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

Methylation at D-aspartyl residues in erythrocytes: Possible step inthe repair of aged membrane proteins

(protein methylation/amino acid racemization/aging/erythrocytes)

PHILIP N. MCFADDEN AND STEVEN CLARKEDepartment 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-man erythrocyte membrane proteins are shown to be in the "un-natural" D configuration. This is demonstrated by treatment ofproteolytically derived aspartic acid fi-[3H]methyl ester with L-and D-amino-acid oxidases and by the resolution ofdiastereomericL-leucyl dipeptides containing either L- or D-aspartic acid j3-methyl ester by ion-exchange chromatography. Based on this ob-servation, we propose a novel role for eukaryotic protein carboxylmethyltransferases (protein 0-methyltransferase; S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24). We sug-gest that these widely distributed enzymes function to recognizeaspartyl residues that have racemized spontaneously for a sub-sequent repair reaction. This repair function is postulated to cou-ple ester hydrolysis with the restoration of the original L config-uration of the aspartyl residue. It is possible that similar types ofracemization repair processes may occur by reversible covalentmodifications at other residues. Other possible functions for D-aspartic acid fl-methyl ester residues in proteins are considered.

A widespread reaction in nature is the posttranslational modi-fication of protein carboxyl groups by methyl ester formation(1). At least two classes of the associated enzyme, protein car-boxyl methyltransferase (protein O-methyltransferase; S-ad-enosyl-L-methionine:protein 0-methyltransferase, EC 2.1.1.24),are known to exist (2). The first of these is a bacterial enzymethat catalyzes the methylation of chemoreceptors at glutamylresidues in an adaptive response to sensory stimuli (2, 3). Thesecond class of enzyme activity, found in both prokaryotes andeukaryotes, shows a much broader substrate specificity andcatalyzes the formation of extremely labile methyl esters, pre-sumably at aspartyl residues (4). The function of this latter pro-cess is not clear.We are interested in determining the function ofmammalian

protein carboxyl methylation and are studying this reaction inthe human erythrocyte. We have shown that specific cytoskel-etal and membrane proteins are reversibly methylated at as-partyl residues (4-6). The carboxyl methylation of these pro-teins is substoichiometric (less than 0.02 methyl groups perpolypeptide chain) in all cases (5, 6), but we have determinedthat this level is consistently higher in older populations oferythrocytes (7). Similarly, experiments with the purified car-boxyl methyltransferase from both erythrocytes and other mam-malian tissues indicate that in vitro substrates are also substoi-chiometrically methylated (8, 9). To understand why aspartylresidues at a given position in a sequence may be only partiallymethylated, we have investigated the nature ofthe aspartic acidresidues that are methylated in the red cell.

It has been shown that racemization of aspartic acid residuesoccurs 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 sim-ilar amino acid racemization might be occurring in aging eryth-rocytes, providing the substrate for the methylation of aspartylresidues in erythrocyte proteins.We report here that all of the methylated aspartic acid that

we have isolated from erythrocyte membrane and cytoskeletalproteins has the uncommon D configuration. We propose thatD-aspartic acid residues can be enzymatically recognized andmodified in a step leading to the repair of aging proteins. Thisprocess may help maintain functional proteins at a metaboliccost that is low compared to that of replacing damaged proteinsby de novo translation.We suspect that this postulated protein repair mechanism is

widely distributed because protein carboxyl methylation occursin all mammalian tissues examined (1, 12). Other varieties ofreversible protein modification reactions, many of which havenot been assigned a function, may also be involved in the repairof racemized or otherwise altered proteins.

MATERIALS AND METHODSMaterials. L-Aspartic acid ,B-methyl ester hydrochloride was

purchased from Vega Biochemicals (Tucson, AZ). D-Asparticacid 3methyl ester hydrochloride was synthesized by themethod ofde Groot and Lichtenstein (13) from 5 g of D-asparticacid (37.6 mmol; Sigma) dissolved in a mixture of 38 ml of an-hydrous methanol (940 mmol) and 5.4 ml of acetyl chloride (75mmol). After recrystallization from diethylether/methanol, 2:1(vol/vol), the product [1.98 g (10.8 mmol); 29% yield] was char-acterized by its melting point (186.5-189.5°C), titrimetric be-havior (1 equivalent of a group with a pKa of 2.2 and 1 equiva-lent of a group with a pKa of 7.6), hydrolysis rate [tl/2 = 89 minat pH 10.5 and 37°C; the literature value for L-aspartic acid /3-methyl ester is 82 min (6)], and migration in thin-layer chro-matography (cf. ref. 4). Amino acid analysis of the product ona Beckman model 120C analyzer revealed up to 10% impurityof aspartic acid. A ninhydrin color constant of 0.20 relative toaspartic acid was calculated for the ester.

Both the L- and D-aspartic acid /8-methyl esters were >99%optically pure as determined by the diastereomer method ofManning and Moore (14); the contaminating aspartic acid hadthe same stereoconfiguration as the parent ester.

L-Proline was from Sigma. Liquid scintillation cocktail(Aquamix) was from West Chem (San Diego, CA), and 7-10volumes were used per 1 volume of aqueous sample.

Isolation of Aspartic Acid fl-[3H]Methyl Ester from HumanErythrocyte Membranes. Membranes containing [3H]methylgroups (referred to hereafter as *membranes) were preparedfrom fresh erythrocytes incubated with L-[methyl-3H]methionine(70-90 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) as describedby Freitag and Clarke (5). These *membranes (5 mg of protein

Abbreviations: LeuCA, L-leucine N-carboxyanhydride; *membranes,membranes containing tritiated methyl groups.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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per ml; 160 pmol of [3H]methyl groups per ml) were digestedfor 16-25 hr at 370C in 2-3 volumes of a Sigma preparation ofbakers' yeast carboxypeptidase Y (2 mg/ml; 100 units/mg ofprotein) containing 8 mg ofcitrate buffer (pH 5) per ml. In someexperiments, radiolabeled aspartic acid /B-methyl ester was iso-lated by ion-exchange chromatography, desalted by SephadexG-15 gel filtration in 0.1 M acetic acid, and concentrated bylyophilization as described by Janson and Clarke (4).

L-Amino-Acid Oxidase Treatment. Carboxypeptidase Y di-gestion of *membranes (0.2 mg of*membrane protein) was ter-minated after 16 hr by the addition of 0.6 mg of phenylmethyl-sulfonyl fluoride (Sigma). The digestion products were mixedwith 4 gnmol of either L- or D-aspartic acid /3-methyl ester and100 ,Amol of Tris HCl (pH 7.5) in a final volume of 0.34 ml.Aliquots (150 ,tl) were incubated with either 20 1.l of water or20 A.l of solution containing 1.6 units of L-amino-acid oxidase(from Crotalus adamanteus venom, Sigma type IV; 8.1 units/mg of protein). These samples were incubated at 370C for 3.5hr and then were quenched with 10 Al of 1 M HC1 and 220 IlIof sodium citrate analyzer sample buffer (pH 2.2; Pierce). Por-tions (350 ,l) were chromatographed on a 0.9 cm X 30 cm col-umn of sulfonated polystyrene analyzer resin (Dionex DC-6A)at 50°C in citrate (pH 3.25; 0.2 M in Na+). The column waseluted at a flow rate of 70 ml/hr with this buffer, followed byelution with 0.2 M NaOH. Fractions (2 min) were collected andanalyzed for ninhydrin-reactive material by the method ofMoore (15) and for radioactivity by liquid scintillation assay.

D-Amino-Acid Oxidase Treatment. D-Amino-acid oxidase(porcine kidney) was obtained from Sigma at a specific activityof 17 units/mg of protein and was dialyzed overnight at 4°Cagainst 500 volumes of 100 mM sodium pyrophosphate buffer,pH 8.3/3 mM EDTA/25 mM NaCl. Isolated aspartic acid /-[3H]methyl ester from the ion-exchange chromatography step(70 Al; 7,000 cpm) was mixed with 10 ,ul of 1 M L-proline and16 ,ul of 0.1 M D-aspartic acid /3-methyl ester. Aliquots (40 ,ul)were treated either with 200 ,l of D-amino-acid oxidase (1 mgof protein) or with 200 ,l of the dialysis buffer for 5 hr at roomtemperature. Each incubation was then quenched with 150 ,lof 8% 5-sulfosalicylic acid. Portions (350 ,ul) ofeach sample werethen analyzed by ion-exchange chromatography as in the L-amino-acid oxidase experiment.

Formation and Separation of L-Leucyl Diastereomeric Di-peptides. L-Leucyl dipeptides were formed as described (14)by reaction of L- and D-aspartic acid /3-methyl esters with L-leu-cine N-carboxyanhydride (LeuCA; 4-(2-methylpropyl)-2,5-ox-azolidinedione). This reagent was synthesized as described byKonopinska and Siemion (16) from N-carbobenzoxy-L-leucine(Sigma) and oxalyl chloride (98%; Aldrich). Amino acids (4-4.5,Amol) were mixed with 1.1 ml of ice-cold 0.45 M sodium boratebuffer (pH 10.2) and immediately transferred to a tube con-taining a 70% molar excess of solid LeuCA. The tube contentswere vigorously mixed at 4°C for 2 min, and the coupling re-action was then quenched with 0.48 ml of 1 M HCl. Thequenched reaction mixture was mixed with an equal volume ofsodium citrate sample buffer (pH 2.2) and applied to a 0.9 cmX 50 cm column of Beckman amino acid analyzer resin (AA-15)equilibrated at 56°C in citrate buffer (pH 3.25; 0.2 M Na+). Thecolumn was then eluted at a flow rate of 70 mVhr with the samebuffer. Fractions (5 min) were collected and analyzed for ra-dioactivity by liquid scintillation counting and for ninhydrincolor by the method of Moore (15).

RESULTSAspartic Acid .3-[3H]Methyl Ester in Membrane Digests Is

Not a Substrate for L-Amino-Acid Oxidase. Radioactive aspar-tic acid 3-methyl ester has been identified in proteolytic digests

of erythrocyte *membrane proteins (4). The radioactive yieldof aspartic acid 83-[3H]methyl ester from the *membranes inthese experiments was 5-10%; much of the remaining radio-activity chromatographed as [3H]methanol and may have beenformed by ester hydrolysis during the digestion step.

Aspartic acid /3-[3H]methyl ester in unfractionated digestswas treated with L-amino-acid oxidase. The radioactive methylester was unaffected by the oxidase, even though a standard ofL-aspartic acid ,3methyl ester added to the mixture was com-pletely degraded (Fig. 1). L-Aspartic acid, which also was pres-ent in the mixture, was a poor substrate for the L-oxidase (17)and provided an internal standard for the ninhydrin analysis.This experiment also was performed with isolated aspartic acid-P[3H]methyl ester mixed with an internal standard of D-as-

partic acid /3-methyl ester. In this case, the L-amino-acid oxi-dase was inactive toward both the radioactive aspartic acid /3-methyl ester and the standard D-aspartic acid /3-methyl ester(data not shown). Overall, the results indicate that L-amino-acidoxidase specifically oxidizes standards of L- and not D-asparticacid /3-methyl ester and that this enzyme does not detectablyoxidize erythrocyte-derived aspartic acid /3-[3H]methyl ester.

Erythrocyte-Derived Aspartic Acid f3-[3H]Methyl Ester Isa Substrate for D-Amino-Acid Oxidase. Radioactive asparticacid P-methyl ester isolated from membrane digests by ion-ex-change chromatography disappeared upon treatment with D-amino-acid oxidase (Fig. 2). Standard D-aspartic acid /3-methylester also disappeared in an enzyme-dependent fashion,whereas an internal ninhydrin standard of L-proline was notaffected by the enzyme. In other experiments, D-amino-acidoxidase was completely inactive towards standard L-asparticacid /3-methyl ester (data not shown).

Resolution of Enantiomers of Aspartic Acid 3-Methyl Esterby Diastereomer Formation: Synthesis of L-Leucyl-D-AsparticAcid fp-[3H]Methyl Ester from Erythrocyte Digests. The pro-cedure (14) for resolving amino acid enantiomers by covalentlycoupling D- and L-amino acids to LeuCA results in the formationof diastereomeric L-leucyl dipeptides, which can be separatedand quantitated by ion-exchange chromatography. By thismethod, L-leucyl-D-aspartic acid /3-methyl ester was separatedfrom L-leucyl-L-aspartic acid /3-methyl ester (see the ninhydrintrace, Fig. 3 Top). The leucyl derivatives of D- and L-asparticacid also were resolved as has been shown previously (ref. 14;ninhydrin trace, Fig. 3 Bottom).When erythrocyte-derived aspartic acid /3-[3H]methyl ester

was coupled to L-leucine by this method, a radioactive productwas formed that coeluted with L-leucyl-D-aspartic acid /3-methyl ester and represented 71% of the total radioactivity.tThe remaining 29% of the radioactivity coeluted with the un-reacted standard or methanol (peak II). No radioactivity wascoeluted with L-leucyl-L-aspartic acid /3-methyl ester. The yieldof products after the LeuCA coupling was about the same forboth the radioactive aspartic acid /-methyl ester starting ma-terial and the standard L- and D-aspartic acid /3-methyl esterstarting materials.A control experiment was performed in which LeuCA was

left out of the reaction mixture (Fig. 3 Middle). In this case, allof the radioactivity remained with the peak of aspartic acid /3-methyl ester (II) as expected.

In another control experiment, the mixture ofradioactive andstandard amino acid esters was hydrolyzed in base before cou-pling them to LeuCA (Fig. 3 Bottom). The radioactive amino

t As is common in high-resolution ion-exchange chromatography sys-tems, the isotope effect due to the three tritium atoms in aspartic acidl3-[3H]methyl ester (90 Ci/mmol) and its derivatives consistently leadsto their slightly early elution as compared with standards (4, 18, 19).

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2462 Biochemistry: McFadden and Clarke

I-

r-

x

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10 20 30 10 20 30Fraction

FIG. 1. L-Amino-acid oxidase treatment of aspartic acid _3-[3H]methyl ester from erythrocyte membranes. Carboxypeptidase Y digests of *mem-branes were incubated either in the absence (A) or presence (B) of L-amino-acid oxidase, and the supernatants were analyzed for radioactive andninhydrin-reactive species by ion-exchange chromatography. Radioactivity in 0.8 ml of each 2.3-ml fraction was measured by the liquid scintillationtechnique. 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, peakof 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 notbeen 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[3H]methanol formed (peak I) and by the parallel loss of radio-activity in peaks II and III. The hydrolysis ofthe standard aminoacid esters was 88% complete as indicated by the increased yieldof dipeptides containing nonesterified aspartic acid.

The results from both the oxidase and diastereomer experi-ments are consistent with the interpretation that all of the iso-lated erythrocyte-derived aspartic acid ,B-[3H]methyl ester hasthe D configuration. No evidence was found for any L-asparticacid P-methyl ester from erythrocyte proteolytic digests.

6

2

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10 20 30Fraction

FIG. 2. D-Amino-acid oxidase treatment of isolated aspartic acid,3[3H]methyl ester. Samples that had been treated in the absence (A)or presence (B) of D-amino-acid oxidase were analyzed by ion-exchangechromatography, and the radioactivity and ninhydrin color of eachfraction were determined as in Fig. 1.

Release of D-Aspartic Acid fl-Methyl Ester by Carboxypep-tidase Y. Carboxypeptidase Y (which may contain small amountsof contaminating endopeptidase activities) was the most effec-tive agent in releasing aspartic acid ,3-[3H]methyl ester fromerythrocyte membrane proteins. Addition of Pronase, subtili-sin, or proteinase K to the digestion mixture lowered yields ofradioactive product; pepsin, papain, elastase, trypsin, chymo-trypsin, or thermolysin had little or no effect (data not shown).Carboxypeptidase Y can hydrolyze peptide bonds on both sidesof D-amino acid residues, although at a low rate (20). We ex-cluded the possibility of artifactual racemization during prep-aration of aspartic acid 8-[3H]methyl ester for several reasons.First, enzymatic or nonenzymatic racemization would produceequal amounts of the L and D forms, yet we only detected D-aspartic acid ,B-methyl ester. Second, no racemization or selec-tive hydrolysis of either D- or L-aspartic acid (3-methyl esterstandards 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 frompoly(L-aspartic acid (3-methyl ester) (data not shown).

DISCUSSIOND-Aspartyl Residues Are Methylated in Erythrocyte Mem-

brane and Cytoskeletal Proteins. Previous work has shown thatintact human erythrocytes incubated with L-[methyl-3H]-methionine synthesize S-adenosyl-L-[methyl-3H]methionine,which serves as the methyl donor for protein methylation re-actions (21). The principal methyl acceptors in the cell are themembrane cytoskeletal protein bands 2.1 and 4.1 and the in-tegral membrane protein band 3 (5). The methylated amino acidresidue has been isolated from carboxypeptidase Y digests ofthese labeled membranes and identified as aspartic acid /3-[3H]methyl ester (4).

In this paper, we present evidence that all ofthe aspartic acid,3 [3H]methyl ester recovered has the uncommon D configu-ration. This configuration of aspartic acid ,B-[3H]methyl esterwas established by the reactivity ofthe amino acid with oxidasesspecific for D-amino acids and by its lack of reactivity with ox-

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aspartic acid (3-[3H]methyl ester to L-leucine to form a diaster-eomeric dipeptide that could be assigned an absolute stereo-configuration. No evidence was found for the presence of anyL-aspartic acid p-[3H]methyl ester.

Our finding of r'-aspartic acid p3-methyl ester in erythrocytesrationalizes many previously poorly understood observations.Racemization in vivo would not be expected to affect the entirepopulation of a protein species; the experimental finding is thatprotein carboxyl methylation is always substoichiometric (5, 8,9). The substrate specificity for protein methylation in vivo isrelatively broad; all proteins are potentially subject to racemi-zation, although sequence or conformational features, or both,as well as the rate of methyl group turnover, might determinethe actual methylation levels observed (5). Proteolytic enzymesare in general specific for L-amino acids; the difficulty of iso-lating aspartic acid P-methyl ester in high yield is likely to resultfrom the inability of most enzymes to digest peptide bonds ofD-amino acid residues (see Results).

Cellular Origin of D-Aspartyl Residues. There is no knownfunction for D-amino acid residues in proteins. The only knownroute by which D-amino acid residues can appear in mammalianproteins is by a spontaneous racemization of the L-amino acidsthat have been biologically assembled into polypeptides. In fact,the racemization of aspartate residues in proteins occurs at agreater rate than that of any other amino acid, except possiblyserine (22, 23). In long-lived tissues, such as lens and toothenamel, the spontaneous rate of racemization can lead to sub-stantial accumulation of D-aspartyl residues during a lifetime(24, 25). The functional consequences of such racemization maybe severe (11).A Model for the Repair of Racemized Proteins. In order to

explain why "abnormal" D-aspartyl residues may be methylatedin the cell, we have proposed a model shown in Fig. 4. We sug-gest that protein carboxyl methylation is involved in the repairof proteins racemized at aspartyl residues. The spontaneousappearance of a potentially damaging D--aspartyl residue resultsin its recognition and methylation by a protein carboxyl meth-yltransferase. The methylation step leads to a postulated "re-pair" step that accompanies ester hydrolysis and results in therestoration of the original L configuration of the aspartyl resi-due. The loss of methanol (and its probable subsequent oxi-dation or excretion) in the repair step ensures that the reversereaction (enzymatic production of D-aspartyl residues) does notoccur.The enzyme-responsible for the esterase/racemase function

postulated in Fig. 4 has not yet been identified. The possibilityalso exists that nonenzymatic hydrolysis of the ester group mayitself lead to racemization at the a carbon and, thus, result ina 50% conversion of the D-amino acid residue back to the orig-inal L configuration in each methylation/demethylation cycle.The reason for this is that the hydrolysis of P-methyl esters ofaspartyl residues is likely to proceed through a cyclic imide in-termediate (cf. refs. 4 and 6 and references therein). Resonancein this five-membered ring system can facilitate the loss of aproton from the a carbon and, thus, promote racemization.

Variations on this model also may be proposed. Our resultshave shown that the product ofcarboxyl methylation is a proteinD-aspartyl P-methyl ester. D-Aspartyl residues could originateeither from L-aspartyl or L-asparaginyl residues because the lat-ter residues may deamidate through a racemization-prone im-ide intermediate (see above). Because the deamidation of as-paragine residues is an age-related process (10), the D-aspartylmethyl ester observed here may be an intermediate in the re-pair of this type ofcellular damage. In this case, the methyl esterwould be replaced by an amide group in the subsequent D-to-L conversion.

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D-Aspartyl residueAbnormal function

H

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H SAMSAH )

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0

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FIG. 4. Proposed pathway for the repair of proteins that have beenspontaneously racemized at aspartyl residues. The previously de-scribed protein carboxyl methyltransferase specifically methylatesonly "aged" aspartyl residues in the D configuration. A second enzymeis postulated to couple ester hydrolysis with the restoration of the nor-mal L configuration of the amino acids. SAM, S-adenosylmethionine;SAH, S-adenosylhomocysteine.

Alternative Models for the Role of D-Aspartyl (i-MethylEster Formation. The racemization repair model is consistentwith past results. However, until the repair pathway is experi-mentally tested, other pathways subsequent to methylation atD-aspartyl residues must be considered. For example, meth-ylation may be a cellular signal that targets a racemized proteinfor destruction. Or, methylation at accumulated D-aspartyl res-idues may be a biological clock that signals the age of the cell.Other schemes may be postulated in which D-amino acids ormodified D-amino acids have specific physiological functions.

Methylation at Protein D-Aspartyl Residues Is Probably aGeneral Phenomenon in Eukaryotic Cells and Tissues. Proteincarboxyl methyltransferase activity similar to that demonstratedin erythrocytes has been measured in extracts of all mammaliantissues examined so far (1, 12). Purified methyltransferases fromthese sources are similar to the erythrocyte enzyme in theircytosolic localization, their polypeptide molecular weight andsubunit structure, their Km values for S-adenosylmethionine,their Ki values for S-adenosylhomocysteine, and their specific-ity for exogenous protein acceptor species (cf. refs. 1 and26-31). Thus, the protein carboxyl methyltransferase reactionwe have been studying in the erythrocyte may be representativeof a widely distributed enzyme and function. It is clear, how-ever, that the protein carboxyl methyltransferase involved inbacterial chemotaxis (2, 3) is distinct from this activity. This lat-ter enzyme methylates residues at stoichiometric levels (32).

Possible Racemization Repair at Other Than Aspartyl Res-idues. The essential feature ofthe repair function proposed hereis that the hydrolysis of the D-aspartyl methyl ester residue canbe coupled to the inversion of the configuration at the a carbon(Fig. 4). In principle, the reversal of any covalent modificationreaction at a protein residue in the D configuration could sim-ilarly be utilized by the cell to thermodynamically drive theconversion to the L configuration. For example, racemizedserine, threonine, or tyrosine residues could be recognized byD-specific protein kinase(s). Inversion ofconfiguration could bedriven in this case by the hydrolysis of the phosphate ester.

Damage to critical cellular components must be repaired.Failure to repair a single lesion in a DNA molecule, for example,may be lethal. Damage to a less critical component, like a ra-

cemized subunit of a hemoglobin molecule, will probably notgreatly disrupt the organism. Nature may supply cells with pro-tein repair systems because many proteins participate in highlycooperative processes. A single damaged protein moleculecould weaken the amplification of a metabolic cascade signal ormight. disrupt the whole structure of a cytoskeletal net. Thus,the recognition of racemized aspartyl (and possibly other) res-idues by specific covalent modification enzymes may be an es-sential function in cells.We are grateful to our colleagues for helpful discussions. This work

was supported by a grant from the National Institutes of Health (GM26020) and a grant-in-aid from the American Heart Association (withfunds contributed in part by the Greater Los Angeles Affiliate). P. N. M.was supported by U.S. Public Health Service Training Grant GM07185.

1. Paik, W. K. & Kim, S. (1980) in Protein Methylation (Wiley, NewYork), pp. 202-231.

2. Clarke, S., Sparrow, K., Panasenko, S. & Koshland, D. E., Jr.(1980) J. Supramot Struct. 13, 315-328.

3. Koshland, D. E., Jr. (1981) Annu, Rev. Biochem. 50, 765-782.4. Janson, C. A. & Clarke, S. (1980)J. BioL Chem. 255, 11640-11643.5. Freitag, C. & Clarke, S. (1981)J. Biol. Chem. 256, 6102-6108.6. Terwilliger, T. C. & Clarke, S. (1981) J. Biol. Chem. 256,

3067-3076.7. Barber, J. R., Brunauer, L. S., O'Connor, C. M. & Clarke, S.

(1981)J. Cell Biol. 91, 260 (abstr.).8. Kim, S. & Li, C. H. (1979) Proc. Natl Acad. Sci. USA 76,

4255-4257.9. Kloog, Y., Flynn, D., Hoffman, A. R. & Axelrod, J. (1980)

Biochem. Biophys. Res. Commun. 97, 1474-1480.10. McKerrow, J. H. (1979) Mech. Ageing Dev. 10, 371-377.11. Masters, P. M., Bada, J. L. & Zigler, J. S., Jr. (1978) Proc. Natl.

Acad. Sci. USA 75, 1204-1208.12. Diliberto, E. J., Jr., & Axelrod, J. (1976) J. Neurochem. 26,

1159-1165.13. de Groot, N. & Lichtenstein, N. (1959) Bull, Res. Counc. Israel

8A, 116.14. Manning, J. M. & Moore, S. (1968) J. Biol. Chem. 243,

5591-5597.15. Moore, S. (1968)J. Biol. Chem. 243, 6281-6283.16. Konopinska, D. & Siemion, I. Z. (1967) Angew. Chem. nt. Ed.

EngI 6, 248.17. Meister, A. & Wellner, D. (1963) in The Enzymes, eds. Boyer,

P. D., Lardy, H. & Myrback, K. (Academic, New York), Vol. 7,2nd Ed., pp. 609-648.

18. Kleene, S. J., Toews, M. L. & Adler, J. (1977)J. Biol. Chem. 252,3214-3218.

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10826-10833.

L-Aspartyl residueNormal function

°%CeoO-CH2

-NH-C-C- SI IIH O

Postulated .}rracemase/ I MeOHesteraseLonfunction

low, spontaneous"aging" reaction

Proc. Natl. Acad. Sci. USA 79 (1982)

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