awaiting renal transplant after loss of renal graft to recurrent renal oxalosis 6 months post-transplant. PH1 with ESKD is a rare disease; simultaneous CLKT offers good quality of life for afflicted children. Graft shortage and renal graft loss to oxalosis challenge the outcome.

The Second Life of Methylarginines as Cardiovascular Targets.

Endogenous methylarginines were proposed as cardiovascular risk factors more than two decades ago, however, so far, this knowledge has not led to the development of novel therapeutic approaches. The initial studies were primarily focused on the endogenous inhibitors of nitric oxide synthases asymmetric dimethylarginine (ADMA) and monomethylarginine (MMA) and the main enzyme regulating their clearance dimethylarginine dimethylaminohydrolase 1 (DDAH1). To date, all the screens for DDAH1 activators performed with the purified recombinant DDAH1 enzyme have not yielded any promising hits, which is probably the main reason why interest towards this research field has started to fade. The relative contribution of the second DDAH isoenzyme DDAH2 towards ADMA and MMA clearance is still a matter of controversy. ADMA, MMA and symmetric dimethylarginine (SDMA) are also metabolized by alanine: glyoxylate aminotransferase 2 (AGXT2), however, in addition to methylarginines, this enzyme also has several cardiovascular protective substrates, so the net effect of possible therapeutic targeting of AGXT2 is currently unclear. Recent studies on regulation and functions of the enzymes metabolizing methylarginines have given a second life to this research direction. Our review discusses the latest discoveries and controversies in the field and proposes novel directions for targeting methylarginines in clinical settings.

AGXT2 and DDAH-1 genetic variants are highly correlated with serum ADMA and SDMA levels and with incidence of coronary artery disease in Egyptians.

Dimethylarginine aminodehydrolase (DDAH1) and alanine glyoxylate aminotransferase2 (AGXT2) are two enzymes that contribute in asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) metabolism. Hence they affect production and bioavailability of eNOS-derived nitric oxide (NO) and consequently healthy blood vessels. The major aims of the current study were to investigate the association of genetic variants of AGXT2 rs37369, AGXT2 rs16899974 and DDAH1 rs997251 SNPs with incidence of coronary artery disease (CAD) in Egyptians and to correlate these variants with the serum levels of ADMA and SDMA. The study included 150 subjects; 100 CAD patients and 50 healthy controls. Genotyping was performed by qPCR while the ADMA and SDMA concentrations were assayed by ELISA. Both serum ADMA and SDMA concentrations were significantly higher in CAD patients compared to controls (both p < 0.0001). Genotype distributions for all studied SNPs were significantly different between CAD patients and controls. Carriers of AGXT2 rs37369-T allele (CT + TT genotypes) and AGXT2 rs16899974-A allele (CA + AA genotypes) had 2.4- and 2.08-fold higher risk of having CAD than CC genotype in both SNPs (p = 0.0050 and 0.0192, respectively). DDAH1 rs997251 TC + CC genotypes were associated with 2.3-fold higher risk of CAD than TT genotype (p = 0.0063). Moreover, the AGXT2 rs37369 TT and AGXT2 rs16899974 AA genotypes were associated with the highest serum ADMA and SDMA while DDAH1 rs997251 CC genotype was associated with the highest ADMA. AGXT2 rs37369-T, AGXT2 rs16899974-A, and DDAH1 rs997251-C alleles represent independent risk factors for CAD in the Egyptians.

Folding Defects Leading to Primary Hyperoxaluria.

Protein misfolding is becoming one of the main mechanisms underlying inherited enzymatic deficits. This review is focused on primary hyperoxalurias, a group of disorders of glyoxylate detoxification associated with massive calcium oxalate deposition mainly in the kidneys. The most common and severe form, primary hyperoxaluria Type I, is due to the deficit of liver peroxisomal alanine/glyoxylate aminotransferase (AGT). Various studies performed in the last decade clearly evidence that many pathogenic missense mutations prevent the AGT correct folding, leading to various downstream effects including aggregation, increased degradation or mistargeting to mitochondria. Primary hyperoxaluria Type II and primary hyperoxaluria Type III are due to the deficit of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) and 4-hydroxy-2-oxoglutarate aldolase (HOGA1), respectively. Although the molecular features of pathogenic variants of GRHPR and HOGA1 have not been investigated in detail, the data available suggest that some of them display folding defects. Thus, primary hyperoxalurias can be ranked among protein misfolding disorders, because in most cases the enzymatic deficit is due to the inability of each enzyme to reach its native and functional conformation. It follows that molecules able to improve the folding yield of the enzymes involved in each disease form could represent new therapeutic strategies.

A Putative Mutation Hotspot of the AGXT Gene Associated with Primary Hyperoxaluria Type 1 in the Chinese Population.

Primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, is characterized by renal stones, nephrocalcinosis, and chronic kidney disease. PH1 is caused by defects in alanine glyoxylate aminotransferase (AGT, 392 amino-acid residues), which is encoded by the alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT) gene. This study aimed to determine the clinical, biochemical, and mutation spectrum of patients with PH1 from mainland China. Four patients (two adults and two children, age range: 1 to 34 years) from four unrelated families were admitted because of kidney stones. The adult patients had chronic kidney disease, while the pediatric patients retained the normal kidney function. Four mutations of the AGXT gene were detected, including one novel mutation, c.1015delG (p.V339Sfs*2). One adult male with late-onset PH1 is a compound heterozygote of the c.815_816insGA (p.S275Rfs*38) and c.1015delG (p.V339Sfs*2) mutations. These frame-shift mutations could result in the production of truncated AGT proteins. Other patients include an adult female who is heterozygous for c.473C>T (p.S158L) and c.815_816insGA mutations and two boys that are respectively homozygous for the c.815_816insGA mutation and for the c.614C>T (p.S205L) mutation. Thus, the c.815_816insGA mutation accounts for 4/8 alleles in the present study; importantly, the position c.815 represents the 5'-end of the consecutive wild-type sequence of GAGAGAGA. In conclusion, we describe one novel mutation, c.1015delG, and a common mutation, c.815_816insGA, of the AGXT gene among four unrelated families with PH1. Moreover, we suggest that the short repeat of the GA dinucleotide may represent a mutation hotspot in the Chinese population.

Effects of alanine:glyoxylate aminotransferase variants and pyridoxine sensitivity on oxalate metabolism in a cell-based cytotoxicity assay.

The hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) is caused by a functional deficiency of the liver-specific, peroxisomal, pyridoxal-phosphate-dependent enzyme, alanine:glyoxylate aminotransferase (AGT). One third of PH1 patients, particularly those expressing the p.[(Pro11Leu; Gly170Arg; Ile340Met)] mutant allele, respond clinically to pharmacological doses of pyridoxine. To gain further insight into the metabolic effects of AGT dysfunction in PH1 and the effect of pyridoxine, we established an "indirect" glycolate cytotoxicity assay using CHO cells expressing glycolate oxidase (GO) and various normal and mutant forms of AGT. In cells expressing GO the great majority of glycolate was converted to oxalate and glyoxylate, with the latter causing the greater decrease in cell survival. Co-expression of normal AGTs and some, but not all, mutant AGT variants partially counteracted this cytotoxicity and led to decreased synthesis of oxalate and glyoxylate. Increasing the extracellular pyridoxine up to 0.3µM led to an increased metabolic effectiveness of normal AGTs and the AGT-Gly170Arg variant. The increased survival seen with AGT-Gly170Arg was paralleled by a 40% decrease in oxalate and glyoxylate levels. These data support the suggestion that the effectiveness of pharmacological doses of pyridoxine results from an improved metabolic effectiveness of AGT; that is the increased rate of transamination of glyoxylate to glycine. The indirect glycolate toxicity assay used in the present study has potential to be used in cell-based drug screening protocols to identify chemotherapeutics that might enhance or decrease the activity and metabolic effectiveness of AGT and GO, respectively, and be useful in the treatment of PH1.

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

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

Multiple mechanisms of action of pyridoxine in primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is a rare hereditary calcium oxalate kidney stone disease caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). About one third of patients are responsive to pharmacological doses of pyridoxine (vitamin B6), but its mechanism of action is unknown. Using stably transformed Chinese Hamster Ovary (CHO) cells expressing various normal and mutant forms of AGT, we have shown that pyridoxine increases the net expression, catalytic activity and peroxisomal import of the most common mistargeted mutant form of AGT (i.e. Gly170Arg on the background of the polymorphic minor allele). These multiple effects explain for the first time the action of pyridoxine in the most common group of responsive patients. Partial effects of pyridoxine were also observed for two other common AGT mutants on the minor allele (i.e. Phe152Ile and Ile244Thr) but not for the minor allele mutant AGT containing a Gly41Arg replacement. These findings demonstrate that pyridoxine, which is metabolised to pyridoxal phosphate, the essential cofactor of AGT, achieves its effects both as a prosthetic group (increasing enzyme catalytic activity) and a chemical chaperone (increasing peroxisome targeting and net expression). This new understanding should aid the development of pharmacological treatments that attempt to enhance efficacy of pyridoxine in PH1, as well as encouraging a re-evaluation of the extent of pyridoxine responsiveness in PH1, as more patients than previously thought might benefit from such treatment.

Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase.

Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects. Gly161 is a highly conserved residue whose mutation to Arg, Cys or Ser is associated with PH1. Here we investigated the molecular bases of the AGT deficit caused by Gly161 mutations with expression studies in a mammalian cellular system paired with biochemical analyses on the purified recombinant proteins. Our results show that the mutations of Gly161 (i) strongly reduce the expression levels and the intracellular half-life of AGT, and (ii) make the protein in the apo-form prone to an electrostatically-driven aggregation in the cell cytosol. The coenzyme PLP, by shifting the equilibrium from the apo- to the holo-form, is able to reduce the aggregation propensity of the variants, thus partly decreasing the effect of the mutations. Altogether, these results shed light on the mechanistic details underlying the pathogenicity of Gly161 variants, thus expanding our knowledge of the enzymatic phenotypes leading to AGT deficiency.

Crystal structure of the S187F variant of human liver alanine: glyoxylate [corrected] aminotransferase associated with primary hyperoxaluria type I and its functional implications.

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5'-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5'-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results.

Unusual cause of cerebral calcifications in an 8-year-old girl.

Key Clinical Message: Genetic counseling and genetic screening for hyperoxaluria should be recommended for children with urinary lithiasis for early management to avoid progression to oxalosis especially if there is a family history of lithiasis.Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT) resulting in overproduction of calcium oxalates. In its later stage, a systemic deposit of calcium oxalates is observed. We present the case of an 8-year-old girl with exceptional neurological involvement secondary to this disease.

A novel mutation of human liver alanine:glyoxylate aminotransferase causes primary hyperoxaluria type 1: immunohistochemical quantification and subcellular distribution.

A novel alanine:glyoxylate aminotransferase (AGT) mutation involved in primary hyperoxaluria type 1 (PH1) was studied in Japanese patients. Two mutations in exon 7, c.751T>A and c.752G>A, lead to a W251K amino acid substitution. Proband 1 (patient 1) was homozygous for the W251K mutation allele (DDBJ Accession No. AB292648), and AGT-specific activity in the patient's liver was very low. To reveal the cause of the low enzymatic activity, the intracellular localization of AGT (W251K) was studied using immunohistochemistry and immunoelectron microscopy. The latter analysis showed that patient 2 had only one-fifth of the normal AGT expression per catalase, suggesting impairment of AGT (W251K) dependent transport into peroxisomes. Peroxisomal transport of human AGT is believed to be dependent on the presence of the type 1 peroxisomal targeting sequence. The C-terminal tripeptide of AGT, KKL is necessary for peroxisomal targeting. In cultured cells, EGFP-AGT (W251K) localized both in the peroxisome and cytosol. These results were consistent with the data obtained from liver analysis of patient 2. The subcellular distribution of AGT (W251K) and the results from a random mutagenesis study suggest that KKL is necessary for peroxisomal targeting of human AGT, but additional signal other than KKL may be necessary.

Primary hyperoxaluria type 1: pathophysiology and genetics.

Primary hyperoxaluria type 1 (PH1) is a rare genetic form of calcium oxalate kidney stone disease. It is caused by a deficiency in the liver-specific enzyme, alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme involved in the metabolism of glyoxylate. The excessive endogenous synthesis of oxalate that ensues leads to hyperoxaluria, and the crystallization of the poorly soluble calcium salt of oxalate is responsible for a severe kidney stone disease, which can progress to end-stage renal disease, systemic deposition of oxalate and death. Knowledge about metabolic precursors of glyoxylate and oxalate, molecular pathology of AGT and analytical methods for diagnosis and clinical assessment have allowed a better understanding of the mechanisms underlying PH1 and opened the door to new therapeutic strategies.

CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1.

AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5'-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates. Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.

Effect of alanine supplementation on oxalate synthesis.

The Primary Hyperoxalurias (PH) are rare disorders of metabolism leading to excessive endogenous synthesis of oxalate and recurring calcium oxalate kidney stones. Alanine glyoxylate aminotransferase (AGT), deficient in PH type 1, is a key enzyme in limiting glyoxylate oxidation to oxalate. The affinity of AGT for its co-substrate, alanine, is low suggesting that its metabolic activity could be sub-optimal in vivo. To test this hypothesis, we examined the effect of L-alanine supplementation on oxalate synthesis in cell culture and in mouse models of Primary Hyperoxaluria Type 1 (Agxt KO), Type 2 (Grhpr KO) and in wild-type mice. Our results demonstrated that increasing L-alanine in cells decreased synthesis of oxalate and increased viability of cells expressing GO and AGT when incubated with glycolate. In both wild type and Grhpr KO male and female mice, supplementation with 10% dietary L-alanine significantly decreased urinary oxalate excretion ~30% compared to baseline levels. This study demonstrates that increasing the availability of L-alanine can increase the metabolic efficiency of AGT and reduce oxalate synthesis.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.