In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Abstract

Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.Deficiencies in the enzyme alanine:glyoxylate aminotransferase (AGT) 3 cause primary hyperoxaluria type I (PH1), a severe autosomal recessive kidney stone disease (1, 2). In humans, AGT is responsible for conversion of glyoxylate to glycine in the liver. Without functional AGT, glyoxylate builds up and is converted to calcium oxalate, which is deposited in the kidneys and can lead to kidney stones and renal failure. In many patients, deficiency of AGT results from one or two amino acid changes that decrease the stability of this enzyme. As a result, AGT may be degraded, become improperly localized, or form nonfunctional aggregates (1, 2).Over 50 different mutations of AGT and two polymorphic variants have been identified (1, 3). The two allelic forms consist of a "wild-type" major allele, AGTma, and a minor allele, AGTmi. The minor allele is present in ϳ20% of European and North American populations and contains P11L and I340M substitutions in the amino acid sequence and a 74-bp duplication in intron 1 (4, 5). The P11L and I340M substitutions, particularly P11L, have several biochemical effects, including decreasing catalytic activity and slowing the dimerization rate, but by themselves are not disease-causing (4, 5). The minor allele of AGT is delete...

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

“…Second, the difference in stability between holo and apo is similar for AGT-Ma and AGT-Mi. This is in apparent contrast with what is recently claimed by Hopper et al (5). These authors state that AGT-Ma is stabilized by PLP to a greater extent than AGT-Mi.…”

“…Recent "in vitro" and "in vivo" studies have established that AGT-Mi is less stable and active than AGT-Ma (5). It has also been reported that some variants (G170R, I244T, F152I) encoded on the background of the minor allele have reduced steady-state protein levels, and it has been hypothesized that this reduction could be largely due to protein destabilization (5). However, although PLP seems to stabilize, even to a different extent, both AGT-Ma and AGT-Mi, it does not appear to have any stabilizing effect on the PH1-causing variants (5).…”

Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

“…We next analyzed alleles of AGT associated with PH1, including a minor allele polymorphic variant (AGTmi, with amino acid substitutions P11L and I340M), and a disease variant associated with the minor allele, I244T (AGTmi I244T ). AGTmi has a mild destabilizing effect, while the AGTmi I244T disease variant has greatly reduced stability (Lumb and Danpure 2000;Hopper et al 2008;Cellini et al 2010a). Using IDESA, we observed robust differences in growth of yeast expressing these variants, with yeast expressing AGTmi and AGTmi I244T showing poorer growth than yeast expressing wild-type AGT and yeast expressing AGTmi I244T showing a temperature-sensitive phenotype at 37° (Figure 1, B and C).…”

“…For proteins with unknown or undefined functions, identification of disease variants that affect activity but not stability can provide clues to their mechanisms of action. We chose 35 disease variants of AGT from PH1 patients (Williams et al 2009) that showed loss of function (,10% of wild-type AGT activity) in a yeast activity assay based on complementation (Hopper et al 2008). Within this set, we identified 12 mutants that showed good stability in IDESA ( Figure 4B, top), but poor activity in the complementation assay ( Figure 4B, bottom).…”

“…To generate methotrexate resistant (MTX r ) DHFR variants, the L22F/F31S amino acid substitutions in DHFR were introduced into pTB3-dhAGT by PCR and integrated using homologous recombination in yeast. Generation of HAtagged AGT variants used in half-life studies ( Figure 2E) is described previously (Hopper et al 2008). To generate pESCLeu-dhAGT constructs used in Figure 3B, the dhfrN-AGT-dhfrC (dhAGT) fusions were excised from pTB3 using BamHI and XbaI and cloned into the plasmid pESCLeu (Agilent Technologies) using BamHI and SalGI sites.…”