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.

[primary hyperoxaluria type 1]

Alanine :glyoxylate aminotransferase (AGT ) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH 1 ). More than 75 PH 1 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 .

Diseases
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Diseases presenting "endogenous non-proteasomal protease" symptom

primary hyperoxaluria type 1

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