Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We have synthesized a series of L-isoaspartyl-containing (isoD) peptides and characterized their interaction with the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77). Our findings indicate that this enzyme interacts with 6 residues extending from the isoD-2 to isoD+3 positions in peptide substrates. Although peptides as simple as G-isoD-G are methylated with low affinity (Km = 17.8 mM), a wide variety of L-isoaspartyl-containing sequences in larger peptides are recognized with high affinity (Km less than 20 microM), the best yet discovered being VYP-isoD-HA, with a Km of 0.29 microM. Only two sequence elements have been found that can interfere with the high affinity binding of peptides of 4 or more residues, these being a prolyl residue in the isoD+1 position and negatively charged residues in the isoD+1, isoD+2, and/or isoD+3 positions. We investigated the effect of higher order structure on binding affinity using several L-isoaspartyl-containing proteins. Although conformation did, in some cases, lower the affinity of the methyltransferase for L-isoaspartyl residues, the range of kinetic constants for the methylation of these proteins was similar to that observed with the synthetic peptides. The L-isoaspartyl/D-aspartyl methyltransferase has been proposed to function in vivo to prevent the accumulation of L-isoaspartyl residues that arise spontaneously as proteins age. To examine whether such a mechanism is feasible given the wide range of substrate Km values observed in vitro, we set up a computer simulation to model the degradation and methylation reactions in aging human erythrocytes. Our results suggest that enough methyltransferase activity exists in these cells to significantly lower the expected number of L-isoaspartyl residues, even when these residues have millimolar Km values for methylation.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation

We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.     

Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin

We have previously shown that the D-aspartyl/L-isoaspartyl protein carboxyl methyltransferase recognizes two major sites in affinity-purified preparations of bovine brain calmodulin that arise from spontaneous degradation reactions. These sites are derived from aspartyl residues at positions 2 and 78, which are located in apparently flexible regions of calmodulin. We postulated that this flexibility was an important factor in the nonenzymatic formation and enzymatic recognition of D-aspartyl and/or L-isoaspartyl residues. Because removal of Ca2+ ions from this protein may also lead to increased flexibility in the four Ca2+ binding regions, we have now characterized the sites of methylation that occur when calmodulin is incubated in buffers with or without the calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid (EGTA). Calmodulin was treated at pH 7.4 for 13 days at 37 degrees C under these conditions and was then methylated with erythrocyte D-aspartyl/L-isoaspartyl methyltransferase isozyme I and S-adenosyl-L-[methyl-3H]methionine. The 3H-methylated calmodulin product was purified by reverse-phase HPLC and digested with various proteases including trypsin, chymotrypsin, endoproteinase Lys-C, clostripain, and Staphylococcus aureus V8 protease, and the resulting peptides were separated by reverse-phase HPLC. Peptides containing Asp-2 and Asp-78, as well as calcium binding sites II, III, and IV, were found to be associated with radiolabel under these conditions. When calmodulin was incubated under the same conditions in the presence of calcium, methylation at residues in the Ca2+ binding regions was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

Distinct C-terminal sequences of isozymes I and II of the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase

We have purified the more acidic major isozyme (II) of the human erythrocyte L-isoaspartyl/D-aspartyl methyltransferase and compared its structure to that of the previously sequenced isozyme I. These isozymes are both monomers of 25,000 molecular weight polypeptides and have similar enzymatic properties, but have isoelectric points that differ by one pH unit. Analysis of 16 tryptic peptides of isozyme II accounting for 89% of the sequence of isozyme I revealed no differences between these enzyme forms. However, analysis of a Staphylococcal V8 protease C-terminal fragment revealed that the last two residues of these proteins differed. The Trp-Lys-COOH terminus of isozyme I is replaced by a Asp-Asp-COOH terminus in isozyme II. Southern blot analysis of genomic DNA suggests that the human genome [corrected] may contain only a single gene encoding the enzyme. We propose that the distinct C-termini of isozymes I and II can arise from the generation of multiple mRNA's by alternative splicing.     

Detection and Characterization of a Protein Isoaspartyl Methyltransferase Which Becomes Trapped in the Extracellular Space During Blood Vessel Injury

Injury to rat blood vessels in vivo was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it becomes trapped. This trapped cohort of PIMT is able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues. As further shown in this study, methylated substrates are detected only at the specific site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. Methylation kinetics, immunoblotting, and the lability of methylated substrates at mild alkaline pH were used to demonstrate that both types of blood vessel contain an endogeneous protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT). At least 50% of the PIMT activity is resistant to nonionic detergent extraction, suggesting that the enzyme activity becomes trapped within or behind the extracellular matrix (ECM). Quantities of lactate dehydrogenase (LDH), another soluble enzyme of presumed intracellular origin, were found to be similarly trapped in the extracellular space of blood vessels.     

Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli

Mammalian tissues contain protein carboxyl methyltransferases that catalyze the transfer of methyl groups from S-adenosylmethionine to the free carboxyl groups of D-aspartyl or L-isoaspartyl residues (EC 2.1.1.77). These enzymes have been postulated to play a role in the repair and/or degradation of spontaneously damaged proteins. We have now characterized a similar activity from Escherichia coli that recognizes L-isoaspartyl-containing peptides as well as protein substrates such as ovalbumin. The enzyme was purified by DEAE-cellulose, hydroxylapatite, Sephadex G-100, polyaspartate, and reversed-phase chromatography and was shown to consist of a single 24-kDa polypeptide chain. The sequence determined for the N-terminal 39 residues was used to design an oligonucleotide probe that allowed the precise localization of its structural gene (pcm) on the physical map of the E. coli chromosome at 59 min. Transformation of E. coli cells with a plasmid containing DNA from this region results in a 3-4-fold overproduction of enzyme activity. The nucleotide sequence determined for the pcm gene and its flanking regions was used to deduce a mature amino acid sequence of 207 residues with a calculated molecular weight of 23,128. This sequence shows 30.8% sequence identity with the human L-isoaspartyl/D-aspartyl methyltransferase and suggests that this enzyme catalyzes a fundamental reaction in both procaryotic and eucaryotic cells.     

Enhanced carboxyl methylation of membrane-associated hemoglobin in human erythrocytes

The alpha- and beta-chains of hemoglobin (Hb) are methylated in intact erythrocytes and in cellular extracts by a protein D-aspartate methyltransferase (EC 2.1.1.77) specific for D-aspartyl and L-isoaspartyl residues. During an 18-h incubation of intact erythrocytes with L-[methyl-3H]methionine, the subfraction of Hb molecules associated with the membrane becomes progressively enriched with methyl esters, reaching a specific activity 10-fold that of cytosolic Hb. The enhanced methylation of membrane Hb in intact cells appears not to result from its methylation at sites with inherently greater stability, since salt-extracted membrane Hb 3H-methyl esters and cytosolic Hb 3H-methyl esters are hydrolyzed at similar rates at pH 8.4 in vitro. Oxidative treatment of column-purified Hb with acetylphenylhydrazine produces an immediate 4-fold increase in its specific methyl-accepting activity coincident with the production of hemichrome forms known to possess a higher affinity for membrane binding sites. Together, the results suggest that the methyltransferase preferentially recognizes partially denatured Hb molecules which possess a higher affinity for membrane binding sites, similar to Hb forms observed in senescent erythrocytes.     

Identification of a site for carboxyl methylation in human alpha-globin

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.     

Specific recognition of altered polypeptides by widely distributed methyltransferases

Protein carboxyl methyltransferase activity has been detected in extracts prepared from bacterial cells (Salmonella typhimurium), amphibian (Xenopus laevis) oocytes, and transformed mammalian cell lines. This activity appears to specifically recognize altered aspartyl residues based on the observation that the synthetic peptide L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala is a good methyl-accepting substrate for the methyltransferase activity, but that the corresponding peptide containing a normal L-aspartyl residue is not. These activities are similar to those of the previously described human erythrocyte and bovine brain enzymes which catalyze the formation of polypeptide D-aspartyl beta-methyl esters and L-isoaspartyl alpha-methyl esters. The wide distribution of these enzymatic activites suggest that the methylation of atypical proteins is an essential function in cells.     

Methylation of microinjected isoaspartyl peptides in Xenopus oocytes. Competition with protein carboxyl methylation reactions

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.     

Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate.

Protein L-isoaspartyl (D-aspartyl) methyltransferases (EC 2.1.1.77) are found in almost all organisms. These enzymes catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of isomerized and racemized aspartyl residues in age-damaged proteins as part of an essential protein repair process. Here, we report crystal structures of the repair methyltransferase at resolutions up to 1.2 A from the hyperthermophilic archaeon Pyrococcus furiosus. Refined structures include binary complexes with the active cofactor AdoMet, its reaction product S-adenosylhomocysteine (AdoHcy), and adenosine. The enzyme places the methyl-donating cofactor in a deep, electrostatically negative pocket that is shielded from solvent. Across the multiple crystal structures visualized, the presence or absence of the methyl group on the cofactor correlates with a significant conformational change in the enzyme in a loop bordering the active site, suggesting a role for motion in catalysis or cofactor exchange. We also report the structure of a ternary complex of the enzyme with adenosine and the methyl-accepting polypeptide substrate VYP(L-isoAsp)HA at 2.1 A. The substrate binds in a narrow active site cleft with three of its residues in an extended conformation, suggesting that damaged proteins may be locally denatured during the repair process in cells. Manual and computer-based docking studies on different isomers help explain how the enzyme uses steric effects to make the critical distinction between normal L-aspartyl and age-damaged L-isoaspartyl and D-aspartyl residues.     

A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.     

Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases

A widely distributed protein methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-methionine to the free carboxyl groups of D-aspartyl and/or L-isoaspartyl derivatives of L-aspartyl and L-asparaginyl residues. This enzyme has been postulated to function in the repair or the catabolism of age-damaged proteins. We present here the complete amino acid sequence of the more basic isozyme I of this enzyme from human erythrocytes. The sequence was determined by Edman degradation and mass spectral analysis of overlapping trypsin, Staphylococcus aureus V8 protease, Pseudomonas fragi endoproteinase Asp-N, cyanogen bromide, and hydroxylamine-generated fragments. The NH2-terminus is modified by acetylation and the protein contains 226 amino acids for a calculated molecular weight of 24,575. This value is in good agreement with the molecular weight determined for the purified protein by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and by gel filtration chromatography under nondenaturing conditions. The identification of 2 different amino acid residues at both positions 22 and 119 may indicate the presence of allelic variants or of two or more closely related structural genes. Finally, comparison of this sequence with those of methyltransferases for RNA, DNA, and small molecules, as well as other S-adenosylmethionine-utilizing enzymes, shows that many of these proteins share elements of three regions of sequence similarity and may be structurally or evolutionarily related.     

Protein repair methyltransferase from the hyperthermophilic archaeon Pyrococcus furiosus. Unusual methyl-accepting affinity for D-aspartyl and N-succinyl-containing peptides.

Protein l-isoaspartate-(d-aspartate) O-methyltransferases (EC ), present in a wide variety of prokaryotic and eukaryotic organisms, can initiate the conversion of abnormal l-isoaspartyl residues that arise spontaneously with age to normal l-aspartyl residues. In addition, the mammalian enzyme can recognize spontaneously racemized d-aspartyl residues for conversion to l-aspartyl residues, although no such activity has been seen to date for enzymes from lower animals or prokaryotes. In this work, we characterize the enzyme from the hyperthermophilic archaebacterium Pyrococcus furiosus. Remarkably, this methyltransferase catalyzes both l-isoaspartyl and d-aspartyl methylation reactions in synthetic peptides with affinities that can be significantly higher than those of the human enzyme, previously the most catalytically efficient species known. Analysis of the common features of l-isoaspartyl and d-aspartyl residues suggested that the basic substrate recognition element for this enzyme may be mimicked by an N-terminal succinyl peptide. We tested this hypothesis with a number of synthetic peptides using both the P. furiosus and the human enzyme. We found that peptides devoid of aspartyl residues but containing the N-succinyl group were in fact methyl esterified by both enzymes. The recent structure determined for the l-isoaspartyl methyltransferase from P. furiosus complexed with an l-isoaspartyl peptide supports this mode of methyl-acceptor recognition. The combination of the thermophilicity and the high affinity binding of methyl-accepting substrates makes the P. furiosus enzyme useful both as a reagent for detecting isomerized and racemized residues in damaged proteins and for possible human therapeutic use in repairing damaged proteins in extracellular environments where the cytosolic enzyme is not normally found.     

Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase

Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoaspartyl and/or D-aspartyl residues but not of normal L-aspartyl residues. In order to identify the origin of these unusual residues which occur in subpopulations of a variety of cellular proteins, we studied the in vitro methylation by the erythrocyte enzyme of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. Methylated glucagon was digested with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg C, and the labeled fragments were separated by high-performance liquid chromatography and identified. In separate experiments, methyl acceptor sites were determined by digesting glucagon first with proteases and then assaying purified glucagon fragments for methyl acceptor activity. Using both approaches, we found that the major site of methylation, accounting for about 62% of the total, was at the position of Asp-9. Chemical analysis of fragments containing this residue indicated that this site represents an L-isoaspartyl residue. A second site of methylation, representing about 23% of the total, was detected at the position of Asn-28 and was also shown to represent an L-isoaspartyl residue. Methyl acceptor sites were not detected at the positions of Asp-15 or Asp-21. Preincubation of glucagon under basic conditions (0.1 M NH4OH, 3 h, 37 degrees C) increased methylation at the Asn-28 site by 4-8-fold while methylation at the Asp-9 site remained unchanged. These results suggest that methylation sites can originate from both aspartyl and asparaginyl residues and that these sites may be distinguished by the effect of base treatment.     

Alternative splicing of the human isoaspartyl protein carboxyl methyltransferase RNA leads to the generation of a C-terminal -RDEL sequence in isozyme II.

We have isolated two cDNA clones that correspond to the mRNAs for two isozymes of the human L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). The DNA sequence of one of these encodes the amino acid sequence of the C-terminal half of the human erythrocyte isozyme I. The other cDNA clone includes the complete coding region of the more acidic isozyme II. With the exception of potential polymorphic sites at amino acid residues 119 and 205, the deduced amino acid sequences differ only at the C-terminus, where the -RWK sequence of isozyme I is replaced by a -RDEL sequence in isozyme II. The latter sequence is identical to a mammalian endoplasmic reticulum retention signal. With the previous evidence for only a single gene for the L-isoaspartyl/D-aspartyl methyltransferase in humans, and with evidence for consensus sites for alternative splicing in corresponding mouse genomic clones, we suggest that alternative splicing reactions can generate the major isozymes previously identified in human erythrocytes. The presence of alternative splicing leads us to predict the existence of a third isozyme with a -R C-terminus. The calculated isoelectric point of this third form is similar to that of a previously detected but uncharacterized minor methyltransferase activity.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.