Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications

Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications

Matrix Biology 21 Ž2002. 39᎐52 Mini review Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical app...

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Matrix Biology 21 Ž2002. 39᎐52

Mini review

Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications Paul A.C. Cloosa,U , Stephan Christgau a a

Nordic Bioscience A r S, Herle¨ Ho¨ edgade 207, DK-2730 Herle¨ , Denmark Received 27 June 2001; accepted 29 October 2001

Abstract Given the complexity of the biosynthetic machinery and the delicate chemical composition of proteins, it is remarkable that cells manage to produce and maintain normally functioning proteins under most conditions. However, it is now well known that proteins are susceptible to various non-enzymatic covalent modifications ŽNECM. under physiological conditions. Such modifications can be of no or little importance to the protein or they can be absolutely detrimental. Often NECM are difficult to study due to the complex and technically demanding methods required to identify many of these modifications. Thus, the role of NECM has not yet been adequately resolved but recent research has allowed a better understanding of such modifications. The present review outlines the various forms of NECM that involve covalent modifications of proteins, and discusses their relevance, biological impact and potential applications in the study of protein turnover and diagnosis of disease. 䊚 2002 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Covalent damage; Conformational damage; Racemization; Isomerization; Oxidation; Cross-linking; Glycation

1. Introduction Proteins are subject to a variety of spontaneous non-enzymatic modifications that may affect their structure, function and stability. The occurrence of these alterations and their localization within a pro-

Abbre¨ iations: AGE, advanced glycation end product; AGEr, advanced glycation end product receptor; CEL, N ␧ Žcarboxyethyl.lysine; CML, N ␧ -Žcarboxymethyl.lysine; Dha, dehydroalanine; HAL, histidinoalanine; IAMT, L-isoaspartylrO-methyltransferase; LAL, lysinoalanine; MSR, methionine sulfoxide reductase; NECM, non-enzymatic covalent modification; PK, protein kinase; PPI, peptidyl-prolyl cis-trans isomerase; ROS, reactive oxygen species; SerŽP., phosphoserine; ThrŽP., phosphothreonine; TyrŽP., phosphotyrosine U Corresponding author. Tel.: q45-44-94-89-00; fax: q45-44-9489-40. E-mail address: [email protected] ŽP.A. Cloos..

tein seem to be determined primarily by protein sequence and structure, the protein microenvironment and half-life of the protein. Non-enzymatic modifications are time-dependent and the effects of individual changes may be cumulative. The term ‘protein fatigue’ has been used to describe this phenomenon ŽGalletti et al., 1995. as an analogy to the so-called ‘metal fatigue’. Non-enzymatic modifications may be subdivided into two general forms ŽVisick and Clarke, 1995.: 1.

Conformational changes Žor conformational damage., i.e. a modification of the three-dimensional structure of the protein without compromising its chemical composition ŽFig. 1a., and 2. Covalent modifications Žor covalent damage. where the primary structure of the protein or peptide in question is altered on the amino

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Fig. 1. Non-enzymatic modifications of proteins. Ža. Conformational modifications. Comprises modifications affecting the three-dimensional structure of proteins and includes unfolding, mis-folding or aggregation of proteins. Molecular chaperones may mitigate conformational changes by restoring proteins to their correct conformation, and constitute a repair pathway for such modifications. Žb. Covalent modifications. Among the most prevalent forms of covalent modifications are: Aspartic acid ŽAsp. isomerization and racemization; Proline ŽPro. isomerization; Methionine ŽMet. oxidation; dephosphorylation; ␤-elimination and formation of advanced glycation end products ŽAGEs.; and various cross-links. Various enzyme systems exist that moderate or relieve the effects of these reactions by repairing, or eliminating the resultant age-modified proteins. These systems include: iso-aspartyl methyl transferase ŽIAMT.; peptidyl-prolyl cis᎐trans isomerases ŽPPIs.; methionine sulfoxide reductase ŽMSR. protein kinases ŽPKs.; and AGE-receptors ŽAGEr.. Damaged proteins can be eliminated by catabolism. Modified and adapted from ŽVisick and Clarke, 1995..

acidrpeptide bond level, by cleavage and formation of covalent bonds ŽFig. 1b.. A clear distinction from one type of modifications to another is not always obvious as covalent changes may cause conformational modifications and vice versa. The present review focuses on the mechanisms resulting in non-enzymatic covalent protein modifications ŽNECM., and the impact of such reactions on protein function. An overview of the various forms of NECM is given in Fig. 1.

Amino acids and peptide bonds are prone to a variety of chemical modifications. Changes in the protein microenvironment such as changes in temperature, pH, or redox potential, etc. may induce or accelerate changes in the primary structure of a protein. Most types of covalent modifications are often restricted to specific susceptible residues or areas of proteins. Table 1 summarizes examples of nonenzymatic covalent modifications. Due to the large number and heterogeneous nature of potential covalent modifications that can occur in proteins, only a broad description of the different

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Table 1 Examples of non-enzymatic covalent modifications of proteins Reaction

Modification

Deamidation

Asn ª Asp Gln ª Glu Peptide bond hydrolysis Asx ª isoAsp Glx ª isoGlu Asx ª D-AspŽD-isoAsp. Glx ª D-GluŽD-isoGlu. Other residues such as Ala, Ser, Tyr, etc. may racemize within the lifespan of human proteins ProT r an s ª ProC i s Met ª methioninesulfoxide His ª Asx Lys ª glycoxidation and lipoxidation products Tyrª ortho-tyrosine, chlorotyrosine, nitrotyrosine Pro, Argª Glu, glutamylsemialdehyde Formation of cross-links Žbityrosine, pentosidine, S᎐S cross-links. Peptide bond scission SerŽP. ª Ser, dehydroalanine ThrŽP. ª Thr, methyl-dehydroalanine Ser, Cystine ª dehydroalanine Thr ª methyl-dehydroalanine Formation of D, L-Ala, Gly Formation of cross-linksŽlysinoalanine, histidinoalanine, etc.. Peptide bond scission Formation of advanced glycation end Products ŽAGEs.: cross-links Žpentosidine, crossline, imidazolium, etc.. Lys ª Amadori products, N-carboalkyl derivatives of Lys Formation of: Pentosidine, crossline, imidazolium Žglycation, oxidation. bityrosine Žoxidation. Lysinoalanine, histidinoalanine, lanthionine and their methylated homologues Ž␤-elimination, dehydration.

Isomerization Racemization

Cis᎐trans isomerization Oxidation

␤-Elimination, dehydration and hydrolysis

Glycation

Cross-linking

modifications will be given with an emphasis on the physiological roles and consequences established from in vitro and in vivo experiments. 1.1. Oxidati¨ e damage Oxidative damage of proteins is caused by the action of free radicals and various other oxidizing compounds on proteins ŽScheme 1.. These compounds include Nitric oxide ŽNO., peroxynitrate, H 2 O 2 or hydroxyl, hydroperoxyl superoxide and lipid peroxyl radicals. A common term for these compounds is reactive oxygen species ŽROS.. ROS are formed by a number of different pathways. Some of these pathways are mediated by specific enzyme systems such as Nitric Oxide Synthetase, Cyclo-Oxygenase and Mono-Amine Oxidase B. Protein modifications induced by these enzyme systems are implicated in several pathological processes as well as in inflammation, tissue healing and other physiological processes. Other mechanisms of inducing oxidative damage are related to environmental effects such as ionizing radi-

ation, reduction of metal ions such as FeŽII. or CuŽI., or chemical compounds. A clear distinction between enzyme catalyzed and non-enzyme catalyzed protein oxidation is difficult to make. Highly reactive ROS radicals are produced by ionizing radiation or as side-products of metabolism ŽFarr and Komoga, 1991. and may in principle attack any amino acid ŽStadtman, 1992.. However, in most proteins certain ‘hot spots’ in the protein seem to be particular susceptible to oxidative damage. Com-

Scheme 1. Amino acid oxidation products. Methionine sulfoxide and ortho-tyrosine have been shown to increase with age in human skin collagen. Chloro-tyrosine, nitro-tyrosine and bityrosine have been identified in lipoproteins from artherosclerotic plaques. Broken lines represent attachment to the peptide backbone.

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pounds formed by oxidation of carbohydrates may react with proteins forming advanced glycation end products ŽAGEs.. Furthermore, ROS-induced changes may induce the formation of covalent cross-links within and between protein molecules. Glycation and cross-linking reactions are reviewed separately in the following sections. Oxidation of amino acid side-chains can give rise to a number of adducts. Many reactions results in the formation of protein carbonyl derivatives such as direct oxidation of arginine or proline to glutamic semialdehyde or lysine to ␣-amino-adipic-semialdehyde ŽStadtman and Berlet, 1998.. Other prevalent modifications comprise: oxidation of Met producing Met sulfoxide ŽFarr and Komoga, 1991.; nitrosylation of tyrosine and tryptophan residues; formation of oxo, hydroxy, nitro and chloro derivatives of amino acids ŽDavies, 1987.; transformation of His to Asx; and of Pro to Glu-adducts ŽStadtman, 1992; Davies, 1987. Žsee Scheme 1.. Furthermore, peptide linkages of the protein backbone may react with ROS resulting in cleavage of the peptide linkage and formation of N-␣-ketoacyl peptides or N-pyrovyl peptides ŽDavies, 1987; Stadtman and Berlet, 1998.. 1.1.1. Repair systems Under normal conditions the oxidative potential of the protein micro-environment is under tight control of a number of balancing systems including antioxidants and free radical scavengers, peroxidases, catalase and superoxide dismutase. These systems can be viewed as protection mechanisms, more than repair systems. Actual repair mechanisms specific for oxidative damage are rare. They include the enzyme methionine sulfoxide reductase ŽMSR., which recognizes methionine sulfoxide in peptides and can convert it back to methionine ŽStadtman et al., 1988. and heme oxygenase 1, which can repair some of the oxidative damages caused by elevated NO levels. 1.1.2. Biological importance Oxidation may have a deleterious effect on protein function and stability. Many enzymes have been shown to loose their biological activity as a consequence of oxidation ŽRattan, 1996.. It has been shown that enzymes isolated from older animals show decreased temperature stability compared to enzymes from young animals where less oxidative damage has occurred. Protein oxidation may be implicated in the pathogenesis of several diseases ŽSmith et al., 1991.. Oxidative damage of proteins often leads to the formation of protein carbonyl compounds as described above. Sensitive assays are available for quantification of such adducts which have been used as an indicator of the oxidative stress exposure of a given tissue or

organism. Increased levels of protein carbonyls have been associated with rheumatoid arthritis, Alzheimer’s disease, Parkinson’s disease, muscular dystrophy and several other serious chronic and debilitating conditions. A direct link has been proposed between oxidative stress and free-radical mediated protein damage and the formation of senile plaques and neurofibrillar tangles in Alzheimer’s disease. The exact role of the oxidized protein adducts in these diseases still needs further elucidation, but the accumulation of such compounds in tissues and cells may result in excessive and damaging aggregation, fragmentation and denaturation of proteins. Oxidation has been reported to alter the susceptibility of proteins towards proteolysis ŽFarr and Komoga, 1991; Davies, 1987.. In some cases proteolytic degradation is increased by the presence of oxidized protein adducts, but in may cases the oxidation of proteins renders them more resistance to proteolysis resulting in the accumulation of oxidized proteins and enzyme forms with increasing age of the tissues and cells. 1.2. Non-enzymatic glycation Incubation of proteins with sugar leads to a complex series of condensation, rearrangement and fragmentation reactions forming a variety of degradation products, collectively termed advanced glycation end products ŽAGEs. ŽScheme 2.. The term glycation is used to discriminate between these spontaneous reactions and the enzyme-catalyzed glycosylation that occur as a highly regulated post-translational processing of many proteins. The reaction schemes for formation of AGEs can be very complex, and are collectively termed Maillard reactions. AGEs are formed when an aldose group of a saccharide condenses with a reactive amino group, typically in lysine side chains of target proteins ŽBaynes, 1991.. These compounds subsequently undergo further reactions, which are referred to as Amadori rearrangements. Other reaction schemes also occur, such as oxidation of Schiff baselinked carbohydrate moieties. Many AGEs are unstable and gradually undergo various dehydration and re-arrangements to produce reactive carbonyl containing compounds as well as covalent intra- and inter-molecular cross-links in the affected proteins. Thus, as a group, AGE reactions result in the formation of very complex and heterogeneous reaction products. Physiological concentrations of monosaccharides such as ribose, fructose and glucose have been shown to induce AGE formation both in vitro and in vivo. These reactions are greatly increased by oxidative stress conditions, i.e. conditions which inducer increase the presence of ROS Žsee previous section.. To stress the link between glycation and oxidation

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proteolytic degradation of the modified proteins. In this context it is noteworthy that AGE modification of proteins can serve to increase their resistance to proteolysis. Thus, proteins containing various AGE adducts can accumulate with age.

Scheme 2. Pathways of non-enzymatic glycation. Physiological concentrations of monosaccharides can react with protein amino groups. Some of the most prevalent products of these covalent reactions is the formation of Ždihydro. imidazolones by the reaction of 3-deoxyglucosome or methylglyoxal with Arg, generation of N-carboalkyl derivatives of Lys as N ␧ -Žcarboxymethyl.lysine ŽCML., N ␧-Žcarboxyethyl.lysine ŽCEL. and pentosidine, crossline and imidazolium cross-links. They are formed via the so-called Maillard reaction, where the ␧-amino group of Lys or the amino acid terminus of a protein reacts with a reducing sugar. The formation proceeds via oxidative cleavage of Schiff bases or Amadori products, or from dicarbonyl compounds under non-oxidizing conditions. Broken lines represent attachment of the residue to the peptide backbone and large ellipses symbolize protein.

resulting in AGE formation, the processes has been referred to as glycoxidation. 1.2.1. Repair systems No enzyme system has been described which can revert the AGE modifications of proteins. However, several ‘AGE receptors’ have been identified, which potentially could be involved in a repair pathway but the role of these receptors has not been fully elucidated ŽSano et al., 1999.. AGE-binding receptors are: scavenger receptor types I and II, the receptor for advanced glycation end-products ŽAGEr.; oligosaccharyl transferase-48 ŽOST-48, AGE-R1.; 80K-H phosphoprotein ŽAGE-R2.; and galectin-3 ŽAGE-R3. ŽThornalley, 1998.. AGE receptors are found in monocytes, macrophages, endothelial cells, pericytes, podocytes, astrocytes and microglia. AGE-modified proteins also bind to lysozyme and lactoferrin. Interaction of AGE modified proteins with these receptors may serve diverse purposes including: endocytosis and disposal of the modified protein; initiation of tissue repair; and regulation of tissue turnover. The main pathways for dealing with AGE modification involve

1.2.2. Biological importance Proteins containing AGEs prepared in vitro have been demonstrated to have several potential pathogenic properties ŽVlassara et al., 1994; Daniels and Hauser, 1992; Yan et al., 1994.. AGEs have been reported to accumulate with age and are implicated in the aging process. This has been shown for instance in human articular cartilage ŽBank et al., 1998. or in cells of the eye lens ŽNagaraj et al., 1991.. AGEs elicit a wide range of cell-mediated responses leading to vascular dysfunction, matrix expansion and atheroand glomerulosclerosis, as well as other pathological processes. A number of studies have implicated the accelerated AGE formation, which occurs in diabetic patients with secondary complications of the disease such as diabetic microangiopathy, rethinopathy and nephropathy ŽSano et al., 1999; Nakamura et al., 1993.. AGE modifications of proteins have also been associated with several neurological disorders such as Alzheimer’s and Parkinson’s disease. The accumulation of AGEs in cartilage has been suggested to contribute to the decrease in the resistance of cartilage to mechanical influence observed in elderly persons ŽBank et al., 1998.. The presence of AGE modified erythrocyte membrane proteins has been reported to result in impairment of red cell function. It is common to measure AGE modified hemoglobin and serum albumin in diabetic patients as an indicator of the glycation-induced secondary complications associated with diabetes ŽIberg and Fluckinger, 1986; ¨ Johnson and Baker, 1986.. AGE modified proteins can become more resistant to proteolytic degradation, and they have a propensity to aggregate, which can result in accumulation of large insoluble protein aggregates, such as the aggregation of neurofilament proteins found in Parkinson’s and Alzheimer’s disease. AGE modified proteins can also activate specific signaling mechanisms. Such activation has been reported to induce production of superoxide and nitric oxide by glial cells and may be considered part of a vicious cycle, which finally leads to neuronal cell death in the substantia nigra in Parkinson’s disease. Immunohistochemical analyses of human atherosclerotic lesions using a monoclonal anti-AGE antibody have demonstrated diffuse extra-cellular AGEdeposition as well as dense intracellular AGE-deposition in macrophage- and vascular smooth muscle cells. Here the deposition of AGE modified proteins may serve not only an indirect role in the formation of

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arthrosclerotic plaques, but also a direct role by interaction with AGE receptors ŽChappey et al., 1997; Vlassara, 1996.. Cellular responses mediated by binding to AGE receptors can result in altered gene expression and cytokine regulation affecting vascular function ŽVlassara, 1996.. AGE modified proteins have also been suggested as playing a role in regulation of the bone turnover process. An immuno-histochemical study using anti-AGE antibody revealed positive immuno-staining for AGEs in bone tissues from elderly subjects ŽMiyata et al., 1996.. AGE-modified proteins were shown to stimulate monocytes and macrophages to secrete cytokines known to affect bone resorption, such as IL-1␤, IL-6 and TNF-␣. Thus, it has been suggested that AGEs enhance osteoclasts-mediated bone resorption. The modification of bone matrices with AGEs might, therefore, play a pathophysiological role not only in the remodeling of senescent bone matrix tissues, but also in dialysis-related amyloidosis or osteoporosis associated with diabetes and aging. 1.3. Cross-linking Covalent cross-links may form in proteins through a range of non-enzymatic pathways, and have been shown to accompany several types of NECM ŽFriedman, 1999; Davies, 1987.. Generally the cross-linking reactions are closely linked to the protein oxidation and glycation reactions described in the two previous sections. A common side-product of glycation is pentosidine, which is formed by reaction of 3-deoxyglucosone or methylglyoxal with arginine side-chains. Also imidazolium cross-links such as glyoxal-lysineand methylglyoxal-lysine-dimers formed by reaction of lysine side chain with various aldose adducts and bityrosine are common products of oxidation and glycation ŽScheme 3.. The cross-links lysinoalanine ŽLAL., histidinoalanine ŽHAL ., lanthionine, methyl-lysinoalanine, methyl-histidinoalanine and methyl-lanthionine, may form spontaneously within certain peptides and proteins ŽScheme 4.. A number of other covalent crosslinks are likely to exist, due to the complex and heterogeneous nature of the chemical reactions involved in cross-link formation. The sites of cross-link formation within proteins are limited, the number and distribution of such sites within a protein are determined by the amino acid composition, accessibility, conformation and chain mobility of affected proteins. 1.3.1. Repair systems No repair systems are known. 1.3.2. Biological importance Cross-links may form both inter- and intra-molecularly. Biological and physical properties of cross-link-

Scheme 3. Some common oxidation and glycation derived crosslinks. The imidazolium cross-links glyoxal-lysine-dimer ŽRs H. and methylglyoxal-lysine-dimer ŽRs CH 3 . are formed by the reaction of glyoxal or methylglyoxal with Lys. Bityrosine is formed by oxidative reaction of two tyrosine residues. Pentosidine joins a Lys and Arg residue through a derivative of a five-carbon monosaccharide Žpentose.. Throughout the figure, broken lines represent attachment of the residue to the peptide backbone.

containing proteins are probably strongly affected by the relative numbers of these cross-links. In most cases cross-linking is assumed to have damaging effects on protein function, but it may also affect protein half-life. The presence of cross-links has been shown to significantly decrease the susceptibility of peptides to proteolytic digestion. Thus, cross-links such as pentosidine, LAL and HAL appear to survive renal and hepatic proteolysis and are found in urine as free or peptide-associated cross-links ŽFujimoto, 1986.. Glycation mediated intermolecular cross-links in the extracellular matrix have been shown to decrease the flexibility and permeability of tissues and reduce turnover. Cardiovascular tissue also contains a significant proportion of the fibrous connective tissue protein elastin, and its properties are similarly modified by glycation and oxidation-mediated cross-link formation. The nature of these glycation cross-links is now being unraveled and this knowledge is crucial in any attempt to inhibit formation of such reactions. LAL cross-links have been detected in bone, dentin, articular cartilage and lens protein ŽShikata et al., 1985; Fujimoto and Roughley, 1984; Kanajama et al., 1986.. In these tissues the content of LAL was very low in young subjects but increased with age. In cartilage, cross-link formation between components of the cartilage matrix has been shown to gradually reduce the flexibility and mechanical properties of the tissue ŽMonnier et al., 1996.. This may suggest that NECM such as LAL formation may be involved in the cartilage deterioration seen in osteoarthritis and other diseases where cartilage is degraded. Cross-link formation in proteins has also been implicated in the pathological processes of various neurological disorders. In Parkinson’s disease, so-called Lewy bodies are composed of densely cross-linked

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Scheme 4. Cross-link formation through ␤-elimination of SerŽP., ThrŽP., Ser, Thr and Cystine. The formation of these cross-links is a two-step process: Initially hydroxyl-ion-induced elimination of cystine, Ser, Thr, or the phosphorylated adducts of the two latter residues form highly reactive dehydroalanine ŽDha. intermediates Žor methyl-dehydroalanine in the case of Thr.. The conjugated carbon᎐carbon double bond of these intermediates may then react with nucleophilic thiol- or amino-groups of other residues as Lys, His, or Cys to form the bifunctional cross-links. Broken lines represent attachment of the residue to the peptide backbone, or participation in a disulfide bond Žcystine..

intracellular protein deposits formed from cytoskeleton components. These complexes accumulate in presymptomatic stages of the disease, and it is supposed that they play an important role in the pathogenesis of the neurological destructions. Recent findings indicate that glycation-generated protein adducts are the major structural cross-linkers that cause the transformation of soluble neurofilament proteins to insoluble Lewy bodies ŽGoedert, 2001.. 1.4. Racemization, deamidation and isomerization Amino acids with the exception of Gly can occur in two stereo-isomeric forms, designated D- and L-enantiomers. The stereo-specificity of the ribosomal biosynthetic system ensures that only L-amino acids are incorporated into mammalian proteins. However, racemization may occur spontaneously at a low rate

causing an accumulation of D-amino acid enantiomers during aging of proteins. A large number of reports have demonstrated an age-dependent increase of Damino acids in human tissues with a low metabolic turnover, such as dentin, bone, dermis, brain, cartilage, eye-lens, etc. The relative racemization rate of the 19 amino acids varies between different proteins but has generally been found to be Asx ) Glx ) Ser ) Ala ) other amino acids. It was originally assumed that the racemization process of free and protein bound amino acids proceeded by direct proton abstraction creating a planar ␣-carbanion, followed by a rapid, random re-protonation leading to one or the other enantiomer, as illustrated by Scheme 5. According to this hypothesis, the ease with which racemization occurs at various amino acids would be dependent on the electrophillic properties of their

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Scheme 5. Racemization of amino acids through direct proton abstractionrre-addition. The racemization of free amino acids proceeds by the removal of a proton from the ␣-carbon atom to form a planar carbanion intermediate. The carbanion, having lost the original chirality, recombines with a proton from the environment to form a chiral amino acid. The re-addition can take place equally well to either side of the carbanion, yielding an equimolar mixture of D- and L-isomers.

side-chains. Indeed, this relationship has been confirmed experimentally for free amino acids. However, the de-protonationrre-protonation model does not adequately describe racemization mechanism of peptide-bound amino acids. As an example, it does not explain the experimental observation that Asp residues racemize faster than Thr and Ser residues, whose side chains are more electrophillic than the aspartyl side chain. The rapid racemization of Asx and Glx within proteins is believed to reflect the propensity of these amino acids for cyclic imide formation ŽGeiger and Clarke, 1987; Radkiewicz et al., 2001. ŽScheme 6.. The involvement of an imide pathway in the racemization of Asx residues has been verified in

Scheme 6. Deamidation, isomerization and racemization of Asx and Glx. Isomerized and racemized Asp residues are derived from L-Asp or L-Asn via succinimide intermediates. Imides are formed by intermolecular cyclization, where the peptide bond nitrogen of the following residue attacks the Asx side chain carbonyl. The subsequent hydrolysis of the succinimide produces either peptides or iso-Asp peptides in both D and L configuration. An alternate degradation pathway involving the attack of the side chain amide nitrogen of Asn on the following peptide bond carbonyl can result in cleavage of the peptide main chain. Gln and Glu residues may be subject to similar reactions, with glutarimide intermediates, yielding isomerised and optically inverted Glu residues.

several studies. The rate of succinimide racemization in the hexapeptide Val-Tyr-Pro-Asu-Gly-Ala Žwhere Asu denotes a succinimide residue. was 117 000 times the rate found in the corresponding Asp-peptide ŽGeiger and Clarke, 1987.. The enhanced susceptibility of the succinimide to racemize is due to the increased acidity of the ␣-carbon of this structure ŽRadkiewicz et al., 2001.. Imide formation also appears to be the central event in two other common spontaneous covalent reactions: deamidation and isomerization. Deamidation refers to the hydrolysis of the side chain amide bond in an Asn or Gln residue to form Asp and Glu, respectively. In isomerization, the peptide backbone is redirected from the ␣-carboxyl group of an Asx or Glx residue through the side chain ␤ or ␥-carboxyl, creating a kink in the peptide backbone. For Asx, and presumably also Glx, all these reactions are presumed to take place through a imide intermediate formed by the nucleophilic attack of the of the side-chain carbonyl on the peptide bond nitrogen, ŽScheme 6.. The imide is highly susceptible to racemization leading to the formation of both racemised and isomerized residues: D-Asp; and D-isoAsp ŽD-Glu and D-isoGlu.. The reaction rates of deamidation, racemization and isomerization are strongly correlated to the ease with which imides are generated. Peptide dihedral angles of ␺ s y120⬚ and ␹ s q120⬚, are optimal for succinimide formation. The reaction is dependent on polypeptide conformation and the nature of the residues immediately preceding or following the Asx residue. Imide formation is facilitated when polypeptides are flexible Žor possess the necessary conformation. and when neighboring residues have small Žnon-bulky. side-chains ŽGeiger and Clarke, 1987; Radkiewicz et al., 2001.. The process of in vivo-racemization is not restricted to Asx and Glx residues. Hence, D-enantiomers of Ser, Tyr, Ala and His have been shown to accumulate in various tissues during human aging ŽDunlop and Neidle, 1997; Luthra et al., 1994; Cloos and Jensen, 2000.. The accumulation of D-Ala observed in some proteins is probably due to the formation of racemic Ala via the intermediates dehydroalanine and pyruvic acid through degradationrre-arrangement of SerŽP., Ser and Cys residues ŽCloos and Jensen, 2000.. 1.4.1. Repair systems There is no known cellular system for the identification and repair of an Asp or Glu residue formed by deamidation of Asn and Gln. However, as mentioned previously, deamidation involves imide formation which also results in formation of D-enantiomeric or isomerized adducts ŽScheme 5.. These ‘unnatural’ adducts may serve to reveal that a

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protein modification has taken place and make enzymatic recognition and degradation possible. The enzyme L-isoaspartylrO-methyltransferase ŽIAMT, EC 2.1.1.77., catalyses the transfer of a methyl group from S-adenosyl-methionine to the ␣-carboxyl group of L-isoAsp residues in a variety of organisms ŽLowenson and Clarke, 1995.. Methylation of a L-isoAsp peptide induces the reformation of a succinimide ring and has been shown to result in the conversion to the L-Asp form of the peptide ŽBrennan et al., 1994; McFadden and Clarke, 1987.. Although the IAMT genes are well conserved from bacteria to humans, the mammalian methyl transferases have the additional ability to recognize D-Asp residues Žbut not D-isoAsp residues., permitting them to repair some racemized residues ŽLowenson and Clarke, 1992.. Presumably the racemization reaction, which proceeds slower than isomerization, may be less detrimental for bacteria, where the protein life span is shorter than in a mammalian cell. No repair systems are known for the handling of isomerized or racemised Glu residues. The enzymes D-amino acid oxidase ŽD-AAO, EC 1.4.3.3. and D-aspartate oxidase ŽD-AspO, EC 1.4.3.1. catalyse the oxidative deamidation of free D-amino acids to their corresponding ␣-keto acids, which in turn can be specifically re-amidated to the L-form ŽD’Aniello et al., 1993.. These enzymes are assumed to have a central role in the metabolism and elimination of D-amino acids accumulated during aging ŽD’Aniello et al., 1993.. 1.4.2. Biological importance Deamidation changes the charge of the affected protein. Racemization and isomerization alter the bonding angles of adjoining peptide bonds. A few racemized or isomerized amino acids may change the dipole moments of a polypeptide chain, thus potentially altering the structure of the whole protein ŽHol et al., 1981.. Such modifications may change protein structure, stability, properties and functions. Mutations of the gene encoding IAMT in Eschericia coli results in mutants which are unusually susceptible to heat shock and survive poorly in a stationary phase, a stage with little or no protein synthesis ŽLi and Clarke, 1992.. Moreover, IAMT deletion mutants have been shown to possess distinct phenotypes ŽKim et al., 1997, 1997b.. IAMT knockout mice exhibit growth retardation, and die of fatal seizures at an average age of 42 days, suggesting that the ability to repair L-isoAsp and D-Asp residues is essential for normal growth and central nervous system function ŽKim et al., 1997, 1997b.. Rats and chickens fed with D-Ala and D-Asp in amounts exceeding the capacity of the D-AAO and D-AspO enzymes exhibit growth re-

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tardation and suppression of protein synthesis ŽD’Aniello et al., 1993.. These observations indicate that systemic accumulation of deamidated, isomerized, or racemized residues is detrimental. 1.5. Dephosphorylation and ␤-elimination Disulfide bonds of Cys residues and hydroxyl groups of Ser and Thr residues and their phosphorylated adducts SerŽP. and ThrŽP. may undergo spontaneous hydroxide ion-induced ␤-elimination reactions to form dehydroalanine wor methyl-dehydroalanine in the case of Thr and ThrŽP.x residues. Phosphorylated Thr and Ser residues may also dephosphorylate by spontaneous hydrolysis to yield a variety of adducts, as outlined in Scheme 7 ŽCloos and Jensen, 2000; Shikata et al., 1985; Fujimoto and Roughley, 1984; Kanajama et al., 1986.. Among the main products of these elimination-addition reactions are bifunctional crosslinks, as lysinoalanine ŽLAL. and histidinoalanine ŽHAL. ŽCloos and Jensen, 2000; Shikata et al., 1985., described in a previous section. Evidence for such decomposition pathways have been reported in various mammalian tissues, including bone and dentin. SerŽP. has been shown to decrease in an age-dependent manner in human dentin, with a concomitant increase in Ala, LAL and HAL ŽCloos and Jensen, 2000.. 1.5.1. Repair systems Protein kinases ŽPKs. may act as repair systems for the spontaneous dephosphorylation of SerŽP., ThrŽP., TyrŽP. through hydrolysis. However, at present no repair or recognition system is known for adducts formed by the elimination reactions Ždehydroalanine, pyruvic acid, cross-links, etc... 1.5.2. Biological importance Phosphorylation is one of the most important biological signal mechanisms controlling the activity of various enzymes, receptors, signaling molecules and other target proteins. Obviously a change in the phosphorylation status may directly affect the function of a protein. In addition, ␤-elimination and hydrolysis of phosphorylated residues alters the charge of affected proteins, influencing protein conformation and function indirectly. Some of the secondary decomposition pathways may cause hydrolysis of the peptide backbone, and formation of bi-functional cross-links, as LAL and HAL ŽScheme 7.. The effect of the presence of ‘unnatural’ residues, dehydroalanine, pyruvic acid and others in proteins has not received great attention. Hence, no examples are described where such modifications are directly involved in pathological processes, but in accordance with the observations made with other types of NECM, they are likely to

48

P.A.C. Cloos, S. Christgau r Matrix Biology 21 (2002) 39᎐52

Scheme 7. Degradation of phosphoserine, serine and cystine in proteins by ␤-elimination, hydrolysis and dehydration. Hydroxide ion-catalyzed ␤-elimination of Ser, SerŽP. and cystine Žand to a lesser extent probably also cysteine. yield highly reactive dehydroalanine ŽDha. intermediates. SerŽP. may also dephosphorylate directly to serine and phosphate. Serine may in turn decompose to Dha or undergo aldol cleavage to form glycine and formaldehyde. The alkene group of the Dha residue may react with available nucleophilic residues to form cross-links as lysinoalanine, histidinoalanine and lathionine. Dha residues may also decompose to pyruvic acid causing hydrolysis of the peptide backbone. Pyruvate may in turn form Ala by transamination with ammonia or amino acids. Note that the Dha and pyruvic acid intermediates have lost their original asymmetry. Ala generated through these reactions will therefore be racemic. In analogy, the formed cross-links will be present as a mixture of stereoisomers. A similar reaction scheme may be drawn for phosphothreonine. The corresponding elimination reaction of threonine produces methyl-dehydroalanine, which may react further to form cross-links as methyl-dehydroalanine and methyl-lanthionine. Broken lines represent attachment of the residue to the peptide backbone.

affect function, structure and stability of the modified proteins. 1.6. Disulfide bond formation and disruption Disulfide bonds linking the sulfohydryl groups of two cysteine residues may be reduced Žunder reducing conditions or by the action of reducing agents. or formed inappropriately under oxidizing conditions Žor by oxidizing agents.. 1.6.1. Repair systems Protein disulfide isomerases, which catalyse disulfide bond formation, are important members of the protein-folding pathway ŽBardwell and Beckwith, 1993.. These proteins have the potential of acting as a repair system by re-oxidizing reduced disulfide bonds in damaged proteins. In analogy, some proteins Ži.e. the cytoplasmic protein thioredoxin reductase. may reduce disulfides to counteract the effects of oxidative stress ŽStein, 1993..

1.6.2. Biological importance Disulfide bond formation is a covalent alteration that clearly influences the higher structures as well as stability of proteins. Incorrectly formed disulfide bonds, or disruption of essential disulfide bonds will change proteins structure and is likely to be detrimental to both the function and stability of the affected protein. 1.7. Proline isomerization Peptide bonds involving Pro can exist in either the ‘normal’ native trans-configuration or the rare cisconformation ŽFisher et al., 1983.. Pro isomerization denotes the conversion of proline trans-linkages to cis-linkages. Pro isomerization occurs spontaneously at a low rate after protein synthesis ŽScheme 8.. Whether such a modification constitutes a covalent modification or a conformational one can be debated ŽStein, 1993.. However, due to the partial double-bond character of the peptide bond and the necessary dis-

P.A.C. Cloos, S. Christgau r Matrix Biology 21 (2002) 39᎐52

Scheme 8. Proline isomerization. Peptidyl-prolyl isomerases ŽPPI. may act as a repair system converting cis-isomers to the trans configuration. Broken lines represent attachment to the polypeptide backbone.

ruption of this bond in the isomerization event, this modification may be classified as a covalent alteration. 1.7.1. Repair systems Peptidyl-prolyl cis᎐trans isomerases ŽPPI., i.e. enzymes capable of catalyzing Pro isomerization, have been identified ŽSchmid et al., 1993.. These proteins probably expedite the initial folding of nascent proteins but can also function in protein repair by the re-conversion of cis-linkages ŽYaron and Naider, 1993; Schmid et al., 1993.. 1.7.2. Biological importance The presence of the altered cis-isomer has been shown to alter the physico-chemical characteristics of the affected peptide. Thus, Pro cis-isomers may delay protein foldingrrefolding, and have also been reported to prevent proteolysis ŽYaron and Naider, 1993.. Proline isomerization has not been implicated in pathological processes, but this issue probably needs further investigation to be properly resolved.

2. Physiological consequences of NECM The presence of non-enzymatic covalent modifications within a protein does not equate functional alteration. However, changes of a single residue of an enzyme or receptor etc. may disrupt activity. As an example, deamidation of CD4 at residue Asn 52 has been shown to decrease its binding to the envelope protein gp120 of human immunodeficiency virus type 1, potentially affecting its usefulness as an AIDS-therapeutic ŽTeshima et al., 1991.. NECM does not necessarily have to involve the active site chemistry of such proteins to cause functional impairment, but can also take place at a site vital for the maintenance of protein structure or stability. Covalent modifications may cause large-scale structural damage such as denaturation and aggregation. For instance, Tomiyama et al. Ž1994. have shown that racemization of Asp 23 of ␤-amyloid protein accelerates aggregation and fibril formation of this protein.

49

In another study, denatured Ribonuclease A was found to re-fold at half the normal rate when isomerized at residue Asp 63 ŽDiDonato et al., 1993.. In analogy, conformational modifications may permit racemization or other covalent modifications in otherwise sterically hindered molecules. Table 2 summarizes effects of various NECMs on protein properties. If NECMs were deleterious to biological function in vivo, some genetic selection minimizing the frequency of ‘susceptible sequences’ within proteins would be anticipated. Indeed some database search studies have indicated a bias against sequences that are prone to succinimide formation ŽWright, 1991; Robinson and Robinson, 1991.. Although selection in most situations may be expected to be against susceptible sequences, it is conceivable that some proteins may have been encoded with specific ‘hot spots’ predisposing them to particular NECM at specific sites to fulfil certain special physiological functions. As an example of this, the process of protein splicing appears to utilize succinimide intermediates ŽBrennan and Clark, 1993; Xu et al., 1994.. Finally, NECM can be present as unwanted side reactions in several industrial processes involving proteins. As an example, oxidative protein damage is a major problem in commercial applications such as food processing, and must be monitored and controlled ŽFriedman, 1999; Fuglsang et al., 1995.. 3. Use of NECM in the study of protein age and turnover The accumulation of NECM within a protein depends both on the age of the protein rate constants for the covalent reactions, and on the rate of protein turnover. Therefore, the extent of these reactions in proteins can potentially be used to determine their turnover rates andror age. Table 2 General effects of various non-enzymatic covalent protein modifications Modification

Racemization AsprGlu isomerization AsnrGln isomerization Pro isomerization Deamidation ␤-elimination AGE-formation Cross-linking Oxidation

Affects proteinrpeptide Charge

Conform-ation

Digestibility

No No Yes No Yes Yes Yes Yesrno Yes

Yes Yes Yes Yes Yesrno Yesrno Yesrno Yes Yes

Yes Yes Yes Yes Yesrno Yesrno Yes Yes Yes

P.A.C. Cloos, S. Christgau r Matrix Biology 21 (2002) 39᎐52

50

If the tissue or protein in question is not catabolized, circumstances are favorable for the accumulation of the modified forms, and covalent reactions may reach equilibrium within a short time ŽCloos and Fledelius, 2000; Cloos and Jensen, 2000.. In contrast, if the rate of turnover is high compared to covalent reactions modified forms cannot accumulate. In some situations, the effect of various enzyme systems Ži.e. repair systems or enzymes promoting the covalent change. must also be taken into consideration, as these may accelerate or retard NECM. If a simple first order irreversible covalent reaction is monitored was the de-phopshorylation of SerŽP.; Cloos and Jensen, 2000x, converting the native molecule x to ‘aged’ molecule y, the following relationship may be deduced ŽCloos, 1995.: w y xt w x xt s

e tŽ k Pr oqk Re m . y 1 k 1 q Rem e tŽ k Pr oqk Rem . k Pro

ž /

Where w x x t and w y x t denote the concentrations of molecules x and y at time t, respectively, and k Pro is the rate constant for the covalent process. Thus, if the w y x trw x x t ratio and the rate constant for the reaction is known, this relationship may be used to estimate the remodeling rate Ž k Rem . for the protein or tissue ŽCloos, 1995.. The corresponding ‘remodeling equation’ for a reversible first order covalent reaction Žas racemization of amino acids other than Asp and Glu ŽCloos and Jensen, 2000. converting molecule x to aged molecule y may be deduced as ŽCloos, 1995.: w y xt w x xt s

e tŽ k Pr oqk Re m . y 1 k q k Rem tŽ k Pr oqk Rem . 1 q Pro e k Pro

ž

/

In more complex reaction mechanisms, such as racemizationrisomerization of Asp, exact solutions cannot be used in the calculation and interpretation of experimental data. In these situations, protein turnover rates may be determined by mathematical modeling ŽCloos and Fledelius, 2000.. In bradytrophic tissues, such as tooth dentin, the contents of ‘aged’ molecules will translate to the age of the individual Žwhen correcting for the time required for the formation of the tissue.. Thus, a large number of studies have demonstrated that the chronological age of an individual can be estimated with high precision through the analysis of the degree of Asp-racemization in dentin, and the method is currently considered the most accurate method to determine ‘age at death’ ŽRitz and Schutz, ¨ 1993.. There is also a close correlation between chronologi-

Fig. 2. Contents of SerŽP. in phosphoprotein from human dentin in relation to age. The amount of SerŽP. was normalized in relation to Leu. The rate of SerŽP. decomposition was: 42.2= 10y3 yearsy1 . The solid line represents the calculated linear regression line; broken lines represent the 95% confidence bands. Cloos and Jensen Ž2000., Age related de-phosphorylation of proteins in dentin: a biological tool for assessment of protein age. Biogerontology, 1, p. 341, Figure 2. Reprinted by kind permission from Kluwer Academic Publishers, Dordrecht, The Netherlands.

cal age and the D-Asp accumulation in the eye lens, bone, tooth enamel, the white matter of the brain and elastin from the lung parenchyma. Other types of NECM may potentially be used for the same purpose, thus we have recently shown ŽCloos and Jensen, 2000. that phosphoserine in human dentin phosphoproteins decomposes Žto serine or dehydro-alanine. in an agedependent manner Žcorrelation to dentine age ) 0.95. ŽFig. 2.. The measurement of NECMs has been facilitated in the later years by introduction of various immunoassays. As an example, we have recently developed immunoassays specific for the authentic form of the type I collagen C-telopeptide as well as assays measuring three aged forms of this molecule containing isomerizedrracemized variants Žiso-Asp, D-Asp and D-isoAsp. of residue Asp-1211 in this part of the protein ŽCloos and Fledelius, 2000; Fledelius et al., 1997.. The immunoassay approach enables quantification of racemization and isomerization of this collagen type I-specific epitope in living individuals through measurements of these molecules in urine or serum ŽCloos and Fledelius, 2000; Christgau et al., 1998.. Interestingly collagen type I C-telopeptide fragments excreted in urine from patients affected with bone metastases or Paget’s disease of bone, have a markedly decreased degree of racemization as compared to healthy adults. Thus, the average Paget’s disease patient has an L-AsprD-Asp ratio more than 20 standard deviations above the mean for healthy controls ŽFig. 3.. The decreased racemization and isomerization of urinary collagen type I C-telopeptide frag-

P.A.C. Cloos, S. Christgau r Matrix Biology 21 (2002) 39᎐52

51

Fig. 3. L-AsprD-Asp ratios measured in urinary degradation products of type I collagen from healthy adults and patients with Paget’s disease. L-Asp and D-Asp forms of urinary degradation products containing the collagen type I-specific sequence EKAHDGGR were quantified using two highly specific immunoassays. The L-AsprD-Asp ratios were determined in healthy adult controls Žmales, pre- and post-menopausal females, n s 87. and in adults with Paget’s disease of bone Ž n s 24.. The shaded area indicates mean " 2 S.D.s for healthy adults. Bars indicate medians.

ments in these pathological conditions is probably explained by the increased turnover of type I collagen in bone ŽCloos and Fledelius, 2000.. 3.1. The role of NECM in pathogenesis An intriguing possibility is that some types of NECM may lead to disease. Several studies have shown that various types of protein damage are hallmarks of aging and characteristics for some chronic diseases of the elderly, such as atherosclerosis, rheumatoid arthritis, Alzheimer’s and diabetes mellitus ŽWelch and Gambetti, 1998; Kim et al., 1997, 1997b; Sano et al., 1999; Daniels and Hauser, 1992; Yan et al., 1994; Tomiyama et al., 1994.. These observations have provided growing support for the involvement of NECM in pathogenesis. However, the question of whether these changes occur as primary or secondary events in these conditions remains to be answered. A major limitation to progress in research on the role of these reactions in aging and disease is related to the measurement of NECM. Most often, measurement of NECM involves hydrolysis of the protein, derivatization and complex and time consuming analytical procedures, such as multi-step HPLC and mass spectroscopy. In this context, immunoassays are becoming increasingly important tools for the detection and measurement of NECM. Immunoassays provide the possibility of assessing NECM directly in tissue extracts ŽGineyts et al., 2000.. Furthermore, immunoassays may be adapted to quantification of NECM non-invasively by measurement of NECM markers in serum or urine ŽCloos and Fledelius, 2000; Christgau et al., 1998..

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