Racemization of aspartic acid in human proteins

Ageing Research Reviews 1 (2002) 43 – 59 www.elsevier.com/locate/arr

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Racemization of aspartic acid in human proteins Stefanie Ritz-Timme a, Matthew J. Collins b,* a

Institut fu¨r Rechtsmedizin der Christian-Albrechts-Uni6ersita¨t zu Kiel, Arnold-Heller-Str. 12, D-24105 Kiel, Germany b Fossil Fuels and En6ironmental Geochemistry (NRG), Drummond Building, Uni6ersity of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, UK Received 19 July 2001; received in revised form 20 July 2001; accepted 20 July 2001

Abstract Aspartic acid racemization (AAR) represents one of the major types of non-enzymatic covalent modification that leads to an age-dependent accumulation of abnormal protein in numerous human tissues. In vivo racemization is an autonomic process during the ‘natural’ ageing of proteins, and correlates with the age of long-lived proteins. Consequently AAR can be used as molecular indicator of protein ageing as well as for the identification of permanent proteins that age with the human organism. Although long-living, structural proteins are mainly affected, AAR may be significant on a time scale also relevant to enzymes and signaling proteins. It may result in a loss of protein function due to proteolysis or due to changes in the molecular structure. In vivo racemization may also increase in pathological conditions. AAR has already been discussed as a relevant pathophysiological factor in the pathogenesis of diseases of old age such as atherosclerosis, lung emphysema, presbyopia, cataract, degenerative diseases of cartilage and cerebral age-related dysfunctions. Although the details of the biological consequences of AAR have to be further elucidated, it is evident that AAR plays a role in the molecular biology of ageing. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aspartic acid; Racemization; Review; Succinimide; Deamidation; Collagen

* Corresponding author. Fax: + 44-191-222-5431. E-mail address: [email protected] (M.J. Collins). 1568-1637/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 6 3 - 3

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1. Introduction: an historical perspective The accumulation of damage is a major component of ageing. One such damage that occurs in proteins is racemization. Proteins are synthesized using only one optically active form of amino acids, laevo- or L-amino acids. However, over time the optical purity of such a solution would be lost by the slow but inevitable isomerization of these amino acids, a process commonly termed racemization. Eventually equilibrium would be achieved at which the relative proportions of D-(dextro) and L-amino acids would remain the same, and the solution would be termed racemic. In Kuhn (1936) recognized that preservation of the optical purity in the human organism was ‘a serious kinetic, thermodynamic, and eventually vital problem’ (Kuhn, 1936, 1958). However, analytical limitations prevented him from testing his hypothesis that there would be a decrease of optical purity with age (Kuhn, 1955, 1958). It was not until the early 1970s that a link between age and racemization was established experimentally, with analytical resources being developed not for biomedical science but in the quest for extra-terrestrial life. It was geochemists, interested in the analysis of chirality in amino acids from moon rock and meteorites, who were the first to investigate the kinetics of amino acid racemization (Kvenvolden et al., 1970). Bada and colleagues began to apply racemization of amino acids to date bone samples (Bada and Protsch, 1973). Most amino acids accumulated D-forms slowly but aspartic acid1 racemized sufficiently rapidly to be detectable not only in archaeological bone but also in living human tissue. Helfman and Bada (1975), Helfman and Bada, (1976) detected an accumulation of D-aspartic acid with increasing age in enamel and dentine of human teeth and observed a close relationship between D-aspartic acid concentration and age in these tissues, especially in dentine (Helfman and Bada, 1975, 1976). They speculated that in vivo racemization of aspartic acid might be ‘a widespread phenomenon in metabolically stable proteins throughout the mammalian body’ and that ‘metabolically inert proteins will be subject to increasing amounts of conformational change with increasing age’ due to the racemization of protein-bound amino acids (Helfman et al., 1977). This group further suggested ‘two uses of amino acid racemization as a metric (1) to estimate ages for living people and (2) to provide a measure for the in vivo lifetimes of structural proteins’. Their ideas have born fruit. An accumulation of D-aspartic acid has been observed in many tissues (Table 1) and the close relationship between aspartic acid racemization (AAR) and age in permanent proteins has been used to develop accurate methods for age estimation in Forensic Medicine (Ogino et al., 1985; Ritz et al., 1993; Mo¨ rnstad et al., 1994; Fu et al., 1995; Ohtani, 1995; Ritz-Timme, 2000). Increasingly, racemization is being used, as Helfman et al., (1977) envisaged, as a measure of the in vivo lifetimes of proteins. Racemization is used to investigate 1

More properly Asx, as asparingyl (Asn), aspartyl (Asp), isoaspartyl (iAsp) and succinimidyl (Asu) residues, are all converted to free aspartic acid during acid hydrolysis, a preperative step in chromatographic amino acid analysis.

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tissue turnover and the pathogenesis of typical diseases of old age such as the chronic emphysema of the lung, atherosclerosis or degenerative disorders of articular cartilage (Shapiro et al., 1991, Powell et al., 1992, Maroudas et al., 1998; Gineyts et al., 2000; Verzijl et al., 2000). Today, we are aware that: 1. AAR is an inevitable process during the ‘natural’ ageing of proteins; it is a widespread phenomenon that occurs spontaneously in numerous human tissues. 2. AAR can be used as a molecular indicator of protein ageing and for the identification of permanent proteins that age with the human organism. 3. AAR affects especially long-living, structural proteins, but may be significant also on a time scale relevant for enzymes and signaling proteins. Table 1 Tissues that contain significant concentrations of long-living proteins that exhibit an accumulation of D-aspartic acid during ageing Tissue

References

Tooth dentine

Helfman and Bada, 1976; Ritz et al., 1993; Mo¨ rnstad et al., 1994; Ohtani, 1995 Helfman and Bada, 1975 Ohtani et al., 1995 Ritz and Schu¨ tz, 1993 Maroudas et al., 1992, 1998 Pfeiffer et al., 1995a; Verzijl et al., 2000 Fujii et al., 1987; Verzijl et al., 2000 Ritz et al., 1994, 1996; Pfeiffer et al., 1995b; Ohtani, 1998; Ohtani et al., 1998a,b; Brady et al., 1999; Gineyts et al., 2000 Masters et al., 1977, 1978; Vandenoetelaar and Hoenders, 1989; Groenen et al., 1990; Fujii et al., 1997, 1999a,b; George et al., 1999 Man et al., 1983; Johnson and Aswad, 1985; Fisher et al., 1986; Shapira and Jen Chou, 1987; Shapira et al., 1988; Payan et al., 1992; Roher et al., 1993; Kenessey et al., 1995; Paranandi and Aswad, 1995; Najbauer et al., 1996; David et al., 1998; Watanabe et al., 1999 Shapiro et al., 1991 Powell et al., 1992 Brunauer and Clarke, 1986; Inaba et al., 1992; Galletti et al., 1995; Perna et al., 1997; Ingrosso and Perna, 1998 Di Salvo et al., 1999 Kinzel et al., 2000

Tooth enamel Tooth cementum Intervertebral discs Articular cartilage (proteoglycans) Cartilage Skin Bone (Type I collagen, telopeptides, osteocalcin)

Ocular lens (aA-crystallin)

Brain (white matter, myelin basic protein, b amyloid protein, tau protein; synapsina, proteoglycansa, tubulina)

Lung parenchyma (elastin) Artery wall (elastin) Membrane proteins of erythrocytes Band 4.1b

Liver (Serine hydroxymethyltransferase)a Cardiac muscle (PKA catalytic subunit) a b

Implicated from presence of isoaspartyl residues. Implicated by deamidation.

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4. AAR may also occur as secondary phenomenon caused by protein degradation, e.g. under pathological conditions. We can further speculate that AAR may be of considerable relevance in the biology of ageing. It may have important pathophysiological consequences and possibly plays a role in the pathogenesis of typical diseases of old age.

2. The mechanism of in vivo AAR in ageing proteins

2.1. The chemistry of AAR Although the rapid racemization of Asx residues relative to other amino acids was initially explained by simple acid–base chemistry at the a-carbon (Neuberger, 1948; Bada, 1984), the explanation is only satisfactory for free amino acids. In proteins, the unusually rapid racemization of Asx residues arises because it occurs prior to peptide bond hydrolysis. Racemization occurs in a five membered succinimide ring (Asu) that forms when the side chain condenses with the N+ 1 peptide bond nitrogen (Clarke, 1987; Geiger and Clarke, 1987; Lowenson and Clarke, 1988; Capasso et al., 1992), because the chiral centre of the succinimide can be easily racemized (Radkiewicz et al., 1996). However, the succinimide is a short lived structure, undergoing rapid hydrolysis at either side of the imide nitrogen to yield both a- and b-linked aspartyl residues; a preferred hydrolysis at the a carbon yields a 3:1 ratio of isoaspartyl to aspartyl residues in flexible sequences (Capasso and Di Cerbo, 2000). The rate of reaction of asparaginyl residues is even more rapid than their aspartyl counterparts, but in this case formation of the succinimide causes deamidation. Consequently, over time the two residues Asn and Asp both decompose to the same four residues, namely L-aspartyl (L-Asp), D aspartyl (D-Asp), L isoaspartyl (L-iAsp) and D isoaspartyl (D-iAsp), all of which are in chemical equilibrium via D- and L-Asu. The range of products arising from succinimide formation is analytically challenging, and most methods do not distinguish all of these forms. Mass spectrometry can detect the mass-shift caused by deamidation (e.g. Robinson and Robinson, 2001a), but none of the aspartyl isomers-although the succinimide is potentially tractable (but see Lehmann et al., 2000). Enzymatic methylation using PIMT (Lowenson and Clarke, 1992) can detect isoaspartyl and D-aspartyl forms, but cannot distinguish between them, and cannot detect residues that are inaccessible to the enzyme (Aswad et al., 2000). Amino acid analysis can resolve the L and D-Asp enantiomers, but not the b linked residues. Only immunological methods can distinguish individual isomeric forms and these are increasingly being used to investigate the role of racemization in the ageing of proteins (e.g. Lehrman et al., 1992; Fledelius et al. 1997; Brady et al. 1999; Fujii et al. 2000; Shimizu et al., 2000). The physicochemical environment of the amino residues determines the rate of in vivo AAR. Since the physical surroundings (e.g. salt concentration, pH and temperature; Capasso et al., 1991; Brennan and Clarke, 1993), are largely constant, the racemization rate (and deamidation rate of Asn) depends mainly on the nature

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Fig. 1. Close relationship between age and the increase in D-Asx in dentine, in lung parenchyma, and in the media of the thoracic aorta. The dentine (3rd molar root) samples were treated as described before (Ritz et al., 1993), the other total tissue samples were minced in fine pieces, washed in a 15% NaCl solution and in ethanol/ether (3:1), and dried. The extent of aspartic acid racemization was determined as described elsewhere (Ritz et al., 1993; Ritz-Timme, 2000) and have here been corrected for the racemization (0.084%) induced during hydrolysis (6 h at 100 °C), to illustrate the initially rapid increase in D-Asx following synthesis. The data for 3rd molar root dentine is plotted relative to age of synthesis not absolute age.

of the adjacent residues (especially the N+ 1 residue; Clarke, 1987; Lowenson and Clarke, 1988; Lura and Schirch, 1988; Wright, 1995; Robinson and Robinson, 2001a), the local environment of the residue within the proteins (Fujii et al. 1996, 1999a) and physical constraint related to higher order structures (Kossiakoff, 1988; Chazin and Kossiakoff, 1995; Van Duin and Collins, 1998; Xie and Schowen, 1999; Robinson and Robinson, 2001b). Many of the sites of succinimide formation occur in the terminal regions of proteins (Roher et al., 1993; Di Salvo et al., 1999; Gineyts et al., 2000), those near the external surface, or in conformationally flexible regions (Clarke, 1987; Lowenson and Clarke, 1988; Potter et al., 1993; Capasso et al., 1996; Fujii et al., 1996, 1999a; Noguchi et al., 1998; Capasso and Salvadori, 1999; Sandmeier et al., 1999). In unconstrained sites with a favourable sequence (notably Asx-Gly; Radkiewicz et al., 2001) racemization is so rapid (t= 1 day– 1 year) that all but the most newly synthesized proteins will carry a significant percentage of D-Asx. This rapid racemization of fast sites explains an otherwise puzzling offset in the intercept of the slope of increase in D-Asx against age in many tissues (Waite and Collins, 2000) (Figs. 1 and 2). For collagen rich tissues, this can be explained by racemization prone sites in the telopeptide (a1(I) Asp1211-Fledelius et al., 1997; Cloos and Fledelius, 2000; a2 (I) Asp82-Brady et al., 1999). Rates of racemization at such flexible sites are slightly lower than those reported for synthetic peptides (e.g. Geiger and Clarke, 1987; Fujii et al., 1996; Robinson and Robinson, 2001b).

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Conformationally constrained sites, and those with primary structures less favourably to succinimide formation will accumulate D-Asx more slowly (c.f. Kossiakoff, 1988), leading to age dependent accumulation (Fig. 1). The local environment may result in D:L equilibrium ratios other than 1.0 (e.g. Asp151 in a A-crystallin, D:L of 5.7, Fujii et al., 1996). However, in the most rigid structures, notably the triple helix of collagen (Van Duin and Collins, 1998), succinimide formation is effectively prevented. No significant accumulation of D-Asx was observed in the collagen rich insoluble fraction of archaeological bone over tens of thousands of years (Matsu’ura and Ueta, 1980).

2.2. A link to deamidation Asparagine deamidation and AAR are closely linked. Deamidation of asparaginyl also occurs by the formation of the succinimide, with concomitant generation of D-Asx and iAsx residues (Geiger and Clarke, 1987; Brennan and Clarke, 1993). Deamidation of Asn–Gly residues via Asu formation can be extremely rapid in flexible sequences, (t=  1 day; Robinson and Robinson, 2001a). The speed of reaction, change in peptide chemistry and the lack of a repair system (PIMT will repair deamidated asparaginyl residues as L-Asp) all lend weight to the view that deamidation can function as a molecular timer (Robinson et al., 1970; Robinson and Robinson, 2001a). A recent analysis of the role of primary structure on deamidation (and hence succinimide formation) rates (Robinson and

Fig. 2. Comparison between the increase in D-Asx in four collagen rich tissues, acid insoluble fraction of dentine (Ritz et al., 1993), acid insoluble fraction of bone (Ritz et al., 1994; Ohtani et al., 1998b), collagen preparations from cartilage (Verzijl et al., 2000) and skin (Verzijl et al., 2000). Data have been corrected for induced racemization.

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Fig. 3. Estimation of in vivo lifetimes of proteins based on the relationship between age and the extent of aspartic acid racemization: Proteins with high turnover (dotted line) do not exhibit an accumulation of modified D-aspartic acid residues with age. Proteins with longer half-life and low turnover (dashed line) can exhibit an accumulation of D-aspartic acid that is not strictly age-dependent because of the existing turnover. An equilibrium between accumulated D-aspartic acid residues and L-aspartic acid residues of newly synthesized proteins may result in elevated D-aspartic acid concentrations at a constant level. Only in permanent proteins without any turnover (solid line) the concentration of D-aspartic acid increases with age in a predictable manner.

Robinson, 2001a; see also Capasso 2000), coupled with approximations for effects of higher order structure now makes it possible to make first-order estimates of deamidation rates of proteins (Robinson and Robinson, 2001b), and by extension racemization rates. The importance of higher order structure identified in this latter study highlights a problem with using high temperature kinetic estimates to predict AAR rates in native proteins. At higher temperatures proteins will denature increasing both the conformational freedom of individual Asx residues and the overall number of Asx residues that can undergo racemization. Consequently these experiments will tend to over-estimate racemization rates in vivo. Collins et al. (1999) were able to use a predictive model to estimate racemization rates in collagen at high temperature but their model greatly over-estimated rates in vivo (c.f. Verzijl et al., 2000).

3. AAR as molecular indicator of protein ageing Autonomous, ‘primary’ AAR (occuring in the absence of pathological conditions, see Section 5.) can be used as a molecular indicator of protein ageing. Using this molecular clock the in vivo lifetimes of extracellular proteins can be estimated, since the relationship between the age of a defined protein and its D-Asx content depends on its turnover (Fig. 3): “ Proteins with high turnover are exchanged before a relevant accumulation of D-aspartic acid can occur. These proteins do not exhibit an accumulation of modified D-Asx residues with age (dotted line in Fig. 3).

50 “

“

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Proteins with longer half-life and low turnover can exhibit an accumulation of D-Asx. However, because of the existing turnover the relationship between aspartic acid racemization and age is not close; an equilibrium between accumulated D-Asx residues and L-Asx residues of newly synthesized proteins may result in elevated D-Asx concentrations at a constant level (dashed line in Fig. 3). Only in permanent proteins without any turnover do we find a very close relationship between the accumulation of altered aspartic acid residues and age: The concentration of D-Asx increases with age in a regular manner (solid line in Fig. 3). This can be the basis for the identification of long-living or permanent proteins that age within the human organism.

4. The occurrence of AAR in ageing human proteins In vivo AAR was first described for tissues with an extremely slow turnover (bradytrophic tissues) such as dentine and enamel of the teeth, intervertebral discs and the ocular lens (Table 1). Meanwhile it has become evident that AAR is a significant phenomenon in non-bradytrophic tissues such as bone or lung parenchyma (Shapiro et al., 1991; Ritz et al., 1994, 1996) and even cellular proteins (Brunauer and Clarke, 1986). Fig. 1 illustrates data for a variety of total tissue AAR. Similar rates of increase in D-Asx do not necessarily indicate similar rates of turnover because different protein compositions or different rates of protein turnover within the same tissue may account for tissue specific differences. The very close relationship between AAR in the analysed total tissue specimens and age (Fig. 1) proves the existence of a significant fraction of long-lived proteins in these tissues (Fig. 3). Table 1 summarizes literature with similar data for diverse tissues and erythrocyte proteins. These data demonstrate that AAR is indeed a widespread phenomenon in the human organism, as first suggested by Helfman et al. (1977). The data presented in Fig. 1 as well as most of the data from the literature in Table 1 are the results of the analysis of total tissue specimens or of incompletely purified protein fractions and do not allow the identification of single permanent and ageing proteins exhibiting AAR. This can only be achieved by the analysis of defined, purified proteins from individuals of different ages. Such a final identification of permanent proteins by AAR has been achieved for elastin in lung parenchyma (Shapiro et al., 1991) and in artery walls (Powell et al., 1992), aA-crystallin of the ocular lens (Fujii et al., 1999b) and in bone osteocalcin (Ritz et al., 1996). The very close relationship between AAR in such permanent proteins and age proves that AAR obviously is an autonomic, inevitable process during the ‘natural’ ageing of proteins. Tissue specific differences in the rate of accumulation of D-Asx in collagen (dentine \ bone \ cartilage \ \skin) indicate that turnover can cause a decrease in the rate of accumulation (Fig. 2). For skin an increase in D-Asp (probably corresponding to racemization of a1(I) Asp1211) is seen to occur following cessation of growth (implying that all the skin collagen in mature individuals has a turnover time in excess of one year; c.f. Gineyts et al., 2000). The slower increase in

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accumulation of Asx in compact bone (femur) collagen when compared with 3rd molar root dentine collagen and the wider scatter of data suggests that there is some limited collagen turnover in compact bone; lower values in cartilage suggest slightly higher rates of turnover again. The data presented in Fig. 2 reveals the potential for Asx as a tool to investigate the rate of protein turnover in different tissues. The results imply an accumulation of small but significant structural damage in the proteins of dentine and bone that may help to explain changes in the mechanical properties of these tissues with age (Zioupos et al., 1999). 5. The occurrence of AAR as epiphenomen of protein degradation under pathological conditions The very close relationship between AAR and age in purified proteins (Shapiro et al., 1991; Powell et al., 1992; Ritz et al., 1996) indicates that the accumulation of D-Asx during ‘natural’ ageing occurs autonomously. However, an association between tissue pathology and elevated D-Asx concentrations has also been observed for example in osteoarthritic cartilage (Maroudas et al., 1992) and in lenses which are either brunescent (Masters et al., 1977) or have been damaged by UV irradiation (Fujii et al., 1997). Since degradation can accelerate racemization, and because in vivo racemization may then lead to further conformational alteration (Fabian et al., 1994; Orpiszewski and Benson, 1999; Mizuno et al., 2000), there is a potential for a vicious circle to emerge; protein degradation“ increased conformational freedom “AAR “ further conformational change“ further degradation. 6. Repair mechanisms and turnover as a protection against molecular damage by AAR One mechanism to control AAR is repair. The biological load of AAR is evidenced by the widespread occurrence of a repair enzyme protein-L-isoaspartate(D-aspartate) O-methyltransferase (PIMT; EC 2.1.1.77; McFadden and Clarke, 1982, 1987; Johnson et al., 1991; Aswad, 1995; Kagan et al., 1997a). The enzyme functions by selectively methlyating L-iAsp (and to a lesser extent D-Asp) residues. The aspartic methyl aspartyl ester product cyclize to a succinimide ring, repeated methylation can shunt residues back to L-Asp. However, the substrate character of D-Asp is low, and there is no mechanism for further correcting deamidated Asn residues. Absence of PIMT impairs survival (Kagan et al., 1997b; Kim et al., 1997; Visick et al., 1998; Yamamoto et al., 1998; Shimizu et al., 2000) and there may be a link between expression of this repair enzyme and ageing (DeVry and Clarke, 1999). PIMT knockout mice die at a mean age of 42 days (Kim et al., 1997; Yamamoto et al., 1998), but this can be extended by localized production of PIMT in the brain (Lowenson et al., 2001). However, it should be noted that PIMT is an intracellular enzyme; modifications of extracellular proteins cannot usually repaired (but see Weber and McFadden, 1997).

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Degradation of damaged proteins is an alternative to repair (Niewmierzycka and Clarke, 1999; Lowenson et al., 2001). Proteins with high turnover will be exchanged before post-translational modifications will become relevant (see Fig. 3), whilst the presence of isoaspartyl residues marks proteins for degradation (Szymanska et al., 1998). Protein turnover can be regarded as a tool to overcome protein ageing; with increasing half-life of the protein the risk of damage by molecular ageing increases. Conversely racemization may act as a molecular timer to initiate protein turnover in a manner analogous to deamidation (Robinson and Robinson, 2001a,b). 7. Pathophysiological relevance of AAR during ageing Even in proteins with a relatively slow rate of AAR relevant stereochemical alterations of the molecule may result. Geiger and Clarke (1987) stressed ‘that a typical protein of a Mr 100.000 may contain 100 aspartyl and asparaginyl residues, and an overall content of 1% damaged residues may reflect a population where every polypeptide chain is altered’. Such a population of altered residues may develop in a relatively short time; in the case of erythrocyte membrane proteins merely 39 days are required to generate 1% of residues in the D-configuration (Brunauer and Clarke, 1986). Pathophysiological consequences of AAR may, therefore, include:

7.1. Loss of protein function due to degradation by (specific) proteolysis It has been assumed that certain intracellular proteases and peptidases may play a role in finding and degrading proteins containing altered aspartic or asparaginyl residues (Geiger and Clarke 1987, Lowenson and Clarke 1988; Szymanska et al., 1998). Extracellular proteins may break down as a result of a molecular instability caused by conformational changes induced by AAR; this may be the reason for a decline in the concentrations of bone osteocalcin (Lian and Gundberg 1988) as well as of elastin in lung parenchyma and the artery wall with increasing age (Powell et al. 1992).

7.2. Loss of protein function due to structural changes AAR has important implications for biological activity, and its’ implications have been recognised in the long-term storage of peptides and proteins (Powell, 1994). A relationship between AAR and an alteration of the biological activities of peptide hormones has been discussed by Masters (1982). If the affected aspartyl or asparaginyl residue lies within an enzyme active or substrate-binding site, racemization could move the b-carboxyl group into a position where it could no longer function as required for enzyme activity (Lowenson and Clarke, 1988). In the case of structural proteins AAR may for example trigger conformational alteration and protein aggregation/precipitation (Masters et al., 1978; Masters, 1982; Roher et al., 1993; Tomiyama et al., 1994; Sandmeier et al., 1999; Shimizu et al., 2000), as demonstrated for a synthetic peptide (Orpiszewski and Benson, 1999).

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There is little direct evidence of pathophysiological consequences of in vivo AAR in natural ageing. Increased levels of AAR are have been linked to reduced anion-transporting activity of erythrocyte membrane proteins in old cells (Lowenson and Clarke, 1988) and abnormal AAR values have been observed in tissues with typical diseases of old age (Masters et al., 1977, 1978; Masters, 1982; Maroudas et al., 1992; Ritz and Schu¨ tz, 1993). A number of authors have considered AAR as a relevant pathophysiological factor in the pathogenesis of diseases of old age including atherosclerosis, lung emphysema, presbyopia, cataract, degenerative diseases of cartilage and cerebral age-related dysfunction (Masters et al., 1977, 1978; Masters, 1982; Man et al., 1983; Shapiro et al., 1991; Powell et al., 1992; Orpiszewski et al. 2000). However, it is extremely difficult to gain concrete data on the effects of AAR on the function of proteins. Racemized proteins have to be separated from native proteins, if racemization is to be correlated to changes in activity or function. Structural changes that lead to increased conformational freedom may also accelerate other degradative reactions. Elevated levels of D-Asx in brunescent cataracts suggest that AAR is either promoting glycation or being accelerated as a consequence of it (Masters et al., 1977).

8. AAR as part of the complex biology of protein ageing AAR obviously is an autonomic, inevitable process during the ‘natural’ ageing of proteins. Its occurrence is determined primarily by the primary and higher order structure of a protein. These molecular characteristics are determined at least by the genetic information. Therefore, AAR may be interpreted as part of a ‘programmed ageing’ process. AAR is only one of several post-translational protein modifications that have been interpreted as manifestations of protein ageing; the most frequently discussed molecular modifications in that context are (besides AAR) oxidation, glycation and deamidation (McKerrow, 1979; Rosenberger, 1991; Wright, 1991; Rattan et al., 1992; Bailey, 2001; Sajdok et al., 2001; Robinson and Robinson 2001a,b). These protein modifications may occur in parallel as reported for aA-crystallin in the human lens (Fujii et al., 1999a) and cartilage and skin collagen (Verzijl et al., 2000). Interestingly AAR as well as oxidation and glycation have been discussed as pathogenetically relevant factors in atherosclerosis, presbyopia and cataract (Rattan et al., 1992; Thornally, 1999, Stadtman and Levine, 2000). Thus, it may be concluded that different posttranslational protein modifications apparently attribute additively to the pathogenesis of diseases of old age. Moreover, ‘secondary’ AAR caused by protein degradation for example in the degenerative diseases of old age may be of pathophysiological relevance. Though many questions remain, it is evident that AAR (and other post-translational protein modifications) plays an important role in the biology of ageing. Without a better understanding of the significance of these protein modifications, our knowledge of the molecular basis of ageing is incomplete and insufficient.

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References Aswad, D.W., 1995. Deamidation and Isoaspartate Formation in Peptides and Proteins. CRC Press, Boca Raton, FL. Aswad, D.W., Paranandi, M.V., Schurter, B.T., 2000. Isoaspartate in peptides and proteins: formation, significance, and analysis. J. Pharm. Biomed. Anal. 21, 1129 – 1136. Bada, J.L., 1984. In vivo racemization in mammalian proteins. Methods Enzymol. 106, 98 – 115. Bada, J.L., Protsch, R., 1973. Racemization reaction of aspartic acid and its use in dating fossil bones. Proc. Natl. Acad. Sci. USA 70, 1331 – 1334. Bailey, A.J., 2001. Molecular mechanisms of ageing in connective tissues. Mech. Ageing Dev. 122, 735–755. Brady, J.D., Ju, J., Robins, S.P., 1999. Isoaspartyl bond formation within N-terminal sequences of collagen type I: implications for their use as markers of collagen degradation. Clin. Sci. 96, 209 – 215. Brennan, T.V., Clarke, S., 1993. Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues-effects of the solvent dielectric. Protein Sci. 2, 331 – 338. Brunauer, L.S., Clarke, S., 1986. Age-dependent accumulation of protein residues which can be hydrolyzed to d-aspartic acid in human erythrocytes. J. Biol. Chem. 261, 12538 – 12543. Capasso, S., 2000. Estimation of the deamidation rate of asparagine side chains. Peptide Res. 55, 224–229. Capasso, S., Salvadori, S., 1999. Effect of the three-dimensional structure on the deamidation reaction of ribonuclease A. J. Peptide Res. 54, 377 – 382. Capasso, S., Di Cerbo, P., 2000. Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues. J. Peptide Res. 56, 382 – 387. Capasso, S., Mazzarella, L., Zagari, A., 1991. Deamidation via cyclic imide of asparaginyl peptides: dependence on salts, buffers and organic solvents. Peptide Res. 4, 234 – 238. Capasso, S., Mazzarella, L., Sica, F., Zagari, A., Salvadori, S., 1992. Spontaneous cyclization of the aspartic acid side chain to the succinimide derivative. J. Chem. Soc. Chem. Comm. 12, 919 – 921. Capasso, S., DiDonato, A., Esposito, L., Sica, F., Sorrentino, G., Vitagliano, L., Zagari, A., Mazzarella, L., 1996. Deamidation in proteins: The crystal structure of bovine pancreatic ribonuclease with an isoaspartyl residue at position 67. J. Mol. Biol. 257, 492 – 496. Chazin, W.J., Kossiakoff, A.A., 1995. The role of secondary and tertiary structures in intramolecular deamidation of proteins. In: Aswad, D.W. (Ed.), Deamidation and Isoaspartate Formation in Peptides and Proteins. CRC Press, Boca Raton, FL, pp. 193 – 206. Clarke, S., 1987. Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int. J. Peptide Protein Res. 30, 808 – 821. Cloos, P.A.C., Fledelius, C., 2000. Collagen fragments in urine derived from bone resorption are highly racemized and isomerized: a biological clock of protein aging with clinical potential. Biochem. J. 345, 473–480. Collins, M.J., Waite, E.R., van Duin, A.C.T., 1999. Predicting protein decomposition: the case of aspartic-acid racemization kinetics. Philos. Trans. R. Soc. London 354 (Ser. B), 51 – 64. David, C.L., Orpiszewski, J., Zhu, X.C., Reissner, K.J., Aswad, D.W., 1998. Isoaspartate in chrondroitin sulfate proteoglycans of mammalian brain. J. Biol. Chem. 273, 32063 – 32070. DeVry, C.G., Clarke, S., 1999. Polymorphic forms of the protein L-isoaspartate (D-aspartate) O-methyltransferase involved in the repair of age-damaged proteins. J. Hum. Genet. 44, 275 – 288. Di Salvo, M.L., Delle Fratte, S., Maras, B., Bossa, F., Wright, H.T., Schirch, V., 1999. Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase. Arch. Bioch. Biophys. 372, 271– 279. Fabian, H., Szendrei, G.I., Mantsch, H.H., Greenberg, B.D., Otvos, L., 1994. Synthetic posttranslationally modified human a-beta peptide exhibits a markedly increased tendency to form beta-pleated sheets in vitro. Eur. J. Biochem. 221, 959 – 964. Fisher, G.H., Garcia, N.M., Payan, I.L., Cadilla-Perzrios, R., Sheremata, W.A., Man, E.H., 1986. D-aspartic acid in purified myelin and myelin basic protein. Biochem. Biophys. Res. Comm. 135, 683–687.

S. Ritz-Timme, M.J. Collins / Ageing Research Re6iews 1 (2002) 43–59

55

Fledelius, C., Johnsen, A.H., Cloos, P.A.C., Bonde, M., Qvist, P., 1997. Characterization of urinary degradation products derived from type I collagen: Identification of a beta-isomerized Asp – Gly sequence within the C-terminal telopeptide (alpha 1) region. J. Biol. Chem. 272, 9755 – 9763. Fu, S.-J., Fan, C.-C., Song, H.-W., Wei, F.-Q., 1995. Age estimation using a modified HPLC determination of ratio of aspartic acid in dentin. Forensic Sci. Int. 73, 35 – 40. Fujii, N., Tamanoi, I., Muraoka, S., Joshima, H., Kashima, M., Harada, K., 1987. D-aspartic acid in aged mouse skin and lens. J. Radiation Res. 28, 117 – 125. Fujii, N., Momose, Y., Harada, K., 1996. Kinetic study of racemization of aspartyl residues in model peptides of aA-crystallin. Int. J. Peptide Protein Res. 48, 118 – 122. Fujii, N., Momose, Y., Ishibashi, Y., Uemura, T., Takita, M., Takehana, M., 1997. Specific racemization and isomerization of the aspartyl residue of aA-crystallin due to UV-B irradiation. Exp. Eye Res. 65, 99 – 104. Fujii, N., Momose, Y., Ishii, N., Takita, M., Akaboshi, M., Kodama, M., 1999a. The mechanism of simultaneous stereoinversion, racemization, and isomerization at specific aspartyl residues of aged lens protein. Mech. Ageing Develop. 107, 347 –358. Fujii, N., Takemoto, L.J., Momose, Y., Matsumoto, S., Hiroko, K., Akaboshi, M., 1999b. Formation of four isomers at the Asp-151 residue of aged human aA-crystallin by natural aging. Biochem. Biophys. Res. Comm. 265, 746 – 751. Fujii, N., Shimo-Oka, T., Ogiso, M., Momose, Y., Kodama, T., Kodama, M., Akaboshi, M., 2000. Localization of biologically uncommon d-beta-aspartate- containing alpha A-crystallin in human eye lens. Mol. Vis. 6, 1 –5. Galletti, P., Ingrosso, D., Manna, C., Clemente, G., Zappia, V., 1995. Protein damage and methylationmediated repair in the erythrocyte. Biochem. J. 306, 313 – 325. Geiger, T., Clarke, S., 1987. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. J. Biol. Chem. 262, 785 – 794. George, J.C., Bada, J., Zeh, J., Scott, L., Brown, S.E., O’Hara, T., Suydam, R., 1999. Age and growth estimates of bowhead whales (Balaena mysticetus) via aspartic acid racemization. Can. J. Zool. Rev. Can. Zool. 77, 571 – 580. Gineyts, E., Cloos, P.A.C., Borel, O., Grimaud, L., Delmas, P.D., Garnero, P., 2000. Racemization and isomerization of type I collagen C-telopeptides in human bone and soft tissues: assessment of tissue turnover. Biochem. J. 345, 481 – 485. Groenen, P.J.T.A., van den Ijssel, P.R.L.A., Voorter, C.E.M., Bloemendal, H., de Jong, W.W., 1990. Site-specific racemization in aging aA-crystalin. FEBS Lett. 269, 109 – 112. Helfman, P.M., Bada, J.L., 1975. Aspartic acid racemization in tooth enamel from living humans. Proc Natl. Acad. Sci. USA 72, 2891 –2894. Helfman, P.M., Bada, J.L., 1976. Aspartic acid racemisation in dentine as a measure of ageing. Nature 262, 279 –281. Helfman, P.M., Bada, J.L., Shou, M.-Y., 1977. Considerations on the role of aspartic acid racemization in the aging process. Gerontology 23, 419 – 425. Inaba, M., Gupta, K.C., Kuwabara, M., Takahashi, T., Benz, E.J., Maede, Y., 1992. Deamidation of human erythrocyte protein 4.1: possible role in ageing. Blood 79, 3355 – 3361. Ingrosso, D., Perna, A.F., 1998. D-amino acids in aging erythrocytes. EXS 85, 119 – 141. Johnson, B.A., Aswad, D.W., 1985. Identification and topography of substrates for protein carboxyl methylrransferase in synaptic membranes and myelin-enriched fractions of bovine and rat brain. J. Neurochem. 45, 1119 –1127. Johnson, B.A., Ngo, S.Q., Aswad, D.W., 1991. Widespread phylogenetic distribution of a protein methyltransferase that modifies L-isoaspartyl residues. Biochem. Int. 24, 841 – 847. Kagan, R.M., McFadden, H.J., McFadden, P.N., Oconnor, C., Clarke, S., 1997a. Molecular phylogenetics of a protein repair methyltransferase. Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 117, 379–385. Kagan, R.M., Niewmierzycka, A., Clarke, S., 1997b. Targeted gene disruption of the Caenorhabditis elegans L- isoaspartyl protein repair methyltransferase impairs survival of dauer stage nematodes. Arch. Biochem. Biophys. 348, 320 –328.

56

S. Ritz-Timme, M.J. Collins / Ageing Research Re6iews 1 (2002) 43–59

Kenessey, A., Yen, S.H., Liu, W.K., Yang, X.R., Dunlop, D.S., 1995. Detection of d-aspartate in tau-proteins associated with Alzheimer paired helical filaments. Brain Res. 675, 183 – 189. Kim, E., Lowenson, J.D., McacLaren, D.C., Clarke, S., Young, S.G., 1997. Deficiency of protein-repair enzyme results in the accumulation of altered proteins, retardation of growth and fatal seizures in mice. Proc. Natl. Acad. Sci. USA 94, 6132 – 6137. Kinzel, V., Konig, N., Pipkorn, R., Bossemeyer, D., Lehmann, W.D., 2000. The amino terminus of PKA catalytic subunit-A site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes. Protein Sci. 9, 2269 – 2277. Kossiakoff, A.A., 1988. Tertiary structure is a principal determinant to protein deamidation. Science 240, 191 –194. Kuhn, W., 1936. Katalytische Erzeugung optisch aktiver Stoffe und chemische Notwendigkeit eines einseitigen Ablaufs biochemischer Vorga¨ nge. Angew. Chem. 49, 215 – 219. Kuhn, W., 1955. Mo¨ gliche Beziehungen der optischen Aktivita¨ t zum Problem des Alterns. Experientia 11, 429 –436. Kuhn, W., 1958. Possible relation between optical activity and aging. Adv. Enzymol. 20, 1 – 29. Kvenvolden, K.A., Lawless, J., Pering, K., Peterson, E., Flores, J., Ponnamperuma, C., Kaplan, I.R., Moore, C., 1970. Evidence for extraterrestrial amino acids and hydrocarbons in the Murchinson meteorite. Nature 228, 923 –926. Lehmann, W.D., Schlosser, A., Erben, G., Pipkorn, R., Bossemeyer, D., Kinzel, V., 2000. Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry. Protein Sci. 9, 2260 – 2268. Lehrman, S.R., Hamlin, D.M., Lund, M.E., Walker, G.A., 1992. Identification and characterization of an anti-isoaspartic acid monoclonal antibody. J. Protein Chem. 11, 657 – 663. Lian, J.B., Gundberg, C.M., 1988. Osteocalcin. Biochemical considerations and clinical applications. Clin. Orthop. Relat. Res. 226, 267 –291. Lowenson, J., Clarke, S., 1988. Does the chemical instability of aspartyl and asparaginyl residues in proteins contribute to erythrocyte aging? Blood Cells 14, 103 – 117. Lowenson, J.D., Clarke, S., 1992. Recognition of d-aspartyl residues in polypeptides by the erythrocyte L-Isoaspartyl/D-Aspartyl Protein Methyltransferase-implications for the repair hypothesis. J. Biol. Chem. 267, 5985 –5995. Lowenson, J.D., Kim, E., Young, S.G., Clarke, S., 2001. Limited accumulation of damaged proteins in L-Isoaspartyl (D-Aspartyl) O-Methyltransferase-deficient mice. J. Biol. Chem. 276, 20695 – 20702. Lura, R., Schirch, V., 1988. Role of peptide conformation in the rate and mechanism of deamidation of asparaginyl residues. Biochemistry US 27, 7671 – 7677. Man, E.H., Sandhouse, M.E., Burg, J., Fisher, G.H., 1983. Accumulation of D-aspartic acid with age in the human brain. Science 220, 1407 – 1408. Maroudas, A., Palla, G., Gilav, E., 1992. Racemization of aspartic acid in human articular cartilage. Connect. Tissue Res. 28, 161 – 169. Maroudas, A., Bayliss, M.T., Uchitel-Kaushansky, N., Schneidermann, R., Gilav, E., 1998. Aggregan turnover in human articular cartilage: Use of aspartic acid racemization as a marker of molecular age. Arch. Biochem. Biophys. 350, 61 – 71. Masters, P.M., 1982. Amino acid racemization in structural proteins. Proc Conf. Non-lethal Biological Markers of Physiological Aging, 19 –20 June 1981 (NIH Publication 82-2221), 120 – 137. Masters, P.M., Bada, J.L., Zigler, J.S., 1977. Aspartic acid racemisation in the human lens during ageing and in cataract formation. Nature 268, 71 – 73. Masters, P.M., Bada, J.L., Zigler, J.S., 1978. Aspartic acid racemization in heavy molecular weight crystallins and water-insoluble protein from normal human lenses and cataracts. Proc. Natl. Acad. Sci. USA 75, 1204 –1208. Matsu’ura, S., Ueta, N., 1980. Fraction dependent variation of aspartic acid racemization age of fossil bone. Nature 286, 883 – 884. McFadden, P.N., Clarke, S., 1982. Methylation of D-aspartyl residues in erythrocytes: Possible step in the repair of aged membrane proteins. Proc. Natl. Acad. Sci. USA 79, 2460 – 2464. McFadden, P.N., Clarke, S., 1987. Conversion of isoaspartyl peptides to normal peptides: Implications for the cellular repair of damaged proteins. Proc. Natl. Acad. Sci. USA 84, 2595 –2599.

S. Ritz-Timme, M.J. Collins / Ageing Research Re6iews 1 (2002) 43–59

57

McKerrow, J.H., 1979. Non-enzymatic, post-translational, amino acid modifications in ageing. A brief review. Mech. Ageing Dev. 10, 371 – 377. Mizuno, A., Fujii, N., Akaboshi, M., Momose, Y., Shimo-oka, T., 2000. Changes of secondary structure detected by laser Raman spectroscopy in model peptides of human alpha A-crystallin due to substitution of D-aspartyl residues for L-aspartyl residues. Jpn. J. Ophthalmol. 44, 354 – 359. Mo¨ rnstad, H., Pfeiffer, H., Teivens, A., 1994. Estimation of dental age using HPLC-technique to determine the degree of aspartic acid racemization. J. Forensic Sci. 39, 1425 – 1431. Najbauer, J., Orpiszewski, J., Aswad, D.W., 1996. Molecular aging of tubulin: Accumulation of isoaspartyl sites in vitro and in vivo. Biochemistry 35, 5183 – 5190. Neuberger, A., 1948. Stereochemistry of Amino Acids. In: Anson, M.L., Edsall, J.T., (Eds.), Advances In Protein Chemistry, Vol. IV. pp. 297 – 83. Niewmierzycka, A., Clarke, S., 1999. Do damaged proteins accumulate in Caenorhabditis elegans Lisoaspartate methyltransferase (pcm-1) deletion mutants? Arch. Biochem. Biophys. 364, 209 – 218. Noguchi, S., Miyawaki, K., Satow, Y., 1998. Succinimide and isoaspartate residues in the crystal structures of hen egg-white lysozyme complexed with tri-N-acetylchitotriose. J. Mol. Biol. 278, 231–238. Ogino, T., Ogino, H., Nagy, B., 1985. Application of aspartic acid racemization to forensic odontology: post mortem designation of age of death. Forensic Sci. Int. 29, 259 – 267. Ohtani, S., 1995. Estimation of age from dentin by using the racemization reaction of aspartic acid. Am. J. Forensic Med. Pathol. 16, 158 – 161. Ohtani, S., 1998. Rate of aspartic acid racemization in bone. Am. J. Forensic Med. Pathol. 19, 284 – 287. Ohtani, S., Sugimoto, H., Sugeno, H., Yamamoto, S., Yamamoto, K., 1995. Racemization of aspartic acid in human cementum with age. Arch. Oral Biol. 40, 91 – 95. Ohtani, S., Matsushima, Y., Kobayashi, Y., Kishi, K., 1998a. Evaluation of aspartic acid racemization ratios in the human femur for age estimation. J. Forensic Sci. 43, 949 – 953. Ohtani, S., Yamamoto, T., Matsushima, Y., Kobayashi, Y., 1998b. Changes in the amount of d-aspartic acid in the human femur with age. Growth Develop. Aging 62 (4), 141 –144. Orpiszewski, J., Benson, M.D., 1999. Induction of beta-sheet structure in amyloidogenic peptides by neutralization of aspartate: A model for amyloid nucleation. J. Mol. Biol. 289, 413 – 428. Orpiszewski, J., Schormann, N., Kluve-Beckerman, B., Liepnieks, J.J., Benson, M.D., 2000. Protein aging hypothesis of Alzheimer disease. FASEB J. 14, 1255 – 1263. Paranandi, M.V., Aswad, D.W., 1995. Spontaneous alterations in the covalent structure of synapsin I during in vitro aging. Biochem. Biophys. Res. Commun. 212, 442 – 448. Payan, I.L., Chou, S.J., Fisher, G.H., Man, E.H., Emory, C., Frey, W.H., 1992. Altered aspartate in alzheimer neurofibrillary tangles. Neurochemical Res. 17, 187 – 191. Perna, A.F., Daniello, A., Lowenson, J.D., Clarke, S., DeSanto, N.G., Ingrosso, D., 1997. D-Aspartate content of erythrocyte membrane proteins is decreased in uremia: Implications for the repair of damaged proteins. J. Am. Soc. Nephrol. 8, 95 – 104. Pfeiffer, H., Mornstad, H., Teivens, A., 1995a. Estimation of chronological age using the aspartic-acid racemization method.1. On human rib cartilage. Int. J. Legal Med. 108, 19 – 23. Pfeiffer, H., Mornstad, H., Teivens, A., 1995b. Estimation of chronological age using the aspartic-acid racemization method.2. On human cortical bone. Int. J. Legal Med. 108, 24 – 26. Potter, S.M., Henzel, W.J., Aswad, D.W., 1993. In vitro aging of calmodulin generates isoaspartate at multiple Asn –Gly and Asp–Gly sites in calcium-binding domains II, III and IV. Protein Sci. 2, 1648– 1663. Powell, M.F., 1994. Peptide stability in aqueous parenteral formulations-prediction of chemical-stability based on primary sequence. ACS Symp. Ser. 567, 100 – 117. Powell, J.T., Vine, N., Crossman, M., 1992. On the accumulation of D-aspartate in elastin and other proteins of the aging aorta. Atherosclerosis 97, 201 – 208. Radkiewicz, J.L., Zipse, H., Clarke, S., Houk, K.N., 1996. Accelerated racemization of aspartic acid and asparagine residues via succinimide intermediates: An ab initio theoretical exploration of mechanism. J. Am. Chem. Soc. 118, 9148 – 9155. Radkiewicz, J.L., Zipse, H., Clarke, S., Houk, K.N., 2001. Neighboring side chain effects on asparaginyl and aspartyl degradation: An ab initio study of the relationship between peptide conformation and backbone NH acidity. J. Am. Chem. Soc. 123, 3499 – 3506.

58

S. Ritz-Timme, M.J. Collins / Ageing Research Re6iews 1 (2002) 43–59

Rattan, S.I.S., Derventzi, A., Clark, B.F.C., 1992. Protein synthesis, posttranslational modifications, and aging. Ann. New York Acad. Sci. 663, 48 – 62. Ritz, S., Schu¨ tz, H.W., 1993. Aspartic acid racemization in intervertebral discs as an aid to post mortem estimation of age at death. J. Forensic Sci. 38, 633 – 640. Ritz, S., Turzynski, A., Schu¨ tz, H.W., 1994. Estimation of age at death based on aspartic acid racemization in non-collagenous bone proteins. Forensic Sci. Int. 69, 149 – 159. Ritz, S., Turzynski, A., Schu¨ tz, H.W., Hollmann, A., Rochholz, G., 1996. Identification of osteocalcin as a permanent aging constituent of the bone matrix: Basis for an accurate age at death determination. Forensic Sci. Int. 770, 13 –26. Ritz, S., Schu¨ tz, H.W., Peper, C., 1993. Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin. Int. J. Legal Med. 105, 289 – 293. Ritz-Timme, S., 2000. Lebensaltersbestimmung aufgrund des Razemisierungsgrades von Asparaginsa¨ ure. Grundlagen, Methodik, Mo¨ glichkeiten, Grenzen, Anwendungsbereiche. In: Berg, S., Brinkmann, B. (Eds.), Arbeitsmethoden der Medizinischen und Naturwissenschaftlichen Kriminalistik, Band 23. Schmidt-Ro¨ mhild, Lubeck. Robinson, N.E., Robinson, A.B., 2001a. Molecular clocks. Proc. Natl. Acad. Sci. USA 98, 944 – 949. Robinson, N.E., Robinson, A.B., 2001b. Prediction of protein deamidation rates from primary and three-dimensional structure. Proc. Natl. Acad. Sci. USA 98, 4367 – 4372. Robinson, A.B., McKerrow, J.H., Cary, P., 1970. Controlled deamidation of peptides and proteins: An experimental hazard and a possible biological timer. Proc. Natl. Acad. Sci. USA 66, 753 – 757. Roher, A.E., Lowenson, J.D., Clarke, S., Wollcow, C., Wang, R., Cotter, R.J., Reardon, I.M., Zurcher-Neely, H.A., Heinrikson, R.L., Ball, M.J., Greenberg, B.D., 1993. Structural alteration in the peptide backbone of b-amyloid core protein may account for its deposition and stability in Alzheimerr’s disease. J. Biol. Chem. 268, 3072 – 3083. Rosenberger, R.F., 1991. Senescence and the accumulation of abnormal proteins. Mutat. Res. 256, 255–262. Sandmeier, E., Hunziker, P., Kunz, B., Sack, R., Christen, P., 1999. Spontaneous deamidation and isomerization of Asn108 in prion peptide 106 – 126 and in full-length prion protein. Biochem. Biophys. Res. Comm. 261, 578 –583. Sajdok, J., Kozak, A., Zidkova, J., Kotrba, P., Pilin, A., Kas, J., 2001. Protein modification during aging of organism. Chemicke Listy 95, 98 – 101. Shapira, R., Jen Chou, C.H., 1987. Differential racemization of aspartate and serine in human myelin and basic protein. Biochem. Biophys. Res. Comm. 146, 1342 – 1349. Shapira, R., Wilkinson, K.D., Shapira, G., 1988. Racemization of individual aspartate residues in human myelin basic protein. J. Neurochemistry 50, 649 – 654. Shapiro, S.D., Endicott, S.K., Province, M.A., Pierce, J.A., Campbell, E.J., 1991. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of d-aspartate and nuclear weaponsrelated radiocarbon. J. Clin. Invest. 87, 1828 – 1834. Shimizu, T., Watanabe, A., Ogawara, M., Mori, H., Shirasawa, T., 2000. Isoaspartate formation and neurodegeneration in Alzheimer’s disease. Arch. Biochem. Biophys. 381, 225 – 234. Stadtman, E.R., Levine, R.L., 2000. Protein oxidation. Ann. New York Acad. Sci. 899, 191 – 208. Szymanska, G., Leszyk, J.D., O’Connor, C.M., 1998. Carboxyl methylation of deamidated calmodulin increases its stability in Xenopus oocyte cytoplasm —Implications for protein repair. J. Biol. Chem. 273, 28516 –28523. Thornally, P.J., 1999. The clinical significance of glycation. Clin. Lab. 45, 263 – 273. Tomiyama, T., Asano, S., Furiya, Y., Shirasawa, T., Endo, N., Mori, N., 1994. Racemization of Asp23 residue affects the aggregation properties of Alzheimer amyloid b-protein analogues. J. Biol. Chem. 269, 10205 –10208. Vandenoetelaar, P.J.M., Hoenders, H.J., 1989. Racemization of aspartyl residues in proteins from normal and cataractous human lenses-an aging process without involvement in cataract formation. Exp. Eye Res. 48, 209 –214. Van Duin, A.C.T., Collins, M.J., 1998. The effects of conformational constraints on aspartic acid racemization. Org. Geochem. 29, 1227 – 1232.

S. Ritz-Timme, M.J. Collins / Ageing Research Re6iews 1 (2002) 43–59

59

Verzijl, N., De Groot, J., Thorpe, S.R., Bank, R.A., Shaw, J.N., Lyons, T.J., Bijlsma, J.W.J., Lafeber, F., Baynes, J.W., Te Koppele, J.M., 2000. Effect of collagen turnover on the accumulation of advanced glycation end products. J. Biol. Chem. 275, 39027 – 39031. Visick, J.E., Cai, H., Clarke, S., 1998. The L-isoaspartyl protein repair methyltransferase enhances survival of aging Escherichia coli subjected to secondary environmental stresses. J. Bacteriol. 180, 2623–2629. Waite, E.R., Collins, M.J., 2000. The interpretation of aspartic acid racemization data of dentine proteins. In: Goodfriend, G.A., Collins, M.J., Fogel, M.L., Macko, S.A., Wehmiller, J.F. (Eds.), Perspectives in Amino Acid and Protein Geochemistry. Oxford University Press, New York, pp. 182– 194. Watanabe, A., Takio, K., Ihara, Y., 1999. Deamidation and isoaspartate formation in smeared tau in paired helical filaments — Unusual properties of the microtubule-binding domain of tau. J. Biol. Chem. 274, 7368 –7378. Weber, D.J., McFadden, P.N., 1997. Injury-induced enzymatic methylation of aging collagen in the extracellular matrix of blood vessels. J. Protein Chem. 16, 269 – 281. Wright, H.T., 1991. Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Crit. Rev. Biochem. Mol. Biol. 26, 1 –52. Wright, H.T., 1995. Amino acid abundance and sequence data: clues to the biological significance of nonenzymatic asparagine and glutamine deamidation in proteins. In: Aswad, D.W. (Ed.), Deamidation and Isoaspartate Formation in Peptides and Proteins. CRC Press, Boca Raton, FL, pp. 229–252. Xie, M.L., Schowen, R.L., 1999. Secondary structure and protein deamidation. J. Pharm. Sci. 88, 8 – 13. Yamamoto, A., Takagi, H., Kitamura, D., Tatsuoka, H., Nakano, H., Kawano, H., Kuroyanagi, H., Yahagi, Y., Kobayashi, S., Koizumi, K., Sakai, T., Saito, K., Chiba, T., Kawamura, K., Suzuki, K., Watanabe, T., Mori, H., Shirasawa, T., 1998. Deficiency in protein L-isoaspartyl methyltransferase results in a fatal progressive epilepsy. J. Neurosci. 18, 2063 – 2074. Zioupos, P., Currey, J.D., Hamer, A.J., 1999. The role of collagen in the declining mechanical properties of aging human cortical bone. J. Biomed. Mater. Res. 45, 108 – 116.