The protein L-isoaspartate (D-aspartate) Omethyltransferase (PCMT1) can initiate the repair of agedamaged aspartyl and asparaginyl residues of intracellular proteins. The human gene PCMT1 encoding this enzyme has at least four polymorphic sites, one of which results in two major isoforms with either an Ile residue or a Val residue at amino acid position 119. The frequencies of the alleles encoding the Ile 119 and Val 119 variants are similar in Caucasian populations, but a predominance of the Ile 119 allele exists in Asian and African populations. Analyses of the enzymatic activities of the Ile 119 and Val 119 variants in red blood cell lysates show that the higher specific activity and thermostability of the Ile 119 isoform is balanced by the potentially compensating higher substrate affinity of the Val 119 isoform. In a preliminary attempt to find an association between genotype frequency at the PCMT1 locus and healthy aging, we compared the distribution of genotypes in a healthy older population of Ashkenazi Jewish individuals with that in a younger ethnically matched control group. We found that 65% of the healthy older population had the heterozygous genotype, greater than the 50% expected by Hardy-Weinberg equilibrium, suggesting a possible selection for having both alleles of the repair methyltransferase in successful aging. Three additional polymorphisms in noncoding regions of the methyltransferase gene were found to be biallelic and demonstrated nonrandom association in a specific haplotype with the codon 119 polymorphism. Finally, we also detected a heterozygous mutation in the splicing branch site of intron 2 that did not appear to affect activity. This study will help define the normal physiological range of activity for this repair methyltransferase and give us a better understanding of its role in the processes of aging and disease.
stability grouping and this polymorphism. The high thermal stability samples were all homozygous Ile, the Protein L-isoaspartyl methyltransferase (PIMT) is low thermal stability samples were all homozygous believed to play an important role in the disposition Val, and the intermediate thermal stability samples of age-damaged proteins by catalyzing the repair of were all heterozygous. Furthermore, this polymorabnormal isoaspartyl linkages resulting from the phism was responsible, in part, for the variation obspontaneous deamidation of asparaginyl residues or served in basal erythrocyte PIMT activity. These reisomerization of aspartyl residues. As a step toward sults will help provide a foundation for future studies testing the hypothesis that human disease-or age-reaimed at correlating levels of PIMT activity, or other lated pathology might be associated with a deficiency properties of this enzyme, with human disease. ᭧ 1997 in PIMT, we investigated basal activity and thermal Academic Press stability of PIMT in erythrocyte lysates from 299 U.S.Key Words: isoaspartate; protein repair; polymorfamily members. Thermal stability was measured bephism. cause it is a sensitive measure of variation in amino acid sequence. Basal activity was normally distributed with a mean { SD of 558 { 43 units/ml erythrocytes. Statistical analysis of the data revealed that basal PIMT activity exhibited a high degree of heritability.Protein L-isoaspartyl methyltransferase (EC 2.1.1.77, Enzyme thermal stability showed a skewed bimodal PIMT) 3 is an ubiquitous enzyme found in bacteria, frequency distribution, and segregation analysis of plants, and animals (1 -5). Its ability to recognize family member pedigrees was consistent with Mende-and methylate the unusual a-carboxyl group on the lian inheritance of two major alleles. No DNA was side chain of isoaspartate in proteins and peptides available from the family samples, so we tested two in vitro is well documented (6 -9) and supports the additional population samples for a known Ile/Val hypothesis that PIMT functions to repair damaged polymorphism at codon 119 and for PIMT activity and proteins. The presence of isoaspartate within a polythermal stability, using blood donated by 25 Norwe-peptide results in the insertion of a methylene group gians and by 20 Koreans. Single-stranded conformainto the peptide backbone, which may lead to loss of tional polymorphism analysis using polymerase chain enzymatic activity or other functional consequences. reaction revealed a 100% correlation between thermalIsoaspartyl residues are generated within susceptible amino acid sequences by deamidation of aspara-
The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to huPrecisely functioning proteins are generated with a man chromosome 6 and multiple transcripts arising high degree of fidelity by the cellular transcriptional through alternative splicing have been identified. Re-and translational machinery. Once synthesized, howstriction enzyme mapping, subcloning, and DNA se-ever, the proteins are immediately subjected to various quence analysis of three overlapping clones from a hu-spontaneous chemical degradative reactions that can man genomic library in bacteriophage P1 indicate that affect their biological activity (1, 2). One such degradathe gene spans approximately 60 kb and is composed tive process results in the deamidation of asparaginyl of 8 exons interrupted by 7 introns. Analysis of intron/ residues and the isomerization and racemization of exon splice junctions reveals that all of the donor and aspartyl residues, giving rise to D-and L-isoaspartyl acceptor splice sites are in agreement with the mam-and D-aspartyl derivatives (3, 4). The presence of these malian consensus splicing sequence. Determination of altered aspartyl residues can lead to protein inactiva- of a succinimidyl intermediate and has been shown to result in the net reconversion to the L-aspartyl residue (9, 10; Fig. 1). The role of this enzyme in protein repair is supported by recent studies showing that the recom-
The sequence of the Candida albicans inositol biosynthetic gene, CaINO1, and its flanking regions is determined in this study. The largest open reading frame has a coding sequence of 1560 base pairs, corresponding to a predicted protein of 521 amino acids. Three primary transcriptional start sites are found 64, 57 and 52 base pairs upstream of the ATG translational start site at position 1374. Five stop codons exist in a cluster at the end of the coding region. Within the upstream region TATA and CAAT eukaryotic regulatory sequences are identified along with regions corresponding to a 10 base pair inositol/choline responsive element consensus sequence. Computer analysis of the DNA sequence shows strong homology to the Saccharomyces cerevisiae INO1 gene. A comparison of the deduced amino acid sequence of the C. albicans INO1 gene product, inositol-1-phosphate synthase, with its homolog in S. cerevisiae shows 64% identity and 77% similarity. The differences between the two proteins are most prominent in the N-terminal regions.
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