title: Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.
abstract: Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.
authors.affiliation: Department of Pediatrics, Children and Women's Health Centre of BC, University of British Columbia, 4500 Oak Street, Vancouver, BC, Canada. marioncm@interchange.ubc.ca
authors.forename: M B
authors.initials: MB
authors.lastname: Coulter-Mackie
authors.forename: Q
authors.initials: Q
authors.lastname: Lian
mesh_terms: D000595:Amino Acid Sequence; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008024:Ligands; D018832:Molecular Chaperones / Q000502:physiology*; D018716:Molecular Mimicry; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense / Q000502:physiology; D010447:Peptide Hydrolases / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D008024:Ligands; D018832:Molecular Chaperones; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000637:Transaminases; C019635:Alanine-glyoxylate transaminase; D010447:Peptide Hydrolases; D014357:Trypsin