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.
Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona, Strada Le Grazie 8 37134 Verona, Italy.
Elisa
E
Oppici
Alessandro
A
Roncador
Riccardo
R
Montioli
Silvia
S
Bianconi
Barbara
B
Cellini
D000818:Animals; D001051:Apoenzymes; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D007124:Immunoenzyme Techniques; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001051:Apoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000637:Transaminases; C019635:Alanine-glyoxylate transaminase
AGT; Alanine:glyoxylate aminotransferase; BS(PEG)5; BSA; CHO; Chinese hamster ovary; DLS; Hereditary metabolic disease; IEM; IFM; PBS; PH1; PLP; PMP; Primary Hyperoxaluria; Primary Hyperoxaluria Type I; Protein aggregation; Protein misfolding; Pyridoxal phosphate; SEC; alanine:glyoxylate aminotransferase; bis-N-succinimidyl-(pentaethylene glycol)ester; bovine serum albumin; dynamic light scattering; immune electron microscopy; immunofluorescence microscopy; phosphate buffered saline; pyridoxal 5′-phosphate; pyridoxamine 5′-phosphate; size-exclusion chromatography