Phenotypic Analysis of Seizure-prone Mice Lackingl-Isoaspartate (d-Aspartate)O-Methyltransferase

Abstract

Within proteins and peptides, both L-asparaginyl and L-aspartyl residues spontaneously degrade, generating isomerized and racemized aspartyl residues. The enzyme protein L-isoaspartate (D-aspartate) O-methyltransferase (E.C. 2.1.1.77) initiates the conversion of Lisoaspartyl and D-aspartyl residues to normal L-aspartyl residues. This "repair" reaction helps to maintain proper protein conformation by preventing the accumulation of damaged proteins containing abnormal amino acid residues. Pcmt1-/-mice manifest two key phenotypes: a fatal seizure disorder and retarded growth. In this study, we characterized both phenotypes and demonstrated that they are linked. Continuous electroencephalogram monitoring of Pcmt1-/-mice revealed that abnormal cortical activity for ϳ50% of each 24-h period, even in mice that had no visible evidence of convulsions. The fatal seizure disorder in Pcmt1-/-mice can be mitigated but not eliminated by antiepileptic drugs. Interestingly, antiepileptic therapy normalized the growth of Pcmt1-/-mice, suggesting that the growth retardation is due to seizures rather than a global disturbance in growth at the cellular level. Consistent with this concept, the growth rate of Pcmt1-/-fibroblasts was indistinguishable from that of wild-type fibroblasts.Under physiological conditions, L-asparaginyl and L-aspartyl residues in polypeptides spontaneously degrade to L-and Disoaspartyl and D-aspartyl residues with half-times ranging from a few hours to hundreds of days (1-5). These abnormal residues can affect both the structure and function of polypeptides and may underlie a portion of the aging-related loss of cellular and tissue function (6 -9). However, many organisms have evolved a strategy for reversing at least some of this damage by an enzymatic methylation reaction that is targeted directly to peptides and proteins containing the major L-isoaspartyl degradation product. The widely distributed cytosolic protein L-isoaspartate (D-aspartate) O-methyltransferase, also termed protein carboxyl methyltransferase 1 (Pcmt1), 1 can initiate the conversion of L-isoaspartyl residues to L-aspartyl residues by forming the methyl ester of the L-isoaspartyl residue (10). This ester is then converted, in a nonenzymatic reaction, to an L-succinimidyl residue. Spontaneous hydrolysis of the L-succinimidyl residue produces either an L-aspartyl or an Lisoaspartyl residue. If an L-isoaspartyl residue is produced, additional rounds of methyl esterification, succinimide formation, and hydrolysis eventually convert it to an L-aspartyl residue.

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

“…These mice exhibited slow growth and a fatal seizure disorder leading to death after an average of 42 days of age (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998). In addition, PIMT-/-mice showed aberrant synaptic transmission in the CA3 region of the hippocampus, associated with an abnormal distribution of synaptic vesicles in the mossy fiber terminals (Ikegaya et al 2001).…”

“…It has been previously reported that PIMT activity is important for the maintenance of the central nervous system since PIMT-deficient mice died from progressive epileptic seizures and showed aberrant synaptic transmission in the hippocampus (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998;Ikegaya et al 2001). This suggests that changes in PIMT activity in the human brain during epilepsy, particularly in the hippocampus which is the major seat of temporal lobe epilepsy, may be involved in this disease.…”

“…These mice showed an accumulation of damaged aspartyl residues in all tissues, particularly in the brain (Kim et al 1997). Such knockout mice (PIMT-/-) also showed significantly slower growth as well as premature death, caused by fatal generalized seizures characteristic of fatal progressive epilepsy (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998). Also, these PIMT-/-mice show aberrant neurotransmission in the synapses of mossy fibers in the CA3 region of the hippocampus, associated with an atypical distribution of synaptic vesicles, abnormal microtubule organization in the dendrites of pyramidal neurons and vacuolar degeneration (Yamamoto et al 1998;Ikegaya et al 2001).…”

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

“…These mice accumulate higher levels of isoaspartate, relative to their wild-type littermates, in all tissues that normally express the enzyme (13)(14)(15). Pcmt1 Ϫ/Ϫ mice have abnormal neuronal excitability, and most die from an epileptic seizure at less than 2 months of age (13,14,16). They experience aberrant mossy fiber-CA3 region synaptic neurotransmission, vacuolar degeneration at the axon hillock of dentate cells, and disorganized microtubules within the dendrites of pyramidal neurons (14,17).…”

A known functional polymorphism (Ile120Val) of the human PCMT1 gene and risk of spina bifida

“…This process helps prevent cells from premature aging. When this gene is ablated in gene targeting studies, nullizygous Pcmt1 knockout mice suffer from epileptic seizures that result in the animal's death at an average of 42 days of age [7]. Farrar and Clarke [8] measured metabolites, including AdoMet and AdoHcy in the brains of Pcmt1 nullizygotes, Pcmt1 heterozygotes, and wildtype mice.…”