Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl
peptide bonds in age-damaged
proteins. It has been reported that
synuclein, a
protein implicated in
neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to
neurodegenerative diseases, we attempted to determine the stoichiometry of
isoaspartate accumulation in
synuclein in vivo and in vitro. Brain
proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl
proteins, and by immunoblotting to confirm the coincident presence of
synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl
proteins, but no signal in the position of
synuclein. This finding was corroborated by immunoprecipitation of
synuclein followed by on-blot (3)H-methylation. To assess the propensity of
synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-
synucleins were aged for two weeks by incubation at pH 7.5 and 37 °C. The stoichiometries of
isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of
isoaspartate per mol of
protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl
protein methyl
ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of
proteins to form isoaspartyl sites and the instability of the (3)H-methyl
esters used to tag them. The results presented here indicate that
synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl
ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in
proteins.