Methylation at D-aspartyl residues in erythrocytes: possible step in the repair of aged membrane proteins.

Abstract

Reversibly methylated aspartyl residues in human erythrocyte membrane proteins are shown to be in the "unnatural" D configuration. This is demonstrated by treatment of proteolytically derived aspartic acid fi-[3H]methyl ester with Land D-amino-acid oxidases and by the resolution ofdiastereomeric L-leucyl dipeptides containing either L-or D-aspartic acid j3-methyl ester by ion-exchange chromatography. Based on this observation, we propose a novel role for eukaryotic protein carboxyl methyltransferases (protein 0-methyltransferase; S-adenosyl-Lmethionine:protein O-methyltransferase, EC 2.1.1.24). We suggest that these widely distributed enzymes function to recognize aspartyl residues that have racemized spontaneously for a subsequent repair reaction. This repair function is postulated to couple ester hydrolysis with the restoration of the original L configuration of the aspartyl residue. It is possible that similar types of racemization repair processes may occur by reversible covalent modifications at other residues. Other possible functions for Daspartic acid fl-methyl ester residues in proteins are considered.A widespread reaction in nature is the posttranslational modification of protein carboxyl groups by methyl ester formation (1). At least two classes of the associated enzyme, protein carboxyl methyltransferase (protein O-methyltransferase; S-adenosyl-L-methionine:protein 0-methyltransferase, EC 2.1.1.24), are known to exist (2). The first of these is a bacterial enzyme that catalyzes the methylation of chemoreceptors at glutamyl residues in an adaptive response to sensory stimuli (2, 3). The second class of enzyme activity, found in both prokaryotes and eukaryotes, shows a much broader substrate specificity and catalyzes the formation of extremely labile methyl esters, presumably at aspartyl residues (4). The function of this latter process is not clear.We are interested in determining the function ofmammalian protein carboxyl methylation and are studying this reaction in the human erythrocyte. We have shown that specific cytoskeletal and membrane proteins are reversibly methylated at aspartyl residues (4-6). The carboxyl methylation of these proteins is substoichiometric (less than 0.02 methyl groups per polypeptide chain) in all cases (5, 6), but we have determined that this level is consistently higher in older populations of erythrocytes (7). Similarly, experiments with the purified carboxyl methyltransferase from both erythrocytes and other mammalian tissues indicate that in vitro substrates are also substoichiometrically methylated (8,9). To understand why aspartyl residues at a given position in a sequence may be only partially methylated, we have investigated the nature ofthe aspartic acid residues that are methylated in the red cell.It has been shown that racemization of aspartic acid residues occurs in aging mammalian proteins (for a review, see ref. 10). For example, D-aspartic acid accumulates in lens proteins at a rate of 0.14% per yr (11). It seemed conceivable to us that similar ami...

Spontaneous Degradation and Enzymatic Repair of Aspartyl and Asparaginyl Residues in Aging Red Cell Proteins Analyzed by Computer Simulation

“…The hypothesis that damaged as partyl and asparaginyl residues might be subject to repair has arisen from the discov ery of an enzyme that transfers a methyl group from the biological methyl donor S-adenosyl-Z.-methionine to the carboxyl group of L-isoaspartyl and D-aspartyl resi dues, but not to normal L-aspartyl residues [McFadden and Clarke, 1982;Aswad, 1984;Murray and Clarke, 1984;Clarke, 1985], This methyltransferase activity has been found in a variety of cell types, including bac teria, plants, and many mammalian tissues [Clarke, 1985;O'Connor, 1987], but prior to 1981, little was known about its substrate specificity or function. This is because al though it can methylate nearly all cellular proteins in vivo and a number of additional proteins in vitro, only a small fraction of each protein species is actually methylated at any one time.…”

“…The first clue to the function of this methyltransferase came when erythrocyte membrane proteins modified in situ with radiolabelled methyl groups were digested with the protease car boxypeptidase Y (CPY). It was found that at least 20% of the radiolabel in the digest was in /)-aspartyl-P-methyl ester, the rest being in methanol and undigested material, suggest ing that £)-aspartyl residues were a major site of méthylation in vivo [McFadden and Clarke, 1982;Lowenson and Clarke, unpubl. results].…”

Localization of D-β-Aspartic Acid–Containing Proteins in Human Eyes

“…Recently, D-Asp was found in the lenses, 1-4 teeth, 5 bones, 6 brains, 7 skin, 8 aortas, 9 erythrocytes, 10 lungs, 11 and ligaments 12 of elderly donors. The presence of D-Asp in aged tissues of the living body is considered the result of racemization of L-Asp in proteins in such metabolically inert tissues.…”

Deficiency in Protein l-Isoaspartyl Methyltransferase Results in a Fatal Progressive Epilepsy

“…This intermediate ring can hydrolyze at either of its two carbonyl groups, yielding both isoaspartyl (isomerization) and normal aspartyl residues (McFadden and C larke, 1987). The methyltransferase activity has been widely detected in various organisms, which transfer the methyl residue to the side chain carboxyl group of L-isoaspartyl and D-aspartyl residues but not to normal L-aspartyl residues (McFadden and C larke, 1982;Aswad, 1984;Clarke, 1985;Ingrosso et al, 1989), and has been designated protein L-isoaspartyl methyltransferase (PIMT) (C larke, 1985). Experiments performed in vitro have demonstrated that the incubation of synthetic L-isoaspartyl-containing peptides with PIMT results in the conversion of at least 50% of the peptide to the normal L-aspartyl-containing form (Johnson et al, 1987;McFadden and Clarke, 1987).…”