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In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase.

Publication ,  Journal Article
Hopper, ED; Pittman, AMC; Fitzgerald, MC; Tucker, CL
Published in: The Journal of biological chemistry
November 2008

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.

Duke Scholars

Published In

The Journal of biological chemistry

DOI

EISSN

1083-351X

ISSN

0021-9258

Publication Date

November 2008

Volume

283

Issue

45

Start / End Page

30493 / 30502

Related Subject Headings

  • Vitamin B Complex
  • Transaminases
  • Saccharomyces cerevisiae
  • Pyridoxal Phosphate
  • Protein Binding
  • Polymorphism, Genetic
  • Oxamic Acid
  • Mutation
  • Ligands
  • Hyperoxaluria, Primary
 

Citation

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MLA
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Hopper, E. D., Pittman, A. M. C., Fitzgerald, M. C., & Tucker, C. L. (2008). In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase. The Journal of Biological Chemistry, 283(45), 30493–30502. https://doi.org/10.1074/jbc.m803525200
Hopper, Erin D., Adrianne M. C. Pittman, Michael C. Fitzgerald, and Chandra L. Tucker. “In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase.The Journal of Biological Chemistry 283, no. 45 (November 2008): 30493–502. https://doi.org/10.1074/jbc.m803525200.
Hopper ED, Pittman AMC, Fitzgerald MC, Tucker CL. In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase. The Journal of biological chemistry. 2008 Nov;283(45):30493–502.
Hopper, Erin D., et al. “In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase.The Journal of Biological Chemistry, vol. 283, no. 45, Nov. 2008, pp. 30493–502. Epmc, doi:10.1074/jbc.m803525200.
Hopper ED, Pittman AMC, Fitzgerald MC, Tucker CL. In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase. The Journal of biological chemistry. 2008 Nov;283(45):30493–30502.

Published In

The Journal of biological chemistry

DOI

EISSN

1083-351X

ISSN

0021-9258

Publication Date

November 2008

Volume

283

Issue

45

Start / End Page

30493 / 30502

Related Subject Headings

  • Vitamin B Complex
  • Transaminases
  • Saccharomyces cerevisiae
  • Pyridoxal Phosphate
  • Protein Binding
  • Polymorphism, Genetic
  • Oxamic Acid
  • Mutation
  • Ligands
  • Hyperoxaluria, Primary