Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

Abstract

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4 -10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 ؎ 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme. Fig. 1, dashed arrows) constitutes a major pathway for spontaneous protein damage at physiological pH and temperature (1-3). The internal ␣-carboxyl characteristic of isoaspartyl sites is selectively methylated by a widely distributed protein L-isoaspartyl O-methyltransferase (PIMT) 2 (4 -6). Considerable evidence suggests that PIMT functions to repair this damage in vivo by the mechanism illustrated in Fig. 1 (solid arrows). PIMT selectively methylates the atypical ␣-carboxyl of the isoAsp site. This is followed immediately by a spontaneous demethylation to form a metastable succinimide. Spontaneous hydrolysis of the succinimide over a time course of 2-4 h generates a normal L-aspartyl site with an efficiency of 15-30%. Purified PIMT catalyzes conversion of isoaspartyl sites to normal Asp-Xaa linkages in synthetic peptides (7-9) and has been shown to restore function to isoaspartate-containing forms of proteins such as calmodulin (10) and the Escherichia coli phosphocarrier protein HPr (11). Pharmacological inhibition of PIMT activity in rat PC12 cells leads to reversible accumulation of isoaspartyl sites in a variety of proteins, including tubulin (12) and histone H2B (13). Formation of isoaspartyl sites (Studies with PIMT-deficient mice further support a repair function for PIMT (14,15). Whereas proteins in extracts from wild-type or heterozygous PIMT mice contain only trace levels of isoaspartate, proteins in extracts from knock-out mice show greatly increased isoaspartate levels in extracts from all tissues examined, w...

Protein Repair in the Brain, Proteomic Analysis of Endogenous Substrates for Protein L-Isoaspartyl Methyltransferase in Mouse Brain

“…Although each cycle through this repair pathway is only 15-30% efficient, recycling of the isoAsp sites through the PIMT reaction results in an overall repair efficiency of 85% or greater. This has been shown in vitro with synthetic isoaspartyl peptides (17,18) and with isoAsp-containing proteins such as calmodulin (19) and synapsin I (20). Moreover, recent studies demonstrating an unusual D/L enantiomer ratio of Asp-25 in histone H2B (an endogenous substrate for PIMT) strongly suggest that repair of isoAsp formation at this site occurs in vivo with an overall efficiency of ϳ87% (21).…”

Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

“…Likewise, isoaspartate-rich and potentially dysfunctional ␣-synuclein, ␤-synuclein, UCHL1, PEBP, clathrin light chains a and b, calreticulin, calmodulin, and synapsin 1 (a protein whose functions include tethering synaptic vesicles to the cytoskeleton (15,21,63)), could drive the aberrant synaptic transmission recorded in these mice (13,14,16,17).…”

“…Recently, Aswad and colleagues have improved the resolution of PIMT substrates (20,21) using Novex NuPage gels, which are cast at pH 6.4 and run with a pH 7.3 buffer. We have now further enhanced this procedure first by coupling isoelectric focusing between pH 4 and 7 to this near neutral pH molecular weight separation, and then by analyzing the radiolabeled methylated proteins transferred to a PVDF membrane using a Microchannel Plate autoradiographic imager (29,32).…”

“…Both techniques produced unrepaired proteins that have been identified by subsequent methylation by exogenous PIMT and radioactive S-adenosylmethionine (AdoMet). In this way, histone H2B (20), synapsin 1 (15,21), tau protein (14), calmodulin (21)(22), and tubulin (23)(24)(25) have thus far been shown to be physiological PIMT substrates.…”

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

“…Examples of pharmaceutical proteins with a tendency to accumulate isoAsp sites at physiological pH and temperature include recombinant human forms of growth hormone [9], tissue plasminogen activator [10], and interleukin 11 [11]. Examples of proteins found to accumulate isoAsp sites in vivo include tubulin [12], histone H2B [13], and the synaptic vesicle-associated protein synapsin I [14]. Quantitation and identification of isoAsp sites in proteins are important in understanding the factors that contribute to protein heterogeneity in both a pharmaceutical and a biological context.…”