Protein L-Isoaspartyl Methyltransferase from the Nematode Caenorhabditis elegans: Genomic Structure and Substrate Specificity

Abstract

We identified a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) in the nematode worm Caenorhabditis elegans. The methylation of abnormal L-isoaspartyl residues by this enzyme can lead to their conversion to L-aspartyl residues and represents a protein repair step for polypeptides damaged by spontaneous reactions during the aging process. We show that the levels of this enzyme increase 2-fold in C. elegans in the dauer larval form, a developmental stage where the organism can survive for extended periods of time. Utilizing degenerate oligonucleotide primers derived from conserved amino acid sequences of mammalian, plant, and bacterial L-isoaspartyl methyltransferases and PCR amplification, we made DNA probes that allowed us to obtain cDNA and genomic DNA clones encoding this enzyme in the nematode. The deduced amino acid sequence is 53% identical to the human enzyme and 29% identical to the Escherichia coli enzyme. Overexpression of the cDNA for the C. elegans enzyme in E. coli gave an active product with micromolar Km values for L-isoaspartyl-containing peptide substrates and for the methyl donor S-adenosyl-L-methionine. No methylation of D-aspartyl-containing peptides was detected under conditions where the human enzyme catalyzed this reaction, suggesting that the ability to methylate D-aspartyl residues in addition to L-isoaspartyl residues was a later evolutionary adaptation of this enzyme. The C. elegans gene for the methyltransferase, designated pcm-1, was mapped to a single site in a 31 kb region in the central portion of chromosome V. The gene is 3.2 kb in length and includes six introns. Although much smaller, its genomic organization is similar to that of the corresponding mouse gene, with identically positioned intron--exon splice junctions at five of seven sites. We propose that this gene plays an important role in facilitating the long term survival of this organisms.

Alternate metabolism during the dauer stage of the nematode Caenorhabditis elegans

“…Two histone H1-like genes showed increased dauer expression and the expression of one histone H1-like gene was reduced, suggesting that dauer chromatin may be altered. The DNA repair endonuclease C47D12.8 (Jones et al, 2001) and the protein repair gene pcm-1 are also upregulated in dauer larvae (Kagan and Clarke, 1995), providing further evidence for the increased emphasis on maintenance and repair processes during dauer diapause.…”

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…Additionally, no activity on D-aspartyl residues in short peptides was found with L-isoaspartyl methyltransferases isolated from E. coli (28), T. maritima (10), Arabidopsis (24), and nematodes (13), suggesting that the ability to methylate D-aspartyl residues might be a special adaptation of the methyltransferases in complex and long-lived mammalian species. Our finding here that the P. furiosus enzyme recognizes D-aspartyl residues in peptides with a 120-fold higher affinity than the human enzyme suggests, however, that the ability of cells to recognize spontaneously damaged proteins containing a Substrates were used at a final concentration of 1 mM.…”

“…The enzyme-mediated methylation reaction is followed by nonenzymatic steps that result in the net conversion of L-isoaspartyl residues to L-aspartyl residues, representing a potentially important mechanism for avoiding the accumulation of damaged proteins as cells age (4 -9). This methyltransferase is found in a wide array of organisms including eubacteria (10), plants (11,12), nematodes (13), insects (14), and mammals (15). Its amino acid sequence is highly conserved (16).…”

“…The K m for corresponding synthetic peptides containing D-aspartyl residues in place of L-isoaspartyl residues can be 700 -10,000-fold higher, however. Additionally, no methylation of D-aspartyl peptides has been detected for the E. coli (28), worm (13), and higher plant (24) enzymes. In mammalian cells, it is possible that the methylation of D-aspartyl residues can lead to eventual L-aspartyl formation in a repair reaction similar to that observed for L-isoaspartyl residues, although considerable amounts of D-isoaspartyl residues would also be expected to accumulate (27).…”

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

“…Thereafter, PIMT (serendipitously fulfilling one of the important criteria essential for successful phage display) has a low turnover rate for substrate release (37,38). The objective of the current work was to identify PIMT target proteins from phage display cDNA libraries normalized for transcripts present in quiescent and germinating Arabidopsis seeds.…”