Abstract
l-Isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1) can initiate the conversion of damaged aspartyl and asparaginyl residues to normal l-aspartyl residues. Mice lacking this enzyme (Pcmt1-/- mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation ("transgenic Pcmt1-/- mice") had brain PCMT1 activity levels that were 6.5-13% those of wild-type mice but had little or no activity in other tissues. The transgenic Pcmt1-/- mice lived, on average, 5-fold longer than nontransgenic Pcmt1-/- mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concentration of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1-/- mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine from Pcmt1-/- mice contained increased amounts of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.
Highlights
The spontaneous chemical modification of proteins by reaction with oxygen, water, sugars, and other abundant metabolites is unavoidable
Nonenzymatic hydrolysis of the succinimide ring readily occurs at either carbonyl to generate both normal aspartyl residues and isoaspartyl residues, in which the peptide backbone proceeds through the -carbonyl rather than the ␣-carbonyl moiety (12)
Quantitation of Damaged Aspartyl Residues—Cellular proteins were incubated at 37 °C for 2 h with 0.8 g of recombinant human L-isoaspartyl methyltransferase (24) in 0.2 M BisTrisHCl, pH 6.0, and 10 M [14C]AdoMet in a final volume of 40 l
Summary
Generation of Pcmt[1] Transgenic Mice—A rat neuron-specific enolase (NSE) promoter was used to direct the expression of mouse Pcmt[1] cDNA in the brains of transgenic mice. The supernatant fraction from homogenized tissues (10 – 60 g of brain, heart, or testis protein; 0.6 – 0.8 mg of erythrocyte protein) was incubated with 0.8 mg of ovalbumin (Sigma, Grade V) in 0.2 M BisTris-HCl, pH 6.0, containing 10 M [14C]AdoMet (53 mCi/mmol; Amersham Pharmacia Biotech) in a final volume of 40 l at 37 °C for 15 min. Incubations containing S-adenosyl-L-[methyl-14C]methionine, ovalbumin, and buffer constituted the “blank” for the assay; the radioactivity in these tubes (typically Ͻ5% of the nonblank samples) was subtracted from total counts in the determination of enzyme activity. Quantitation of Damaged Aspartyl Residues—Cellular proteins were incubated at 37 °C for 2 h with 0.8 g of recombinant human L-isoaspartyl methyltransferase (specific activity, 10,000 pmol of methyl esters formed on ovalbumin at 37 °C/min/mg protein) (24) in 0.2 M BisTrisHCl, pH 6.0, and 10 M [14C]AdoMet in a final volume of 40 l. The value of standard deviation here represents differences between individual mice; the variation of the assay within a single tissue extract was generally very low (Ͻ5%)
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