title: Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.
abstract: 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.
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Montioli
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Oppici
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.
authors.forename: Mirco
authors.initials: M
authors.lastname: Dindo
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Roncador
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gotte
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
authors.affiliation: Department of Life and Reproduction Sciences, University of Verona, Verona, Italy. Electronic address: carla.borrivoltattorni@univr.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Borri Voltattorni
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000483:Alleles; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D057075:Enzyme Assays; D015870:Gene Expression; D006038:Glyoxylates / Q000737:chemistry / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D011736:Pyridoxine / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D006038:Glyoxylates; D020035:Holoenzymes; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000637:Transaminases; C023067:glyoxylate aminotransferase; C031150:glyoxylic acid; D011736:Pyridoxine; D000409:Alanine
keywords: Alanine:glyoxylate aminotransferase; Pathogenic variant; Primary Hyperoxaluria Type 1; Pyridoxal 5′-phosphate; Pyridoxine treatment