Methylation at D-Aspartyl Residues in Erythrocytes

Proc. Natl Acad. Sci. USA Vol. 79, pp. 2460-2464, April 1982 Biochemistry

Methylation at D-aspartyl residues in erythrocytes: Possible step in the repair of aged membrane proteins (protein methylation/amino acid racemization/aging/erythrocytes) PHILIP N. MCFADDEN AND STEVEN CLARKE Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90024 Communicated by Paul D. Boyer, December 23, 1981

ABSTRACT Reversibly methylated aspartyl residues in hu- rate of 0.14% per yr (11). It seemed conceivable to us that sim- man erythrocyte membrane proteins are shown to be in the "un- ilar amino acid racemization might be occurring in aging eryth- natural" D configuration. This is demonstrated by treatment of rocytes, providing the substrate for the methylation ofaspartyl proteolytically derived aspartic acid fi-[3H]methyl ester with L- residues in erythrocyte proteins. and D-amino-acid oxidases and by the resolution ofdiastereomeric We report here that all of the methylated aspartic acid that L-leucyl dipeptides containing either L- or D-aspartic acid j3- we have isolated from erythrocyte membrane and cytoskeletal methyl ester by ion-exchange chromatography. Based on this ob- proteins has the uncommon D configuration. We propose that servation, we propose a novel role for eukaryotic protein carboxyl acid residues can be methyltransferases (protein 0-methyltransferase; S-adenosyl-L- D-aspartic enzymatically recognized and methionine:protein O-methyltransferase, EC 2.1.1.24). We sug- modified in a step leading to the repair ofaging proteins. This gest that these widely distributed enzymes function to recognize process may help maintain functional proteins at a metabolic aspartyl residues that have racemized spontaneously for a sub- cost that is low compared to that ofreplacing damaged proteins sequent repair reaction. This repair function is postulated to cou- by de novo translation. ple ester hydrolysis with the restoration of the original L config- We suspect that this postulated protein repair mechanism is uration of the aspartyl residue. It is possible that similar types of widely distributed because protein carboxyl methylation occurs racemization repair processes may occur by reversible covalent in all mammalian tissues examined (1, 12). Other varieties of modifications at other residues. Other possible functions for D- reversible protein modification reactions, many ofwhich have aspartic acid fl-methyl ester residues in proteins are considered. not been assigned a function, may also be involved in the repair of racemized or otherwise altered proteins. A widespread reaction in nature is the posttranslational modification of protein carboxyl groups by methyl ester formation MATERIALS AND METHODS (1). At least two classes of the associated enzyme, protein car- Materials. L-Aspartic acid ,B-methyl ester hydrochloride was boxyl methyltransferase (protein O-methyltransferase; S-ad- purchased from Vega Biochemicals (Tucson, AZ). D-Aspartic enosyl-L-methionine:protein 0-methyltransferase, EC 2.1.1.24), acid 3methyl ester hydrochloride was synthesized by the are known to exist (2). The first of these is a bacterial enzyme method ofde Groot and Lichtenstein (13) from 5 g ofD-aspartic that catalyzes the methylation of chemoreceptors at glutamyl acid (37.6 mmol; Sigma) dissolved in a mixture of 38 ml of an- residues in an adaptive response to sensory stimuli (2, 3). The hydrous methanol (940 mmol) and 5.4 ml ofacetyl chloride (75 second class ofenzyme activity, found in both prokaryotes and mmol). After recrystallization from diethylether/methanol, 2:1 eukaryotes, shows a much broader substrate specificity and (vol/vol), the product [1.98 g (10.8 mmol); 29% yield] was char- catalyzes the formation ofextremely labile methyl esters, pre- acterized by its melting point (186.5-189.5°C), titrimetric be- sumably at aspartyl residues (4). The function ofthis latter pro- havior (1 equivalent of a group with a pKa of 2.2 and 1 equiva- cess is not clear. lent ofa group with a pKa of7.6), hydrolysis rate [tl/2 = 89 min We are interested in determining the function ofmammalian at pH 10.5 and 37°C; the literature value for L-aspartic acid /3- protein carboxyl methylation and are studying this reaction in methyl ester is 82 min (6)], and migration in thin-layer chro- the human erythrocyte. We have shown that specific cytoskel- matography (cf. ref. 4). Amino acid analysis of the product on etal and membrane proteins are reversibly methylated at as- a Beckman model 120C analyzer revealed up to 10% impurity partyl residues (4-6). The carboxyl methylation of these pro- of aspartic acid. A ninhydrin color constant of 0.20 relative to teins is substoichiometric (less than 0.02 methyl groups per aspartic acid was calculated for the ester. polypeptide chain) in all cases (5, 6), but we have determined Both the L- and D-aspartic acid /8-methyl esters were >99% that this level is consistently higher in older populations of optically pure as determined by the diastereomer method of erythrocytes (7). Similarly, experiments with the purified car- Manning and Moore (14); the contaminating aspartic acid had boxyl methyltransferase from both erythrocytes and other mam- the same stereoconfiguration as the parent ester. malian tissues indicate that in vitro substrates are also substoi- L-Proline was from Sigma. Liquid scintillation cocktail chiometrically methylated (8, 9). To understand why aspartyl (Aquamix) was from West Chem (San Diego, CA), and 7-10 residues at a given position in a sequence may be only partially volumes were used per 1 volume of aqueous sample. methylated, we have investigated the nature ofthe aspartic acid Isolation ofAspartic Acid fl-[3H]Methyl Ester from Human residues that are methylated in the red cell. Erythrocyte Membranes. Membranes containing [3H]methyl It has been shown that racemization ofaspartic acid residues groups (referred to hereafter as *membranes) were prepared occurs in aging mammalian proteins (for a review, see ref. 10). from fresh erythrocytes incubated with L-[methyl-3H]methionine For example, D-aspartic acid accumulates in lens proteins at a (70-90 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) as described by Freitag and Clarke (5). These *membranes (5 mg ofprotein The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: LeuCA, L-leucine N-carboxyanhydride; *membranes, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. membranes containing tritiated methyl groups. 2460 Downloaded by guest on September 27, 2021 Biochemistry: McFadden and Clarke Proc. Natl. Acad. Sci. USA 79 (1982) 2461 per ml; 160 pmol of [3H]methyl groups per ml) were digested of erythrocyte *membrane proteins (4). The radioactive yield for 16-25 hr at 370C in 2-3 volumes of a Sigma preparation of of aspartic acid 83-[3H]methyl ester from the *membranes in bakers' yeast carboxypeptidase Y (2 mg/ml; 100 units/mg of these experiments was 5-10%; much of the remaining radio- protein) containing 8 mg ofcitrate buffer (pH 5) per ml. In some activity chromatographed as [3H]methanol and may have been experiments, radiolabeled aspartic acid /B-methyl ester was iso- formed by ester hydrolysis during the digestion step. lated by ion-exchange chromatography, desalted by Sephadex Aspartic acid /3-[3H]methyl ester in unfractionated digests G-15 gel filtration in 0.1 M acetic acid, and concentrated by was treated with L-amino-acid oxidase. The radioactive methyl lyophilization as described by Janson and Clarke (4). ester was unaffected by the oxidase, even though a standard of L-Amino-Acid Oxidase Treatment. Carboxypeptidase Y di- L-aspartic acid ,3methyl ester added to the mixture was com- gestion of*membranes (0.2 mg of*membrane protein) was ter- pletely degraded (Fig. 1). L-Aspartic acid, which also was pres- minated after 16 hr by the addition of0.6 mg ofphenylmethyl- ent in the mixture, was a poor substrate for the L-oxidase (17) sulfonyl fluoride (Sigma). The digestion products were mixed and provided an internal standard for the ninhydrin analysis. with 4 gnmol of either L- or D-aspartic acid /3-methyl ester and This experiment also was performed with isolated aspartic acid 100 ,Amol of Tris HCl (pH 7.5) in a final volume of 0.34 ml. -P[3H]methyl ester mixed with an internal standard of D-as- Aliquots (150 ,tl) were incubated with either 20 1.l of water or partic acid /3-methyl ester. In this case, the L-amino-acid oxi- 20 A.l of solution containing 1.6 units of L-amino-acid oxidase dase was inactive toward both the radioactive aspartic acid /3- (from Crotalus adamanteus venom, Sigma type IV; 8.1 units/ methyl ester and the standard D-aspartic acid /3-methyl ester mg of protein). These samples were incubated at 370C for 3.5 (data not shown). Overall, the results indicate that L-amino-acid hr and then were quenched with 10 Al of 1 M HC1 and 220 IlI oxidase specifically oxidizes standards of L- and not D-aspartic of sodium citrate analyzer sample buffer (pH 2.2; Pierce). Por- acid /3-methyl ester and that this enzyme does not detectably tions (350 ,l) were chromatographed on a 0.9 cm X 30 cm col- oxidize erythrocyte-derived aspartic acid /3-[3H]methyl ester. umn of sulfonated polystyrene analyzer resin (Dionex DC-6A) Erythrocyte-Derived Aspartic Acid f3-[3H]Methyl Ester Is at 50°C in citrate (pH 3.25; 0.2 M in Na+). The column was a Substrate for D-Amino-Acid Oxidase. Radioactive aspartic eluted at a flow rate of 70 ml/hr with this buffer, followed by acid P-methyl ester isolated from membrane digests by ion-ex- elution with 0.2 M NaOH. Fractions (2 min) were collected and change chromatography disappeared upon treatment with D- analyzed for ninhydrin-reactive material by the method of amino-acid oxidase (Fig. 2). Standard D-aspartic acid /3-methyl Moore (15) and for radioactivity by liquid scintillation assay. ester also disappeared in an enzyme-dependent fashion, D-Amino-Acid Oxidase Treatment. D-Amino-acid oxidase whereas an internal ninhydrin standard of L-proline was not (porcine kidney) was obtained from Sigma at a specific activity affected by the enzyme. In other experiments, D-amino-acid of 17 units/mg of protein and was dialyzed overnight at 4°C oxidase was completely inactive towards standard L-aspartic against 500 volumes of 100 mM sodium pyrophosphate buffer, acid /3-methyl ester (data not shown). pH 8.3/3 mM EDTA/25 mM NaCl. Isolated aspartic acid /- Resolution of Enantiomers ofAspartic Acid 3-Methyl Ester [3H]methyl ester from the ion-exchange chromatography step by Diastereomer Formation: Synthesis ofL-Leucyl-D-Aspartic (70 Al; 7,000 cpm) was mixed with 10 ,ul of 1 M L-proline and Acid fp-[3H]Methyl Ester from Erythrocyte Digests. The pro- 16 ,ul of 0.1 M D-aspartic acid /3-methyl ester. Aliquots (40 ,ul) cedure (14) for resolving amino acid enantiomers by covalently were treated either with 200 ,l of D-amino-acid oxidase (1 mg coupling D- and L-amino acids to LeuCA results in the formation of protein) or with 200 ,l ofthe dialysis buffer for 5 hr at room of diastereomeric L-leucyl dipeptides, which can be separated temperature. Each incubation was then quenched with 150 ,l and quantitated by ion-exchange chromatography. By this of 8% 5-sulfosalicylic acid. Portions (350 ,ul) ofeach sample were method, L-leucyl-D-aspartic acid /3-methyl ester was separated then analyzed by ion-exchange chromatography as in the L- from L-leucyl-L-aspartic acid /3-methyl ester (see the ninhydrin amino-acid oxidase experiment. trace, Fig. 3 Top). The leucyl derivatives of D- and L-aspartic Formation and Separation of L-Leucyl Diastereomeric Di- acid also were resolved as has been shown previously (ref. 14; peptides. L-Leucyl dipeptides were formed as described (14) ninhydrin trace, Fig. 3 Bottom). by reaction ofL- and D-aspartic acid /3-methyl esters with L-leu- When erythrocyte-derived aspartic acid /3-[3H]methyl ester cine N-carboxyanhydride (LeuCA; 4-(2-methylpropyl)-2,5-ox- was coupled to L-leucine by this method, a radioactive product azolidinedione). This reagent was synthesized as described by was formed that coeluted with L-leucyl-D-aspartic acid /3- Konopinska and Siemion (16) from N-carbobenzoxy-L-leucine methyl ester and represented 71% of the total radioactivity.t (Sigma) and oxalyl chloride (98%; Aldrich). Amino acids (4-4.5 The remaining 29% of the radioactivity coeluted with the un- ,Amol) were mixed with 1.1 ml ofice-cold 0.45 M sodium borate reacted standard or methanol (peak II). No radioactivity was buffer (pH 10.2) and immediately transferred to a tube con- coelutedwith L-leucyl-L-aspartic acid /3-methyl ester. The yield taining a 70% molar excess of solid LeuCA. The tube contents of products after the LeuCA coupling was about the same for were vigorously mixed at 4°C for 2 min, and the coupling re- both the radioactive aspartic acid /-methyl ester starting ma- action was then quenched with 0.48 ml of 1 M HCl. The terial and the standard L- and D-aspartic acid /3-methyl ester quenched reaction mixture was mixed with an equal volume of starting materials. sodium citrate sample buffer (pH 2.2) and applied to a 0.9 cm A control experiment was performed in which LeuCA was X 50 cm column of Beckman amino acid analyzer resin (AA-15) left out of the reaction mixture (Fig. 3 Middle). In this case, all equilibrated at 56°C in citrate buffer (pH 3.25; 0.2 M Na+). The of the radioactivity remained with the peak of aspartic acid /3- column was then eluted at a flow rate of 70 mVhr with the same methyl ester (II) as expected. buffer. Fractions (5 min) were collected and analyzed for ra- In another control experiment, the mixture ofradioactive and dioactivity by liquid scintillation counting and for ninhydrin standard amino acid esters was hydrolyzed in base before cou- color by the method of Moore (15). pling them to LeuCA (Fig. 3 Bottom). The radioactive amino RESULTS t As is common in high-resolution ion-exchange chromatography sys- Aspartic Acid .3-[3H]Methyl Ester in Membrane Digests Is tems, the isotope effect due to the three tritium atoms in aspartic acid Not a Substrate for L-Amino-Acid Oxidase. Radioactive aspar- l3-[3H]methyl ester (90 Ci/mmol) and its derivatives consistently leads tic acid 3-methyl ester has been identified in proteolytic digests to their slightly early elution as compared with standards (4, 18, 19). Downloaded by guest on September 27, 2021 2462 Biochemistry: McFadden and Clarke Proc. Natl. Acad. Sci. USA 79 (1982)

I-

r- "0 x a- to 1t

10 20 30 10 20 30 Fraction FIG. 1. L-Amino-acid oxidase treatment of aspartic acid _3-[3H]methyl ester from erythrocyte membranes. Carboxypeptidase Y digests of *membranes were incubated either in the absence (A) or presence (B) of L-amino-acid oxidase, and the supernatants were analyzed for radioactive and ninhydrin-reactive species by ion-exchange chromatography. Radioactivity in 0.8 ml of each 2.3-ml fraction was measured by the liquid scintillation technique. Ninhydrin color was quantitated by the absorbance at 570 nm from 0.4 ml of each fraction in a total volume of 0.7 ml. Open arrow, peak of aspartic acid 13 [3H]methyl ester (V); solid arrow, initiation of elution with 0.2 M NaOH. The radioactive material in peaks I, III, and IV has not been identified; peak II contains [3H]methanol; and peak VI contains undigested proteins (4). Amino acid standards were eluted as indicated. acid esters were 86% hydrolyzed as judged by the amount of Release of D-Aspartic Acid fl-Methyl Ester by Carboxypep- [3H]methanol formed (peak I) and by the parallel loss of radio- tidase Y. Carboxypeptidase Y (which may contain small amounts activity in peaks II and III. The hydrolysis ofthe standard amino of contaminating endopeptidase activities) was the most effec- acid esters was 88% complete as indicated by the increased yield tive agent in releasing aspartic acid ,3-[3H]methyl ester from of dipeptides containing nonesterified aspartic acid. erythrocyte membrane proteins. Addition of Pronase, subtili- The results from both the oxidase and diastereomer experi- sin, or proteinase K to the digestion mixture lowered yields of ments are consistent with the interpretation that all of the iso- radioactive product; pepsin, papain, elastase, trypsin, chymo- lated erythrocyte-derived aspartic acid ,B-[3H]methyl ester has trypsin, or thermolysin had little or no effect (data not shown). the D configuration. No evidence was found for any L-aspartic Carboxypeptidase Y can hydrolyze peptide bonds on both sides acid P-methyl ester from erythrocyte proteolytic digests. of D-amino acid residues, although at a low rate (20). We ex- cluded the possibility of artifactual racemization during preparation of aspartic acid 8-[3H]methyl ester for several reasons. First, enzymatic or nonenzymatic racemization would produce equal amounts of the L and D forms, yet we only detected D- aspartic acid ,B-methyl ester. Second, no racemization or selec-

6 tive hydrolysis of either D- or L-aspartic acid (3-methyl ester standards occurred during carboxypeptidase Y digestion. Fi- nally, enzymatic digestion under conditions described here re- sulted in the formation of L-aspartic acid (3-methyl ester from 2 poly(L-aspartic acid (3-methyl ester) (data not shown).

- "0 DISCUSSION x Are in Mem- E 8 uz D-Aspartyl Residues Methylated Erythrocyte c. . brane and Cytoskeletal Proteins. Previous work has shown that intact human erythrocytes incubated with L-[methyl-3H]- cm 6 methionine synthesize S-adenosyl-L-[methyl-3H]methionine, 4 which serves as the methyl donor for protein methylation reactions (21). The principal methyl acceptors in the cell are the 2 membrane cytoskeletal protein bands 2.1 and 4.1 and the in- tegral membrane protein band 3 (5). The methylated amino acid 0 residue has been isolated from carboxypeptidase Y digests of 10 20 30 these labeled membranes and identified as aspartic acid /3- Fraction [3H]methyl ester (4). In this paper, we present evidence that all ofthe aspartic acid FIG. 2. D-Amino-acid oxidase treatment of isolated aspartic acid ,3 [3H]methyl ester recovered has the uncommon D configu- ,3[3H]methyl ester. Samples that had been treated in the absence (A) acid ester or presence (B) of D-amino-acid oxidase were analyzedby ion-exchange ration. This configuration of aspartic ,B-[3H]methyl chromatography, and the radioactivity and ninhydrin color of each was established by the reactivity ofthe amino acid with oxidases fraction were determined as in Fig. 1. specific for D-amino acids and by its lack of reactivity with ox- Downloaded by guest on September 27, 2021 Biochemistry: McFadden and Clarke Proc. Natl. Acad. Sci. USA 79 (1982) 2463 aspartic acid (3-[3H]methyl ester to L-leucine to form a diastereomeric dipeptide that could be assigned an absolute stereoconfiguration. No evidence was found for the presence of any L-aspartic acid p-[3H]methyl ester. Our finding of r'-aspartic acid p3-methyl ester in erythrocytes rationalizes many previously poorly understood observations. Racemization in vivo would not be expected to affect the entire population of a protein species; the experimental finding is that protein carboxyl methylation is always substoichiometric (5, 8, 9). The substrate specificity for protein methylation in vivo is relatively broad; all proteins are potentially subject to racemization, although sequence or conformational features, or both, as well as the rate of methyl group turnover, might determine the actual methylation levels observed (5). Proteolytic enzymes are in general specific for L-amino acids; the difficulty of iso- lating aspartic acid P-methyl ester in high yield is likely to result from the inability of most enzymes to digest peptide bonds of D-amino acid residues (see Results). Cellular Origin of D-Aspartyl Residues. There is no known 210 function for D-amino acid residues in proteins. The only known 20 route by which D-amino acid residues can appear in mammalian proteins is by a spontaneous racemization of the L-amino acids that have been biologically assembled into polypeptides. In fact, 15 Asp-a3-methyl ester the racemization of aspartate residues in proteins occurs at a 0. greater rate than that of any other amino acid, except possibly 10~~~~~~~~~~~~~~~~. serine (22, 23). In long-lived tissues, such as lens and tooth Asp)' enamel, the spontaneous rate of racemization can lead to sub- stantial accumulation of D-aspartyl residues during a lifetime may 0.0 (24, 25). The functional consequences of such racemization

0 be severe (11). C A Model for the Repair of Racemized Proteins. In order to explain why "abnormal" D-aspartyl residues may be methylated in the cell, we have proposed a model shown in Fig. 4. We sug- gest that protein carboxyl methylation is involved in the repair of proteins racemized at aspartyl residues. The spontaneous A mxre 2025tyleser(op10 203 oftadad-L-LeU-L-Asp05 07 2.0 appearance of a potentially damaging D--aspartyl residue results in its recognition and methylation by a protein carboxyl meth- 15,0 cL-Leu-D-Aspw it L-UeU yltransferase. The methylation step leads to a postulated "repair" step that accompanies ester hydrolysis and results in the restoration of the original L configuration of the aspartyl resi- 10 mslp due. The loss of methanol (and its probable subsequent oxi- dation or excretion) in the repair step ensures that the reverse reaction (enzymatic production of D-aspartyl residues) does not occur. The enzyme-responsible for the esterase/racemase function postulated in Fig. 4 has not yet been identified. The possibility also exists that nonenzymatic hydrolysis of the ester group may itself lead to racemization at the a carbon and, thus, result in a 50% conversion of the D-amino acid residue back to the original L configuration in each methylation/demethylation cycle. Awslf The reason for this is that the hydrolysis of P-methyl esters of 5.8-mlf~~~raction.Mdl)Cnrleprmn~ nwihL aspartyl residues is likely to proceed through a cyclic imide in- FIoutReoutoteculnpreduatre.Rdoarctdivepetierof of3asparwas mixe termediate (cf. refs. 4 and 6 and references therein). Resonance with eser Aeachur(Top) of D-sadrand L-asparticacid,&ehlete.Rdoc ring system can facilitate the loss of a tiviyreacionwastmersured0withith euAwas2.5nIml,Fractionaiaciiywarfespefrctiveyond purifiednlzeConthrolsi in this five-membered cyexperimentidhcractionvwithtymeasurdbylquidsinwaseuaspartic acidp_[3Hmethyltilationactiyestersws(100ILI inmnlyfearadio-~l proton from the a carbon and, thus, promote racemization. Variations on this model also may be proposed. Our results macieeteriamth sntanderslproucts(2.0ri an wer2.aobrD-esoleradanvuifeery2.4ionexcangofL-stromatoreabase absrc10.2 befor the Radpio- have shown that the product ofcarboxyl methylation is a protein hydrolze freatvt18.5 meinboasuedbyfe D-aspartyl P-methyl ester. D-Aspartyl residues could originate the-mfrcincspcpm/ml (ideontrolwere experimentcoupleacdstoinwhchove.waLeuIndeendTen uonreatersartiongolf either from L-aspartyl or L-asparaginyl residues because the lat- mthesrialtand proucs weraiedrsle bycopion-echnerychromatogdraphy. ter residues may deamidate through a racemization-prone im- by before withs5hestructure2spoeifchwasofor obtinedL-aminoan acidstiIdpendent(pling confir.madionaofthe-cuping ide intermediate (see above). Because the deamidation of as- paragine residues is an age-related process (10), the D-aspartyl methyl ester observed here may be an intermediate in the re- pairofthis type ofcellulardamage. In this case, the methyl ester would be replaced by an amide group in the subsequent D-to- L conversion. Downloaded by guest on September 27, 2021 2464 Biochemistry: McFadden and Clarke Proc. Natl. Acad. Sci. USA 79 (1982) L-Aspartyl residue Normal function cemized subunit of a hemoglobin molecule, will probably not greatly disrupt the organism. Nature may supply cells with pro- °%CeoO- tein repair systems because many proteins participate in highly CH2 cooperative processes. A single damaged protein molecule -NH-C-C- Slow, spontaneous could weaken the amplification of a metabolic cascade signal or I II "aging" reaction H O D-Aspartyl residue might. disrupt the whole structure of a cytoskeletal net. Thus, Abnormal function the recognition of racemized aspartyl (and possibly other) residues by specific covalent modification enzymes may be an es- H sential function in cells. -NH-C-C- Postulated .}r I II racemase/ I MeOH CH2 0 We are grateful to our colleagues for helpful discussions. This work esteraseLon was a function 1C; supported by grant from the National Institutes of Health (GM 0 -O 26020) and a grant-in-aid from the American Heart Association (with H SAM contributed in the SAH ) funds part by Greater Los Angeles Affiliate). P. N. M. -NH-C-C- was supported by U.S. Public Health Service Training Grant GM CH2 D-Aspartyl protein methyltransferase 07185.

0 1. Paik, W. K. & Kim, S. (1980) in Protein Methylation (Wiley, New CH3 York), pp. 202-231. D-Aspartyl-p-methyl ester residue 2. Clarke, S., Sparrow, K., Panasenko, S. & Koshland, D. E., Jr. (1980) J. Supramot Struct. 13, 315-328. FIG. 4. Proposed pathway for the repair of proteins that have been 3. Koshland, D. E., Jr. (1981) Annu, Rev. Biochem. 50, 765-782. spontaneously racemized at aspartyl residues. The previously de- 4. Janson, C. A. & Clarke, S. (1980)J. BioL Chem. 255, 11640-11643. scribed protein carboxyl methyltransferase specifically methylates 5. Freitag, C. & Clarke, S. (1981)J. Biol. Chem. 256, 6102-6108. only "aged" aspartyl residues in the D configuration. A second enzyme 6. Terwilliger, T. C. & Clarke, S. (1981) J. Biol. Chem. 256, is postulated to couple ester hydrolysis with the restoration of the nor- 3067-3076. mal L configuration of the amino acids. SAM, S-adenosylmethionine; 7. Barber, J. R., Brunauer, L. S., O'Connor, C. M. & Clarke, S. SAH, S-adenosylhomocysteine. (1981)J. Cell Biol. 91, 260 (abstr.). 8. Kim, S. & Li, C. H. (1979) Proc. Natl Acad. Sci. USA 76, Alternative Models for the Role of D-Aspartyl (i-Methyl 4255-4257. Ester Formation. The racemization repair model is 9. Kloog, Y., Flynn, D., Hoffman, A. R. & Axelrod, J. (1980) consistent Biochem. Biophys. Res. Commun. 97, 1474-1480. with past results. However, until the repair pathway is experi- 10. McKerrow, J. H. (1979) Mech. Ageing Dev. 10, 371-377. mentally tested, other pathways subsequent to methylation at 11. Masters, P. M., Bada, J. L. & Zigler, J. S., Jr. (1978) Proc. Natl. D-aspartyl residues must be considered. For example, meth- Acad. Sci. USA 75, 1204-1208. ylation may be a cellular signal that targets a racemized protein 12. Diliberto, E. J., Jr., & Axelrod, J. (1976) J. Neurochem. 26, for destruction. Or, methylation at accumulated res- 1159-1165. D-aspartyl 13. idues may a de Groot, N. & Lichtenstein, N. (1959) Bull, Res. Counc. Israel be biological clock that signals the age of the cell. 8A, 116. Other schemes may be postulated in which D-amino acids or 14. Manning, J. M. & Moore, S. (1968) J. Biol. Chem. 243, modified D-amino acids have specific physiological functions. 5591-5597. Methylation at Protein D-Aspartyl Residues Is Probably a 15. Moore, S. (1968)J. Biol. Chem. 243, 6281-6283. General Phenomenon in Eukaryotic Cells and Tissues. Protein 16. Konopinska, D. & Siemion, I. Z. (1967) Angew. Chem. nt. Ed. carboxyl methyltransferase activity similar to that EngI 6, 248. demonstrated 17. A. in erythrocytes has been measured in extracts ofall mammalian Meister, & Wellner, D. (1963) in The Enzymes, eds. Boyer, P. D., Lardy, H. & Myrback, K. (Academic, New York), Vol. 7, tissues examined so far (1, 12). Purified methyltransferases from 2nd Ed., pp. 609-648. these sources are similar to the erythrocyte enzyme in their 18. Kleene, S. J., Toews, M. L. & Adler, J. (1977)J. Biol. Chem. 252, cytosolic localization, their polypeptide molecular weight and 3214-3218. subunit structure, their Km values for S-adenosylmethionine, 19. Klein, P. D. & Szczepanik, P. A. (1967) Anal. Chem. 39, their values for and their 1276-1281. Ki S-adenosylhomocysteine, specific- 20. Y. ity for exogenous Hayashi, R., Bai, & Hata, T. (1975) J. Biochem. 77, 69-79. protein acceptor species (cf. refs. 1 and 21. Kim, S., Galletti, P. & Paik, W. K. (1980) J. Biol. Chem. 255, 26-31). Thus, the protein carboxyl methyltransferase reaction 338-341. we have been studying in the erythrocyte may be representative 22. Masters, P. M. & Friedman, M. (1980) in Chemical Deteriora- of a widely distributed enzyme and function. It is clear, how- tion of Proteins, eds. Whitaker, J. R.; & Fujimike, M. (Am. ever, that the protein carboxyl methyltransferase involved in Chem. Soc., Washington, DC), pp. 165-194. bacterial chemotaxis (2, 3) is distinct from this activity. This lat- 23. Whitaker, J. R. (1980) in Chemical Deterioration of Proteins, ter eds. Whitaker, J. R. & Fujimike, M. (Am. Chem. Soc., Wash- enzyme methylates residues at stoichiometric levels (32). ington, DC), pp. 145-163. Possible Racemization Repair at Other Than Aspartyl Res- 24. Helfman, P. M. & Bada, J. L. (1975) Proc. Natl. Acad. Sci. USA idues. The essential feature ofthe repair function proposed here 72, 2891-2894. is that the hydrolysis ofthe D-aspartyl methyl ester residue can 25. Masters, P. M., Bada, J. L. & Zigler, J. S., Jr. (1977) Nature be coupled to the inversion ofthe configuration at the a carbon (London) 268, 71-73. (Fig. 4). In principle, the reversal of any 26. Kim, S. (1974) Arch. Biochem. Biophys. 161, 652-657. covalent modification 27. E. reaction at a Polastro, T., Deconinck, M. M., Devogel, M. R., Mailier, E. protein residue in the D configuration could sim- L., Looza, Y. B., Schnek, A. G. & Leonis, J. (1978) Biochem. ilarly be utilized by the cell to thermodynamically drive the Biophys. Res. Commun. 81, 920-927. conversion to the L configuration. For example, racemized 28. Iqbal, M. & Steenson, T. (1976)J. Neurochem. 27, 605-608. serine, threonine, or tyrosine residues could be recognized by 29. Oliva, A., Galletti, P., Zappia, V., Paik, W. K. & Kim, S. (1980) D-specific protein kinase(s). Inversion ofconfiguration could be Eur. J. Biochem. 104, 595-602. 30. driven in this case by the hydrolysis of the phosphate ester. Kim, S. (1973) Arch. Biochem. Biophys. 157, 476-484. 31. Diliberto, E. J., Jr., & Axelrod, J. (1974) Proc. Nat. Acad. Sci. Damage to critical cellular components must be repaired. USA 71, 1701-1704. Failure to repair a single lesion in a DNA molecule, for example, 32. Stock, J. B. & Koshland, D. E., Jr. (1981) J. Biol. Chem. 256, may be lethal. Damage to a less critical component, like a ra- 10826-10833. Downloaded by guest on September 27, 2021