Roger J.W. Truscott's research while affiliated with University of Wollongong and other places
Recent discoveries may change the way that multiple sclerosis (MS) is viewed, particularly with regard to the reasons for the untoward immune response. The fact that myelin proteins are long-lived, and that by the time we are adults, they are extensively degraded, alters our perspective on the reasons for the onset of autoimmunity and the origin of MS. For example, myelin basic protein (MBP) from every human brain past the age of 20 years, is so greatly modified, that it is effectively a different protein from the one that was laid down in childhood. Since only a subset of people with such degraded MBP develop MS, a focus on understanding the mechanism of immune responses to central nervous system (CNS) antigens and cerebral immune tolerance appear to be worthwhile avenues to explore. In accord with this, it will be productive to examine why all people, whose brains contain large quantities of a “foreign antigen”, do not develop MS. Importantly for the potential causation of MS, MBP from MS patients breaks down differently from the MBP in aged controls. If the novel structures formed in these MS-specific regions are particularly antigenic, it could help explain the origin of MS. If verified, these findings could provide an avenue for the rational synthesis of drugs to prevent and treat MS.
The breakdown of long-lived proteins is associated with aging, as well as disease, however our understanding of the molecular processes involved is still limited. Of particular relevance, crosslinked proteins are often reported in aged tissues but the mechanisms for their formation are poorly understood. In this study, sites of protein crosslinking in human ocular lenses were characterized using proteomic techniques. In long-lived lens proteins, several sites of crosslinking were found to involve the addition of Lys to Asp or Asn residues. Using model peptides containing Asp or Asn, a mechanism was elucidated that involves a succinimide intermediate. Succinimides formed readily from Asn at neutral pH, whereas a higher rate of formation from Asp peptides was observed at more acidic pHs. Succinimides were found to be relatively stable in the absence of nucleophiles. Since racemization of Asp residues, as well as deamidation of Asn, involves a succinimide intermediate, sites of D-Asp and isoAsp in long-lived proteins should also be considered as potential sites of protein covalent crosslinking.
Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer’s, Parkinson’s, and cataract are characterized by the accumulation of protein aggregates.
Background: The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues. Methods: Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation. Results: In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys. Conclusions: This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG. General significance: Our finding demonstrates a novel +144.0535Da PTM arising from the breakdown of oxidised glutathione.
Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemised, truncated and covalently crosslinked. One reaction responsible for age-related protein crosslinking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence was found for this in a number of lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent crosslinks than others. Foremost among them was Cys 5 in βA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a pre-requisite for DHA formation and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys 5. Modelling and direct investigation of the N-terminal sequence of βA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form inter-molecular crosslinks with Lys and Cys. In the lens, the most abundant crosslink involved Cys 5 of βA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent crosslinks and the role of nearby amino acids in creating "hotpsots" for the spontaneous processes responsible for protein degradation in aged tissues.
Membrane lipid composition is altered in the brain during the pathogenesis of several age-related neurodegenerative diseases, including Alzheimer’s disease. The entorhinal cortex is one of the first regions of the brain to display the neuropathology typical of Alzheimer’s disease, yet little is known about the changes that occur in membrane lipids within this brain region during normal aging (i.e., in the absence of dementia). In the present study, the phospholipid composition of mitochondrial and microsomal membranes from human entorhinal cortex was examined for any changes over the adult lifespan (18–98 years). Overall, changes in several molecular phospholipids were seen with age in the entorhinal cortex across both membranes. The proportion of total phosphatidylcholine within the mitochondrial fraction increased within the entorhinal cortex with age, while total mitochondrial phosphatidylethanolamine decreased. Many mitochondrial phosphatidylethanolamines containing docosahexaenoic acid increased with age; however, this did not translate into an overall age-related increase in total mitochondrial docosahexaenoic acid. The most abundant phospholipid present within the human brain, PC 16:0_18:1, also increased with age within the mitochondrial membranes of the entorhinal cortex. When compared to other regions of the brain, the phospholipid composition of the entorhinal cortex remains relatively stable in adults over the lifespan in the absence of dementia.
Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic. Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0348-x) contains supplementary material, which is available to authorized users.
Long wavelength solar UV radiation is implicated in photodamage to the human eye. The human lens contains multiple tryptophan-derived compounds that have significant absorbance bands in the UVA region (λ 315 - 400 nm) that act as efficient physical filters for these wavelengths. The concentrations of many of these UV filter compounds decrease with increasing age, resulting in diminished protection, increased oxidative damage and the accumulation of modified proteins implicated in nuclear cataract formation. This damage may arise via formation of α,β-unsaturated carbonyls from the UV filter compounds, adduction to lens proteins and subsequent action as photosensitizers, and/or via the reactions of redox-active transition metal ions that accumulate in aged human lenses. The latter may promote the oxidation of free, or protein-bound, o-aminophenols, such as the UV filter compounds 3-hydroxykynurenine (3OHKyn) and 3-hydroxyanthranilic acid (3OHAA). It is shown here that Cu(II), and to a lesser extent Fe(III), enhance oxidation of free 3OHKyn, 3OHAA and 3OHKyn bound to specific amino acids and lens proteins, with this resulting in increased cross-linking of lens proteins. These data indicate that elevated levels of transition metal ions in aging lenses can enhance the loss of protective UV filter compounds, and contribute to the formation of high-molecular-mass dysfunctional crystallin proteins in a light-independent manner. These reactions may contribute to the formation of lens cataracts in humans.
It has only recently been appreciated that the human body contains many long-lived proteins (LLPs). Their gradual degradation over time contributes to human aging and probably also to a range of age-related disorders. Indeed, the role of progressive damage of proteins in aging may be indicated by the fact that many neurological diseases do not appear until after middle age. A major factor responsible for the deterioration of old proteins is the spontaneous breakdown of susceptible amino acid residues resulting in racemization, truncation, deamidation, and crosslinking. When proteins decompose in this way, their structures and functions may be altered and novel epitopes can be formed that can induce an autoimmune response.
: The degradation of long-lived proteins in the body is an important aspect of aging and much of the breakdown is due to the intrinsic instability of particular amino acids. In this study, peptides were examined to discover if spontaneous non-enzymatic reactions could be responsible for the composition of Alzheimer's (AD) plaque in the human brain. The great majority of AD plaque consists of N-terminally truncated versions of Aβ(1-40/1-42), with the most abundant peptide com-mencing with Glu (residue 3 in Aβ1-40/1-42) that is present as pyroGlu. Several Asp residues are racemised in Aβ plaque, with residue 1 being predominantly L-isoAsp and peptide bond cleavage next to Ser 8 is also evident. In peptides, loss of the two N-terminal amino acids as a diketopiperazine was demonstrated at pH 7. For the Aβ N-terminal hexapeptide, AspAlaG-luPheArgHis, this resulted in the removal of AspAla diketopiperazine and the generation of Glu as the new N-terminal resi-due. The Glu cyclised readily to pyroGlu. This pathway was altered significantly by zinc, which promoted pyroGlu formation, but decreased AspAla diketopiperazine release. Zinc also facilitated cleavage on the N-terminal side of Ser 8. Racemisation of the original N-terminal Asp to L-isoAsp, was also detected, and loss of one amino acid from the N-terminus. These data are therefore entirely consistent with plaque in human brain forming from deposition of Aβ(1-40/1-42) and, over time, de-composing spontaneously. Since amyloid plaque is present in the human brain for years prior to the onset of AD, gradual spontaneous changes to the polypeptides within it will alter its properties, and those of the oligomers that can diffuse from it. Such incremental changes in composition may therefore contribute to the origin of AD-associated cytotoxicity.
Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long-lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N-terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser- and Thr-containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N-terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co-ordinates zinc and that once zinc is co-ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long-lived proteins, for example truncation on the N-terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long-term stability of cellular proteins.
One of the biggest risk factors for developing Alzheimer's disease is advanced age. Despite several studies examining changes to phospholipids in the hippocampus during the pathogenesis of Alzheimer's disease, little is known regarding changes to phospholipids in this region during normal adult aging. This study examined the phospholipid composition of the mitochondrial and microsomal membranes of the human hippocampus from post-mortem tissue of neurologically normal subjects aged between 18 and 104 years. Many of the age-related changes found were in low-to-moderately abundant phospholipids in both membrane fractions, with decreases with age being seen in many phospholipids containing either adrenic or arachidonic acid. The most abundant phospholipid of this type was phosphatidylethanolamine 18:0_22:4, which decreased in both the mitochondrial and microsomal membranes by approximately 20 % from ages 20 to 100. Subsequent decreases with age were seen in total adrenic and arachidonic acid in the phospholipids of both membrane fractions, but not in either fatty acid specifically within the phosphatidylethanolamine class. Increases with age were seen in the hippocampus for mitochondrial phosphatidylserine 18:0_22:6. This is the first report of changes to molecular phospholipids of the human hippocampus over the adult lifespan, with this study also providing a comprehensive profile of the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids of the human hippocampus.
It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification. Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues. Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015. Published by Elsevier B.V.
Membrane phospholipids make up a substantial portion of the human brain, and changes in their amount and composition are thought to play a role in the pathogenesis of age-related neurodegenerative disease. Nevertheless, little is known about the changes that phospholipids undergo during normal adult aging. This study examined changes in phospholipid composition in the mitochondrial and microsomal membranes of human dorsolateral prefrontal cortex over the adult lifespan. The largest age-related changes were an increase in the abundance of both mitochondrial and microsomal phosphatidylserine 18:0_22:6 by approximately one third from age 20 to 100, and a 25% decrease in mitochondrial phosphatidylethanolamine 18:0_20:4. Generally, increases were seen with age in phospholipids containing docosahexaenoic acid across both membrane fractions, while phospholipids containing either arachidonic or adrenic acid decreased with age. These findings suggest a gradual change in membrane lipid composition over the adult lifespan.
Proteomics may have enabled the root cause of a major human blinding condition, age-related cataract, to be established. Cataract appears to result from the spontaneous decomposition of long-lived macromolecules in the human lens, and recent proteomic analysis has enabled both the particular crystallins, and the specific sites of amino acid modification within each polypeptide, to be identified. Analysis of proteins from cataract lenses has demonstrated that there are key sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. Proteomic analysis, using mass spectrometry (MS), revealed that the most abundant posttranslational modification of aged lens proteins is racemization. This is somewhat ironic, since structural isomers can be viewed as the 'Achilles heel' of mass spectrometry and there are typically few, if any, differences in the MS/MS spectra of tryptic peptides containing one D-amino acid. It is proposed that once a certain level of spontaneous posttranslational modification at key sites occurs, that protein-protein interactions are disrupted, and binding of complexes to cell membranes takes place that impairs cell-to-cell communication. These findings may apply more widely to age-related human diseases, in particular where the deterioration of long-lived proteins is a crucial component in the etiology. This article is protected by copyright. All rights reserved.
Supplementary Figure 1B: Truncation at N-terminal side of Ser as a function of time. Cleavage was calculated based on the amount of Ac-AA formed compared to the amount of each peptide present at time = 0. Peptides were incubated in sodium borate buffer (100 mM, pH 12.5) at 37 °C (B) = Ac-AASAA and Ac-AALSAA.
Supplementary Figure 1A: Truncation at N-terminal side of Ser as a function of time. Cleavage was calculated based on the amount of Ac-AA formed compared to the amount of each peptide present at time = 0. Peptides were incubated in sodium borate buffer (100 mM, pH 12.5) at 37 °C. (A) = Ac-AASAA and Ac-AAPSAA.
Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of L-serine to D-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins.
Non-enzymatic posttranslational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC-tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty four of thirty six sites identified on fifteen proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β-elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein-protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Non-disulfide crosslinks were identified for the first time in lens tissue between βB2- & βB2-, βA4- & βA3-, γS -& βB1- and βA4- & βA4-crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long-lived proteins and that DHA and DHB-mediated protein crosslinking may the source of the long sought after non-disulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long-lived proteins of other tissues leading to protein aggregation diseases. This article is protected by copyright. All rights reserved.
Age-dependent deterioration of long-lived proteins in humans may have wide-ranging effects on health, fitness and diseases of the elderly. To a large extent, denaturation of old proteins appears to result from the intrinsic instability of certain amino acids, however these reactions are incompletely understood. One method to investigate these involves exposing peptides to elevated temperatures at physiological pH. Incubation of PFHSPSY, which corresponds to a region of human αB-crystallin that is susceptible to age-related modification, resulted in the appearance of a major product. NMR spectroscopy confirmed that this novel peptide had formed via racemisation of the N-terminal Pro. This phenomenon was not confined to Pro, because peptides with N-terminal Ser and Ala residues also underwent racemisation. Since N-terminal racemisation occurred at 37 o C, a long-lived protein was examined. LC-MS/MS analysis revealed that approximately one third of aquaporin 0 polypeptides in the centre of aged human lenses had been racemised at the N-terminal methionine. © 2013 The Authors Journal compilation © 2013 FEBS.
A common modification of human long-lived proteins is spontaneous isomerisation of aspartate residues, and its biological importance can be inferred from the ubiquitous presence of protein isoaspartate methyl transferase (PIMT), that repairs this damage. Cyclisation of L-Asp residues yields four isomers: L-Asp, L-isoAsp, D-Asp and D-isoAsp, however little is known about their rate of formation or interconversion. This is important because PIMT is inactive towards D-isoAsp. Peptides containing the four Asp isoforms corresponding to a susceptible site (Asp 151) in the chaperone, αA-crystallin, were examined for their interconversion at pH 7. D-Asp formed from L-Asp readily, whereas L-isoAsp was not detected until significantly later. D-isoAsp formed very slowly, with just 1% present after 8 days at 60(0)C. These findings can be used to rationalise the substrate specificity of PIMT. In addition, both the D-isoAsp and L-isoAsp peptides were found to be remarkably stable, showing little conversion to other isomers, even after weeks of incubation. Therefore L-isoAsp and D-isoAsp appear to represent "terminal" stages of L-Asp modification. If PIMT is present, L-isoAsp may be reverted to L-Asp, however there appears to be no prospect of reversing D-isoAsp formation in aged proteins. Interestingly, Asp 151 in recombinant αA crystallin isomerised more rapidly than the L-Asp peptide.
Racemisation is one of the most abundant modifications in long-lived proteins. It has been proposed that the accumulation of such modifications over time could lead to changes in tissues and ultimately human age-related diseases. Serine is one of the main amino acids involved in racemisation, however the site of D-Ser in any aged protein has yet to be reported. In this study racemisation of two residues, Ser 59 and Ser 62, has been demonstrated in an unstructured region of the small heat shock protein, αA crystallin. αA Crystallin is also the most abundant structural protein in the human lens. D-Ser increased linearly with age in normal lenses, until it accounted for approximately 35% of the Ser at both sites by age 75. In agreement with a possible role in human age-related disease, levels were significantly higher in cataract lenses. It is likely that such prevalent age-related changes contribute to the denaturation of α crystallin, and therefore its ability to act as a chaperone. Racemisation of amino acids, such as serine, in flexible regions of long-lived proteins, could be associated with the development of human age-related conditions such as cataract.
Racemisation of amino acids is one of the most abundant modifications in long-lived proteins. In this study racemisation of Asp 58 in the small heat shock protein, αA crystallin, was investigated. In normal human lenses, levels of L-isoAsp, D-isoAsp and D-Asp increased with age, such that by age 70 they accounted for approximately half of the total Asp at this site. Levels of D-isoAsp were significantly higher in all cataract lenses than age-matched normal lenses. The introduction of D-isoAsp in αA crystallin could therefore be associated with the development of cataract. Its more rapid formation in cataract lenses may represent an example of accelerated protein aging leading to a human age-related disease.
Long-lived proteins exist in a number of tissues in the human body however little is known about the reactions involved in their degradation over time. Lens proteins which do not turnover, provide a useful system to examine such processes. Using a combination of Western blotting and proteomic methodology, age-related changes to a major protein, γS crystallin, were studied. By teenage years insoluble intact γS crystallin was detected, indicative of protein denaturation. This was not the only change however, as blots revealed evidence of significant cross-linking, as well as cleavage of γS crystallin in all adult lenses. Cleavage at a serine residue near the C-terminus was a major reaction that caused the release a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. Several other crystallin-derived peptides with double basic residues also lodged in the cell membrane fraction. Model studies showed that once cleaved from γS crystallin, SPAVQSFRRIVE adopts a markedly different shape from that in the intact protein. Further, the acquired helical conformation may explain why the peptide seems to affect water permeability. This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may therefore yield peptides with untoward biological activity.
The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long-term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N-terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N-terminal Ser represented one-quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or 'laddering' of amino acid residues from newly exposed N- and C-termini. As N-terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long-lived proteins.
Human aging is associated with the deterioration of long-lived proteins. Gradual cumulative modifications to the life-long proteins of the lens may ultimately be responsible for the pronounced alterations to the optical and physical properties that characterize lenses from older people. γS crystallin, a major human lens protein, is known to undergo several age-dependent changes. Using proteomic techniques, a site of deamidation involving glutamine 92 has been characterized and its time course established. The proportion of deamidation increased from birth to teen-age years and then plateaud. Deamidation at this site increased again in the eighth decade of life. There was no significant difference in the extent of deamidation between cataract and age-matched normal lenses. Gln92 is located in the linker region between the two domains, and the introduction of a negative charge at this site may alter the interaction between the two regions of the protein. Gln170, which is located in another unstructured part of γS crystallin, showed a similar deamidation profile to that of Gln92. As the other Gln residues in β-sheet regions of γS crystallin appear to remain as amides, modification of Gln92 and Gln170 thus conforms to a pattern whereby deamidation is localized to the unstructured regions of long-lived proteins.
Long-lived proteins are widespread in man, yet little is known about the processes that affect their function over time, or their role in age-related diseases. Racemization of two proteins from normal and cataract human lenses were compared with age using tryptic digestion and LC/mass spectrometry. Asp 151 in αA crystallin and Asn 76 in γS crystallin were studied. Age-dependent profiles for the two proteins from normal lenses were different. In neither protein did the modifications increase linearly with age. For αA crystallin, racemization occurred most rapidly during the first 15 years of life, with approximately half of L-Asp 151 converted to D-isoAsp, L-isoAsp, and D-Asp in a ratio of 3:1:0.5. Values then changed little. By contrast, racemization of Asn 76 in γS crystallin was slow until age 15, with isoAsp accounting for only 5%. Values remained relatively constant until age 40 when a linear increase (1%/year) took place. When cataract lenses were compared with age-matched normal lenses, there were marked differences in the time courses of the two crystallins. For αA crystallin, there was no significant difference in Asp 151 racemization between cataract and normal lenses. By contrast, in γS crystallin the degree of conversion of Asn 76 to isoAsp in cataract lenses was approximately double that of normals at every age. Modification of Asn and Asp over time may contribute to denaturation of proteins in the human lens. An accelerated rate of deamidation/racemization at selected sites in proteins, such as γS crystallin, may contribute to cataract formation.
A heat map showing relative changes in major proteins that were tightly bound to lens membranes. Four geometric regions were dissected from each of two lenses aged 20 and 52. The average abundance in the young lens for each region was compared with the same region of the older lens. Red represents an overall relative protein decrease, blue an overall increase and white no change between the two age groups. Proteins with a spectral count less than 15 were not included in the data set.
It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. Fiber cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea, and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an NH₂-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure.
The human lens nucleus is formed in utero, and from birth onwards, there appears to be no significant turnover of intracellular proteins or membrane components. Since, in adults, this region also lacks active enzymes, it offers the opportunity to examine the intrinsic stability of macromolecules under physiological conditions. Fifty seven human lenses, ranging in age from 12 to 82 years, were dissected into nucleus and cortex, and the nuclear lipids analyzed by electrospray ionization tandem mass spectrometry. In the first four decades of life, glycerophospholipids (with the exception of lysophosphatidylethanolamines) declined rapidly, such that by age 40, their content became negligible. In contrast the level of ceramides and dihydroceramides, which were undetectable prior to age 30, increased approximately 100-fold. The concentration of sphingomyelins and dihydrosphingomyelins remained unchanged over the whole life span. As a consequence of this marked alteration in composition, the properties of fiber cell membranes in the centre of young lenses are likely to be very different from those in older lenses. Interestingly, the identification of age 40 years as a time of transition in the lipid composition of the nucleus coincides with previously reported macroscopic changes in lens properties (e.g., a massive age-related increase in lens stiffness) and related pathologies such as presbyopia. The underlying reasons for the dramatic change in the lipid profile of the human lens with age are not known, but are most likely linked to the stability of some membrane lipids in a physiological environment.
A number of tissues and organs in the human body contain abundant proteins that are long-lived. This includes the heart, lung, brain, bone and connective tissues. It is proposed that the accumulation of modifications to such long-lived proteins over a period of decades alters the properties of the organs and tissues in which they reside. Such insidious processes may affect human health, fitness and ultimately may limit our lifespan. The human lens, which contains proteins that do not turnover, is used to illustrate the impact of these gradual deleterious modifications. On the basis of data derived from the lens, it is postulated that the intrinsic instability of certain amino acid residues, which leads to truncation, racemisation and deamidation, is primarily responsible for the age-related deterioration of such proteins. Since these post-translational modifications accumulate over a period of many years, they can only be studied using organisms that have lifespans measured in decades. One conclusion is that there may be important aspects of human aging that can be studied only using long-lived animals.
Long-lived proteins are found at several sites in the body and they undergo numerous changes as a result of prolonged exposure to physiological conditions. Truncation is a common modification and many cleavages appear to be non-enzymatic, however little is known about the processes involved. In this study we demonstrate, using synthetic peptides that incorporate the sequence of a protein that is known to cleave in older lenses, that truncation on the N-terminal side of serine residues can occur at neutral pH. A mechanism that incorporates an N,O-acyl shift, which is analogous to intein cleavage, is proposed. Such cleavages may explain the origin of abundant peptides derived from crystallins in aged human lenses. KeywordsOld proteins–Age–Hydrolysis–Posttranslational modification–Human lens
ASTRACT: Several amino acids were found to undergo progressive age-dependent racemisation in the lifelong proteins of normal human lenses. The two most highly racemised were Ser and Asx. By age 70, 4.5% of all Ser residues had been racemised, along with >9% of Asx residues. Such a high level of inversion, equivalent to between 2 and 3 D- amino acids per polypeptide chain, is likely to induce significant denaturation of the crystallins in aged lenses. Thr, Glx and Phe underwent age-dependent racemisation to a smaller degree. In model experiments, D- amino acid content could be increased simply by exposing intact lenses to elevated temperature. In cataract lenses, the extent of racemisation of Ser, Asx and Thr residues was significantly greater than for age-matched normal lenses. This was true, even for cataract lenses removed from patients at the earliest ages where age-related cataract is observed clinically. Racemisation of amino acids in crystallins may arise due to prolonged exposure of these proteins to ocular temperatures and increased levels of racemisation may play a significant role in the opacification of human lenses.
The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.
The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.
Not only are human lenses different in many ways from those of non-primates, they also undergo dramatic changes with age. These age-dependent alterations lead to perturbations in the properties of older lenses, and ultimately to disturbances in visual function, which typically become apparent at middle age. Recent data suggest that many, if not all, of these age-dependent features can be traced to the lack of macromolecular turnover in the lens and to the inexorable modifications to proteins and membrane components over a period of decades. Exposure of lenses to heat can reproduce many of these alterations, suggesting that long-term incubation at body temperature may be an important factor in aging the human lens. Two conclusions flow from this. Firstly, the human lens may be an ideal tissue for studying macromolecular aging in man. Secondly, it will be extremely challenging to examine the origin of human age-related conditions, such as presbyopia and nuclear cataract, using traditional laboratory animals. Characterising the unfolding and decomposition of long-lived macromolecules appears to provide the key to understanding the two most common human lens disorders: presbyopia and age-related nuclear cataract.
Little is known about the rate of denaturation of proteins within the human body. To monitor this decline, human eye lenses were dissected into discrete regions that were formed at different stages of life and assayed for activity of lactate dehydrogenase (LDH) and a particularly stable enzyme, glutathione reductase (GR). Activity was highest for both enzymes in the most recently synthesized outer part of the lens, decreased further into the lens, and, for LDH, was barely detectable in nuclear regions that consist of proteins that were synthesized in utero. For LDH, 95% of total lens activity was found in the outer half of the adult lens at all ages. Activity was unchanged in the outermost part of the lens as a function of age, suggesting that the ability of humans to synthesize the two enzymes is not impaired, even up to the tenth decade. After age of 40, LDH activity declined steadily in the interior of the lens at the rate of 8.3% per decade. GR activity diminished more slowly, and western blotting indicated that both denaturation of the enzyme and truncation were responsible. These data support the view that few, if any, metabolic pathways remain in the center of older lenses. Exposure of the enzymes to physiological pH and temperature over a period of decades is presumably sufficient to cause denaturation. The center of older human lenses is a unique environment in which the accumulation of untoward posttranslational modifications to proteins can be studied in the absence of significant enzymatic amelioration.
To examine the physical properties of human lens cell membranes as a function of age. The environment of the phospholipid head groups in fiber cell membranes from human lenses, aged 22 to 83 years, was assessed with Laurdan and two-photon confocal microscopy. The effect of mild thermal stress on head group order was studied with lens pairs in which one intact lens was incubated at 50 °C. Dihydrosphingomyelin vesicles were preloaded with Laurdan, α-, β-, or γ-crystallin was added, and surface fluidity was determined. The membrane head group environment became more fluid with age as indicated by increased water penetration. Furthermore, these changes could be replicated simply by exposing intact human lenses to mild thermal stress; conditions which decreased the concentration of soluble α- and β-crystallins. Vesicle binding experiments showed that α- and β-, but not γ-, crystallins markedly affected head group order. The physical properties of cell membranes in the lens nucleus change substantially with age, and α- and β-crystallins may modulate this effect. β-Crystallins may therefore play a role in lens cells, and cells of other tissues, apart from being simple structural proteins. Age-dependent loss of these crystallins may affect membrane integrity and contribute to the dysfunction of lenses in older people.
Phospholipids are a major component of lens fiber cells and influence the activity of membrane proteins. Previous investigations of fatty acid uptake by the lens are limited. The purpose of the present study was thus to determine whether exogenous fatty acids could be taken up by the rat lens and incorporated into molecular phospholipids. Lenses were incubated with fluorescently labeled palmitic acid and then analyzed by confocal microscopy. Concurrently, lenses incubated with either fluorescently labeled palmitic acid or the more physiologically relevant (13)C(18)-oleic acid were sectioned into nuclear and cortical regions and analyzed by highly sensitive and structurally selective electrospray ionization tandem mass spectrometry techniques. The detection of fluorescently labeled palmitic acid, even after 16 hours of incubation, was limited to approximately the outer 25% to 30% of the rat lens. Mass spectrometry also revealed the presence of free (13)C(18)-oleic acid in the cortex but not the nucleus. No evidence could be found for incorporation of fluorescently labeled palmitic acid into phospholipids; however, a low level of (13)C(18)-oleic acid incorporation into phosphatidylethanolamine (PE), specifically PE (PE 16:0/(13)C(18) 18:1) was detected in the lens cortex after 16 hours. These data demonstrate that uptake of exogenous (e.g., dietary fatty acids) by the lens and their incorporation into phospholipids is minimal, most likely occurring only during de novo synthesis in the outermost region of the lens. This finding adds support to the hypothesis that once synthesized there is no active remodeling or turnover of fiber cell phospholipids.
The lipid composition of the human lens is distinct from most other tissues in that it is high in dihydrosphingomyelin and the most abundant glycerophospholipids in the lens are unusual 1-O-alkyl-ether linked phosphatidylethanolamines and phosphatidylserines. In this study, desorption electrospray ionization (DESI) mass spectrometry-imaging was used to determine the distribution of these lipids in the human lens along with other lipids including, ceramides, ceramide-1-phosphates, and lyso 1-O-alkyl ethers. To achieve this, 25 μm lens slices were mounted onto glass slides and analyzed using a linear ion-trap mass spectrometer equipped with a custom-built, 2-D automated DESI source. In contrast to other tissues that have been previously analyzed by DESI, the presence of a strong acid in the spray solvent was required to desorb lipids directly from lens tissue. Distinctive distributions were observed for [M + H](+) ions arising from each lipid class. Of particular interest were ionized 1-O-alkyl phosphatidylethanolamines and phosphatidylserines, PE (18:1e/18:1), and PS (18:1e/18:1), which were found in a thin ring in the outermost region of the lens. This distribution was confirmed by quantitative analysis of lenses that were sectioned into four distinct regions (outer, barrier, inner, and core), extracted and analyzed by electrospray ionization tandem mass spectrometry. DESI-imaging also revealed a complementary distribution for the structurally-related lyso 1-O-alkyl phosphatidylethanolamine, LPE (18:1e), which was localized closer to the centre of the lens. The data obtained in this study indicate that DESI-imaging is a powerful tool for determining the spatial distribution of human lens lipids.
The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens sections were initially analyzed by MALDI mass spectrometry imaging. A distinct annular distribution of the dihydrosphingomyelin, DHSM (d18:0/16:0), in the barrier region was observed in 64- and 70-year-old lenses but not in a 23-year-old lens. An increase in the dihydroceramide, DHCer (d18:0/16:0), in the lens nucleus was also observed in the older lenses. These findings were supported by ESI mass spectrometry analysis of lipid extracts from lenses dissected into outer, barrier, and nuclear regions. A subsequent analysis of 18 lenses ages 20-72 years revealed that sphingomyelin levels increased with age in the barrier region until reaching a plateau at approximately 40 years of age. Such changes in lipid composition will have a significant impact on the physical properties of the fiber cell membranes and may be associated with the formation of a barrier.
Deamidation is a common posttranslational modification in human lens crystallins and may be a key factor in the age-related denaturation of such lifelong proteins. The aim of this study was to identify the sites of deamidation in older lenses. High-performance liquid chromatography/mass spectrometry of tryptic digests was used to identify sites of deamidation in the major human lens crystallins. Older normal and age-matched cataractous lenses were compared with fetal lenses. Approximately equal numbers of glutamine and asparagine residues were deamidated in older lenses; however, the extent of deamidation of Asn was three times greater than that of Gln (Asn, 22.6% +/- 3.6%; Gln, 6.6% +/- 1.3%). Individual crystallins differed markedly in their extent of deamidation, and deamidated residues were typically localized within discrete regions of the polypeptides. A large percentage (42%) of the sites of deamidation were characterized by the presence of a basic amino acid one residue removed from the original Gln or Asn. At nine such sites, the extent of Asn deamidation averaged 50% in aged lenses. There were few differences in deamidation between crystallins of aged normal and nuclear cataractous lenses. Equal numbers of Asn and Gln residues are deamidated in crystallins from aged normal and cataractous lenses. Deamidation of Asn/Gln in lifelong proteins, such as those in the lens, may be governed to a significant degree by base-catalyzed processes.
In a study first reported more than 25 years ago, the investigators detected electrical currents outside the lens that depended on the activity of lens ion transporters.1 Based on these ionic currents and measures of lens electrical impedance, a model was developed that postulated the existence of fluid flow through the lens fiber cell cytoplasm (hereafter referred to as the fluid circulation model [FCM]).2,3 Although the FCM is more than 20 years old, direct evidence to support it is still lacking. Our analysis suggests that the FCM, as previously described, has conceptual shortcomings and does not appear to be consistent with published data. Contrary to the postulates of the FCM, it is unlikely that fluid circulation through the fiber cell cytoplasm is needed to maintain the metabolism of fiber cells that have degraded their organelles. We suggest that, if water did flow through the cytoplasm of fiber cells from the lens center to its periphery in the manner outlined in the FCM, it would be harmful to lens transparency. We accept the initial observation that ion currents, generated by active transport, flow around and through the lens. However, we argue that these ion movements do not create a microcirculatory system in which water flows from the lens center to its periphery.
With age, large amounts of crystallins become associated with fiber cell membranes in the human lens nucleus, and it has been proposed that this binding of protein may lead to the obstruction of membrane pores and the onset of a barrier to diffusion. This study focused on membrane binding within the barrier region and the outermost lens cortex. Human lenses across the age range were used, and the interaction of crystallins with membranes was examined using sucrose density gradient centrifugation, two-dimensional gel electrophoresis, and amine-reactive isobaric tagging technology. Lipids were quantified using shotgun lipidemics. Binding of proteins to cell membranes in the barrier region was found to be different from that in the lens nucleus because in the barrier and outer cortical regions, only one high-density band formed. Most of the membrane-associated protein in this high-density band was α-crystallin. Mild thermal stress of intact young lenses led to pronounced membrane binding of proteins and yielded a sucrose density pattern in all lens regions that appeared to be identical with that from older lenses. α-Crystallin is the major protein that binds to cell membranes in the barrier region of lenses after middle age. Exposure of young human lenses to mild thermal stress results in large-scale binding of α-crystallin to cell membranes. The density gradient profiles of such heated lenses appear to be indistinguishable from those of older normal lenses. The data support the hypothesis that temperature may be a factor responsible for age-related changes to the human lens.
There are a number of sites in the body where proteins are present for decades and sometimes for all of our lives. Over a period of many years, such proteins are subject to two types of modifications. The first results from the intrinsic instability of certain amino acid residues and leads to deamidation, racemization, and truncation. The second type can be traced to relentless covalent modification of such ancient proteins by reactive biochemicals produced during cellular metabolism.The accumulation of both types of posttranslational modifications over time may have important consequences for the properties of tissues that contain such proteins. It is proposed that the age-related decline in function of organs such as the eye, heart, brain, and lung, as well as skeletal components, comes about, in part, from the posttranslational modification of these long-lived proteins. Examples are provided in which this may be an important factor in the etiology of age-related conditions. As the properties of these proteins alter inexorably over time, the molecular changes contribute to a gradual decline in the function of individual organs and also tissues such as joints. This cumulative degeneration of old proteins at multiple sites in the body may also constrain the ultimate life span of the individual. The human lens may be particularly useful for discovering which reactive metabolites in the body are of most importance for posttranslational modification of long-lived proteins.
To characterize age-related changes to proteins in the center of the human lens. Human lenses of different ages were dissected using trephines. Sucrose density gradient centrifugation was used to separate the proteins from two defined nuclear regions. Densitometry of Coomassie-stained protein bands was compared with lipid analysis with the use of mass spectrometry. A profound change in the density gradient profiles of lenses occurred at approximately age 40. As soluble crystallins decreased, four higher density bands appeared that were absent in younger lenses. These four bands contained crystallins, as well as membrane lipids, and appear to have resulted from the interaction of denatured crystallins with fiber cell membranes. Changes in lens proteins and membranes can be detected in each decade of life; however, major changes to the lens crystallins of the nucleus take place between age 40 and 50, after the loss of free soluble alpha crystallin. These alterations are consistent with large-scale binding of crystallin aggregates to fiber cell membranes after middle age.
The human lens is ideal for the study of macromolecular aging because cells in the centre, along with their constituent proteins, are present for our entire lives. We examined the major membrane protein, aquaporin 0 (AQP0), in regions of the lens formed at different times during our lifespan, to determine if similar changes could be detected and if they were progressive. Membrane fractions from three concentric lens regions were examined by SDS-PAGE coupled with densitometry, and Western blotting, to assess the time course of truncation. The overall extent of modification was also examined by MALDI mass spectrometry of the undigested proteins. In all regions, AQP0 became progressively more truncated, specifically by the loss of a 2kDa intracellular C-terminal peptide. The proteolysis increased steadily in all regions such that half of the AQP0 in the barrier region (that part of the lens formed immediately after birth) had been cleaved by age 40-50. MALDI mass spectrometry revealed that in all regions, AQP0 not only was shortened, it also became progressively more heterogeneous with age. Since the lens interior is devoid of active enzymes, it is very likely that the cleavage of AQP0 is chemically induced. We speculate that the loss of this C-terminal peptide 'spacer' may allow occlusion of AQP0 pores on the cytoplasmic face of the fibre cell membranes. Once a significant proportion of AQP0 has been cleaved, this occlusion may contribute to the formation of the lens permeability barrier that develops at middle age.
Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophosphoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our knowledge have not previously been observed in human tissue. Isomeric 1-O-alk-1'-enyl ethers were absent or in low abundance. Examination of the double bond position within the phospholipids using OzID revealed that several positional isomers were present, including sites of unsaturation at the n-9, n-7, and even n-5 positions. Tandem CID/OzID experiments revealed a preference for double bonds in the n-7 position of 1-O-ether linked chains, while n-9 double bonds predominated in the ester-linked fatty acids [e.g., GPEtn(11Z-18:1e/9Z-18:1) and GPSer(11Z-18:1e/9Z-18:1)]. Different combinations of these double bond positional isomers within chains at the sn-1 and sn-2 positions point to a remarkable molecular diversity of ether-lipids within the human lens.
All people will be presbyopic by age 50, and we now understand something of the basis for this condition. It turns out to be a direct consequence of two features; first the design of the transparent lens and the way it must change shape to enable focussing by the human eye, and second the instability of proteins over a very long time period. The incremental changes that take place in the lens to render the central region inflexible by middle age and, as a consequence the person presbyopic, may also promote the subsequent development of cataract. Based on the most recent data, heat-induced denaturation of proteins in the lens appears to be a worthy topic for future investigation. Understanding such processes may allow us to glimpse the origin both of presbyopia and age-related nuclear cataract.
The most complete proteome of human lenses has been compiled using 2-D LC-MS/MS analysis of foetal, aged normal and advanced nuclear cataract lenses. A total of 231 proteins were identified across all lens groups, including 112 proteins that have not been reported previously. Proteins were grouped according to their PANTHER molecular function classification in order to facilitate comparisons. Previously unreported N-terminal acetylation was detected in a number of proteins, with the majority being associated with the prior removal of a methionine residue. This pattern of proteolysis may indicate that methionine aminopeptidase activity is present in human lenses. Acetylation is likely to aid in the stability of proteins that are present in the lens for many decades. Protein sequences were also used to interrogate the three human lens cDNA libraries publicly available. Surprisingly, 84 proteins we identified were not present in the cDNA libraries.
Loss of protein thiols is a key feature associated with the onset of age-related nuclear cataract (ARNC), however, little is known about the specific sites of oxidation of the crystallins. We investigated cysteine residues in ARNC lenses and compared them with age-matched normal lenses. Proteomic analysis of tryptic digests revealed ten cysteine residues in older normal lenses that showed no significant oxidation compared to foetal counterparts (Cys 170 in betaA1/3-crystallin, Cys 32 in betaA4-crystallin, Cys 79 in betaB1-crystallin, Cys 22, Cys 78/79, C153 in gammaC-crystallin and Cys 22, Cys 24 and Cys 26 in gammaS-crystallin). Although these thiols were not oxidised in normal lenses past the 6th decade, they were present largely as disulphides in the ARNC lenses. By contrast, two cysteine residues, Cys 41 in gammaC-crystallin and Cys 18 in gammaD-crystallin, were not oxidised, even in advanced ARNC lenses. These cysteines are buried deep within the protein and any unfolding associated with cataract must be insufficient to expose them to the oxidative environment present in the centre of advanced ARNC lenses. The vast majority of the loss of protein thiol observed in such lenses is due to disulphide bond formation.
Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague–Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens.
To analyze free and total water in human normal and cataractous lenses. Thermogravimetric analysis was used to determine total water, and differential scanning calorimetry was used for free water. In normal human lenses, the total water content of the nucleus remained unchanged with age, but the state of the water altered. The ratio of free to bound water increased steadily throughout adult life. In a 20-year-old person, there was approximately one bound water molecule for each free water molecule in the lens center, whereas in a 70- to 80-year-old person, there were two free water molecules for each bound water molecule. This conversion of bound to free water does not appear to be simply a consequence of the aggregation of soluble crystallins into high molecular weight aggregates because studies with intact pig lenses, in which such processes were facilitated by heat, did not show similar changes. The region of the lens in which the barrier to diffusion develops at middle age corresponds to a transition zone in which the protein concentration is intermediate between that of the cortex and the nucleus. In cataractous lenses, the free-to-bound water ratio was not significantly different from that of age-matched normal lenses; however, total water content in the center of advanced nuclear cataractous lenses was slightly lower than in normal lenses. As the human lens ages, bound water is progressively changed to free water. Advanced nuclear cataract may be associated with lower total hydration of the lens nucleus.
Electrospray ionisation tandem mass spectrometry has allowed the unambiguous identification and quantification of individual lens phospholipids in human and six animal models. Using this approach ca. 100 unique phospholipids have been characterised. Parallel analysis of the same lens extracts by a novel direct-insertion electron-ionization technique found the cholesterol content of human lenses to be significantly higher (ca. 6 times) than lenses from the other animals. The most abundant phospholipids in all the lenses examined were choline-containing phospholipids. In rat, mouse, sheep, cow, pig and chicken, these were present largely as phosphatidylcholines, in contrast 66% of the total phospholipid in Homo sapiens was sphingomyelin, with the most abundant being dihydrosphingomyelins, in particular SM(d18:0/16:0) and SM(d18:0/24:1). The abundant glycerophospholipids within human lenses were found to be predominantly phosphatidylethanolamines and phosphatidylserines with surprisingly high concentrations of ether-linked alkyl chains identified in both classes. This study is the first to identify the phospholipid class (head-group) and assign the constituent fatty acid(s) for each lipid molecule and to quantify individual lens phospholipids using internal standards. These data clearly indicate marked differences in the membrane lipid composition of the human lens compared to commonly used animal models and thus predict a significant variation in the membrane properties of human lens fibre cells compared to those of other animals.
Surgical evidence suggests that nuclear cataract lenses are generally harder than normal lenses. We examined this quantitatively using dynamic mechanical analysis of cataract lenses removed during surgery and compared the results with data from normal lenses. Stiffness of the lens centre was found to depend on the type of cataract and the age of the patient. Nuclear cataract lenses were generally stiffer than those extracted from patients with predominantly cortical cataract, with some in the latter group appearing not to differ significantly from age-matched normals. At age 40-50, the nuclear region of advanced nuclear cataract lenses was found to be approximately 46 times harder than that of normal lenses of the same age. By age 70-80 the stiffness of advanced nuclear cataract lenses had doubled, however, by this age, normal lenses had also increased significantly in stiffness so that the difference between cataract and normal lenses was much less pronounced, being a factor of approximately 2.5.
When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).
The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.
Presbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, alpha-crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble alpha-crystallin from the centre of the lens. These alterations in alpha-crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 degrees C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for alpha-crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of alpha-crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.
The urea-soluble proteins from the nucleus of two young, two aged, and two early-stage nuclear cataract lenses were subjected to tryptic digestion and analysis by 2D LC-MS/MS. Several novel post-translational modifications were identified. Deamidation was, by far, the most common modification. A number of differences were found in cataract compared to normal lenses, most notably an increase in the number of oxidized tryptophan residues. Semiquantitative analysis revealed that there appeared to be a trend toward increased levels of deamidation with age; however, there was no apparent increase upon the onset of nuclear cataract. This is in contrast to Trp oxidation, where an increase in the extent of modification was apparent in cataract lenses when compared to aged normal lenses. These findings suggest Trp oxidation may be involved in nuclear cataract development.
In human cataract lenses the UV filters, 3-hydroxykynurenine glucoside (3OHKG) and kynurenine (Kyn) were found to be covalently bound to proteins and the levels in the nucleus were much higher than in the cortex. The levels of the bound UV filters in cataract nuclei were much lower than those in age-matched normal lenses. 3-Hydroxykynurenine could not be detected in cataract lenses. As with normal lenses, protein-bound 3OHKG in cataract lenses was found at the highest levels followed by Kyn. Free UV filter concentrations were also markedly reduced in cataract lenses. This feature may well contribute to the lower protein-bound levels; however, there was no clear relationship between free and bound UV filter contents when individual lenses were examined. We propose that since cysteine is a major site for UV filter binding, the well-documented oxidation of protein sulfhydryl groups during the progression of nuclear cataract may account, in part, for the pronounced decrease in bound UV filters in cataract lenses.
Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.
4-(2-Aminophenyl)-4-oxobutanoic acid, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid and glutathionyl-kynurenine have been identified as novel metabolites in normal and cataractous human lenses following total synthesis and comparison with authentic human lens samples. Their structures are consistent with those derived from the major human lens UV filters kynurenine and 3-hydroxykynurenine, and it is proposed that these compounds also play a role as UV filters. These metabolites were isolated in pmol/mg levels (dry mass) in lenses. 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid and glutathionyl-kynurenine were found to be unstable at physiological pH. Other potential metabolites, glutathionyl-3-hydroxykynurenine, kynurenine yellow and 3-hydroxykynurenine yellow, were not detected in either normal or cataractous lenses.
To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total protein-bound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people.
At first sight, the lens of the eye would appear to be an ideal environment for amyloid fibril formation. The protein concentration is the highest of any tissue in the body, the proteins are very long-lived, present in slightly acidic conditions and subjected over time to extensive truncation and post-translational modification. In addition, it has been demonstrated that lens crystallins can readily be induced to form amyloid fibrils in vitro. The situation may be further exacerbated after the onset of age-related nuclear cataract, which is characterized by massive oxidation of cysteine and methionine residues, accompanied by protein unfolding. Despite this, there is as yet no evidence for amyloid fibril formation in either the aged or the cataract human lens. Paradoxically, the reason may have to do with the supramolecular ordered β-sheet array that crystallins adopt once they are packed into mature fiber cells. This extended matrix in normal lenses displays some of the classic features normally associated with amyloid, for example, staining with Congo red and thioflavine T.
UV filters protect the human lens and retina from UV light-induced damage. Here, we report the identification of a new UV filter, cysteine-l-3-hydroxykynurenine O-beta-d-glucoside, which is present in older normal human lenses. Its structure was confirmed by independent synthesis. It is likely this novel UV filter is formed in the lens by nucleophilic attack of cysteine on the unsaturated ketone derived from deamination of 3-hydroxykynurenine O-beta-d-glucoside. Quantitation studies revealed considerable variation in normal lens levels that may be traced to the marked instability of the cysteine adduct. The novel UV filter was not detected in advanced nuclear cataract lenses.
Indoleamine 2,3-dioxygenase is the first and rate limiting enzyme of the kynurenine pathway of tryptophan metabolism, has potent effects on cell proliferation and mediates antimicrobial, antitumorogenic, and immunosuppressive effects. As a potent cytotoxic effector, the mechanisms of indoleamine 2,3-dioxygenase inhibition deserve greater attention. The work presented here represents the first systematic study exploring the mechanisms by which low levels of hydrogen peroxide (10-100 microM) inhibit indoleamine 2,3-dioxygenase in vitro. Following brief peroxide exposure both enzyme inhibition and structural changes were observed. Loss of catalysis was accompanied by oxidation of several cysteine residues to sulfinic and sulfonic acids, observed by electrospray and MALDI mass spectrometry. Enzyme activity could in part be preserved in the presence of sulfhydryl containing compounds, particularly DTT and methionine. However, these structural alterations did not prevent substrate (l-tryptophan) binding. Some enzyme activity could be recovered in the presence of thioredoxin, indicating that the inhibitory effect of H(2)O(2) is at least partially reversible in vitro. We present evidence that cysteine oxidation represents one mechanism of indoleamine 2,3-dioxygenase inhibition.
Major UV filters have been identified in the lens of the 13 lined ground squirrel (Spermophilus tridecemlineatus). These were found to be N-acetyl-3-hydroxykynurenine and N-acetyl-kynurenine, in addition to a small quantity of 3-hydroxykynurenine. The level of N-acetyl-3-hydroxykynurenine measured in the ground squirrel lens, 8.2mM, is approximately 11 times the concentration of 3-hyroxykynurenine glucoside reported previously for the human lens. Two additional UV filters of related structure were also present; however, their structures are still under investigation. HPLC elution profiles indicated that the ground squirrel lens cortex and nucleus contained comparable amounts of alpha-, beta(H)-, beta(L)-, and gamma-crystallins. Levels of GSH in the cortex and nucleus were 12.4 and 7.4mM, respectively. Such high concentrations of GSH may act to inhibit oxidation of the 3-hydroxykynurenine and N-acetyl-3-hydroxykynurenine. N-Acetylated kynurenines are less labile than those with free alpha-amino groups since N-acetyl-alpha-amino groups do not undergo spontaneous deamination. This modification thus stabilises the squirrel UV filters. In addition, because deamination is prevented, the decomposition products will not be involved in binding to lens proteins. Because of the similarity of the UV filters present in the ground squirrel to those in man, this species may be a suitable animal model for investigating the effects of UV radiation on cataract, and other ocular diseases, thought to involve exposure to light.
Age-related nuclear (ARN) cataract is a major cause of world blindness. With the onset of ARN cataract, the normally transparent and colorless lens becomes opaque and can take on colors ranging from orange, brown, and even black. The molecular basis for this remarkable transformation is unknown. ARN cataract is also characterized by extensive oxidation, insolubilization, and cross-linking of polypeptides, particularly in the nucleus of the lens. It has been postulated that 3-hydroxykynurenine (3OHKyn) may be involved in these changes. This endogenous tryptophan metabolite is readily oxidized and is involved in the tanning of moth cocoons and the formation of pigments in the eyes of butterflies. 3OHKyn is a component of our primate-specific UV-filter pathway, and the brownish hue of ARN cataract lenses is also unique to humans. Because numerous colored compounds can be produced by autoxidation of 3OHKyn, this process could provide an explanation for the variety of lens colors and other changes seen in ARN cataract. For such a theory to be tenable, it needs to be demonstrated that 3OHKyn is bound to proteins in the human lens. Here, we show that all normal lenses older than 50 have 3OHKyn covalently attached to the nuclear proteins, most likely via cysteine residues. If indeed 3OHKyn is implicated in ARN cataract, a reduction in the levels that are bound in cataract, compared to normal lenses, would be expected. In agreement with this hypothesis, no bound 3OHKyn could be detected in proteins isolated from ARN cataract lenses.
The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.
A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.
Presbyopia, the inability to accommodate, affects almost everyone at middle age. Recently, it has been shown that there is a massive increase in the stiffness(1) of the lens with age and, since the shape of the lens must change during accommodation, this could provide an explanation for presbyopia. In this review, we propose that presbyopia may be the earliest observable symptom of age-related nuclear (ARN) cataract. ARN cataract is a major cause of world blindness. The genesis of ARN cataract can be traced to the onset of a barrier within the lens at middle age. This barrier restricts the ability of small molecules, such as antioxidants, to penetrate into the centre of the lens leaving the proteins in this region susceptible to oxidation and post-translational modification. Major protein oxidation and colouration are the hallmarks of ARN cataract. We postulate that the onset of the barrier, and the hardening of the nucleus, are intimately linked. Specifically, we propose that progressive age-dependent hardening of the lens nucleus may be responsible for both presbyopia and ARN cataract.
The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: approximately 70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and approximately 30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 188.8.131.52), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 184.108.40.206) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -- with H2O2 as co-substrate -- indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.
Age is by far the biggest risk factor for cataract, and it is sometimes assumed that cataract is simply an amplification of this aging process. This appears not to be the case, since the lens changes associated with aging and cataract are distinct. Oxidation is the hallmark of age-related nuclear (ARN) cataract. Loss of protein sulfhydryl groups, and the oxidation of methionine residues, are progressive and increase as the cataract worsens until >90% of cysteine and half the methionine residues are oxidised in the most advanced form.
1H-NMR spectroscopic studies of bovine eye lens β-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three β-crystallin aggregates are dominated by resonances from the βB2 subunit, particularly from the N- and C-terminal extensions of this subunit. Resonances from other β subunits, which all have terminal extensions, are, in general, absent from spectra of the β-crystallin aggregates. Therefore, the βB2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other β-crystallin subunits. Furthermore, resonances arising from the C-terminal extension of βB2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal extension of βB2 and the hydrophobic βB1 subunit, which is only found in the octomer, is discussed. At higher temperatures (45°C) in the octomer, partial exposure of the C-terminal extension of βB2 occurs indicating that the octomer may be starting to break up into smaller aggregates.
1H-NMR spectroscopic studies of a 46-kDa homodimer, βB2-crystallin, from bovine eye lens are presented. βB2-crystallin has terminal extensions extending from globular N- and C-terminal domains that are well resolved in the NMR spectra, whereas, in the main, resonances from the bulk of the protein are not observed. Using two-dimensional NMR methods on βB2-crystallin, its synthesised terminal extensions and a proteolysed sample of βB2-crystallin with a portion of its C-terminus removed, it was possible to assign resonances to most of the amino acids in the terminal extensions. One-dimensional experiments at various pH values provided H-2 chemical shifts for the three terminal extension histidines from which their pKa values were measured. It is concluded that the terminal extensions appear to be of little ordered conformation, are accessible to solvent and flex freely from the main body of the protein. The results of the NMR spectroscopic studies of βB2-crystallin are in excellent agreement with those for the X-ray crystal structure [Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P. F., Mahadevan, D., Blundell, T. L. & Slingsby, C. (1990) Nature 347, 776–780]. No change in the spectrum of βB2-crystallin was observed in the presence of calcium, suggesting that the termini are not involved in calcium binding.
To determine the stiffness of different regions of human lenses as a function of age, and to correlate the biophysical measurements in the lens center with nuclear water content. A custom made probe fitted to a dynamic mechanical analyzer was employed to measure stiffness values at 1 mm increments across equatorial sections of individual human lenses. Thermogravimetric analysis was used to determine the percentage water content in the nuclei of human lenses. There was a pronounced increase in lens stiffness over the age range from 14 to 78. In the nucleus, stiffness values varied almost 1,000 fold over this age range, with the largest change observed in lenses between the ages of 20 to 60. Nuclear stiffness values increased on average by a factor of 450. By contrast, in the cortex the average increase in stiffness was approximately 20 fold over this same time period. In lenses younger than age 30, the nucleus was found to be softer than the cortex. This was true for all six lenses examined. In contrast all lenses older than 30 were characterized by having nuclear values higher than those of the cortex. In lenses over the age of 50, the lens nucleus was typically an order of magnitude more rigid than that of the cortex. The crossover age, when the cortical and nuclear stiffness values were similar, was in the 30s. There was no significant change in the water content of the human lens nucleus from age 13 to age 82. There is a marked increase in the stiffness of the human lens with age. This is most pronounced in the nucleus. Since in vivo data indicate that the nucleus must change shape significantly during accommodation, it is highly likely that these measured changes in physical properties will markedly diminish the ability of the lens to accommodate, and thus may be a major contributing factor to presbyopia. Since there was no measurable difference in the water contents of the nuclear regions of the lenses, this marked increase in stiffness is not due to compaction of the lens nucleus.
To determine the levels of free UV filters and selected amino acids in cataract lenses compared with normal lenses. Indian cataract lenses (n=39) and normal lenses (n=6) were examined by HPLC to quantify levels of UV filter compounds, the UV filter precursor amino acid tryptophan (Trp), as well as tyrosine (Tyr) and uric acid. The levels of the two major primate UV filters, 3-hydroxykynurenine glucoside (3OHKG) and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG), in cataract lenses were markedly decreased compared with levels in normal lenses. By contrast, the levels of Trp were greatly increased. Mean Trp concentrations were an order of magnitude higher than in normal lenses, with 86% of dark-colored cataract lens nuclei having Trp concentrations greater than the mean level in the normal lenses. The concentrations of Tyr were also higher in cataract lenses. The levels of Kyn, however, were unchanged, and the uric acid levels were substantially lower. The levels of the free UV filter compounds 3OHKG and AHBG, and also of Trp, Tyr, and uric acid were different in cataract lenses compared to normal lenses. These data suggest that the metabolism of a large proportion of patients with cataract may be substantially different than in persons with normal lenses. Although the mechanism of such metabolic defects are unknown, the authors speculate that an amino acid transporter system may be upregulated in patients with cataract. Because kynurenine levels in cataract were not significantly different from those of normal lenses, there may be a defect in the lenticular UV filter pathway at one, or both, of the steps that convert kynurenine to 3OHKG.
Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.
The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases. Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO). Significant formation of inclusion bodies was noted at the growth temperature of 37 degrees C, with reduced formation at 30 degrees C. The addition of the natural biosynthetic precursor of protoporphrin IX, delta-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 degrees C and purification by nickel-agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 micromol of kynurenine/h/mg of protein (both identical to native human IDO). The protein was homogeneous in terms of electrophoretic analysis. Yields of soluble protein (3-5 mg/L of bacterial culture) and heme content are greater than previously reported.
Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long-term preservation of lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (QO2) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors on (QO2) and partial pressure of oxygen (PO2). The distribution of mitochondria was mapped in living lenses by 2-photon microscopy. We found that a steep gradient of PO2 was maintained within the tissue, leading to PO2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens; however, PO2 gradients extended beyond the boundaries of the mitochondria-containing cell layer, indicating the presence of non-mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion-consumption model. Typical values were 3 x 10(-5) cm(2) s(-1) for the effective diffusion coefficient and a 5 min time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibres (DF) that contained mitochondria and mature fibres (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease in (QO2) and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen-rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of postsurgical nuclear cataract.
Posttranslational modification by UV filters is a key event in human lenses that appears to be largely responsible for normal age-dependent yellowing. It has been proposed that subsequent reactions of these covalently bound UV filters may also be involved in the genesis of age-related nuclear cataract. To examine this hypothesis, the levels of kynurenine-lysine and kynurenine-histidine were measured in both normal and cataractous human lenses. Proteins isolated from the nuclei of normal lenses and lenses with and types I to IV nuclear cataract were hydrolyzed in 6 M HCl, and the levels of kynurenine-lysine and kynurenine-histidine were determined by HPLC. The content of kynurenine-lysine and kynurenine-histidine decreased substantially with the progression of age-related nuclear cataract. On average, levels of both kynurenine adducts were four times lower in advanced cataract (type IV) than in normal lenses. Simple autoxidation of the derivatives did not appear to be responsible for this decrease, because incubation in the presence of oxygen or H(2)O(2) did not affect adduct stability. Although protein-bound kynurenine accumulates over time in normal lenses, the levels attached to the proteins decrease significantly with the progression of age-related nuclear cataract. This finding suggests that in cataract there is a breakdown of the protein-bound adducts. Such further reactions of bound UV filters may contribute to the etiology of age-related nuclear cataract.
UV light has often been investigated as a risk factor for the most common cause of blindness, human age-related cataract. One mechanism whereby UV light could induce cataract is via the action of photosensitisers. In this regard, xanthurenic acid has recently been highlighted since it has been reported to be present in the human lens and, in model studies, it markedly enhances the photo-oxidation of proteins by wavelengths of light that penetrate the cornea. In this study we used HPLC and mass spectrometry to examine whether xanthurenic acid is indeed present in human lenses and, if so, the effect of age on its lenticular concentration. Xanthurenic acid could be formed artefactually by incubation of 3-hydroxykynurenine (3OHKyn) yellow, a known autoxidation product of the lenticular UV filter, 3OHKyn, in the presence of air and light, however, it could not be detected in any human lenses studied. Therefore, it appears unlikely that xanthurenic acid plays a role in lens aging or human cataract.
Age-related cataract is the leading cause of world blindness. Until recently, the biochemical mechanisms that result in human cataract formation have remained a mystery. In the case of nuclear cataract, it is becoming apparent that changes that take place within the lens at middle age may be ultimately responsible. The centre of the lens contains proteins that were synthesised prior to birth and while these crystallins are remarkably stable, it appears that an antioxidant environment may be necessary in order for them to remain soluble and for lens transparency. Once an internal barrier to the movement of small molecules, such as antioxidants, develops in the normal lens at middle age, the long-lived proteins in the lens centre become susceptible both to covalent attachment of reactive molecules, such as UV filters, and to oxidation. These processes of protein modification may, over time, lead inevitably to lens opacification and cataract.
Monkey lenses were incubated with 35S-L-cysteine for various times and the movement of label within the lens followed by autoradiography. Cysteine appeared to enter primarily at the germinative region of the lens. No evidence was found for major transport through either the anterior or posterior faces of the lens. The movement of cysteine within different parts of the lens was followed over time. The data suggest that, for cysteine, the major pathway for transport within the lens involves entry at the germinative region followed by movement along the fibre cells. The data were consistent with orthogonal movement across the fibres in the equatorial plane but little or no movement across the fibres at the anterior pole or posterior faces of the lens. Such a scenario is in accord with the distribution of connexons, indicating that this pattern of entry may also be observed for other small molecules. The finding of high permeability at the lens germinative region is in accord with the anatomy of the eye, since this is the lens surface in contact with the posterior chamber. Thus, cysteine secreted by the ciliary body into the aqueous humor would come into contact initially with the region of the lens best able to absorb this amino acid. Although this aspect was not addressed in the current study, the same phenomenon may also be observed with other lens nutrients.
L-Tryptophan is the least abundant essential amino acid in humans. Indoleamine 2,3-dioxgyenase (IDO) is a cytosolic heme protein which, together with the hepatic enzyme tryptophan 2,3-dioxygenase, catalyzes the first and rate-limiting step in the major pathway of tryptophan metabolism, the kynurenine pathway. The physiological role of IDO is not fully understood but is of great interest, because IDO is widely distributed in human tissues, can be up-regulated via cytokines such as interferon-gamma, and can thereby modulate the levels of tryptophan, which is vital for cell growth. To identify which amino acid residues are important in substrate or heme binding in IDO, site-directed mutagenesis of conserved residues in the IDO gene was undertaken. Because it had been proposed that a histidine residue might be the proximal heme ligand in IDO, mutation to alanine of the three highly conserved histidines His16, His303, and His346 was conducted. Of these, only His346 was shown to be essential for heme binding, indicating that this histidine residue may be the proximal ligand and suggesting that neither His303 nor His16 act as the proximal ligand. Site-directed mutagenesis of Asp274 also compromised the ability of IDO to bind heme. This observation indicates that Asp274 may coordinate to heme directly as the distal ligand or is essential in maintaining the conformation of the heme pocket.
Tryptophan-derived UV filters (kynurenine and 3-hydroxylkynurenine glucoside) have recently been shown to bind to human lens proteins. These UV filter adducts increase in amount with age and appear to be mainly responsible for the yellowing of the lens in man. On the basis of research performed in other tissues, it has been assumed that indoleamine 2,3-dioxygenase (IDO) may be the first and probably rate-limiting enzyme in UV filter biosynthesis. In this study, 25 human lenses were examined by a reliable and sensitive assay method with a monoclonal antibody specific for IDO. IDO activity was detected in all lenses ranging from 26 to 80 years, and there was no clear relationship of IDO activity with age. The mean activity was 0.85 + 0.49 nmol of kynurenine/h/lens. The level in the iris/ciliary body was negligible (<0.05 nmol of kynurenine/h). The lens IDO activity is consistent with UV filter turnover values obtained previously. These findings indicate that IDO is the first enzyme in the UV filter pathway and that UV filter biosynthesis is active even in aged lenses. Yellowing of the aged lens may therefore be preventable by drug-induced suppression of IDO activity.
Indoleamine 2,3-dioxygenase (IDO), the first enzyme of the kynurenine pathway of tryptophan metabolism, has been implicated in numerous disease states. Site-directed mutagenesis was undertaken using the expression plasmid pQE9-IDO, to incorporate a stop codon at lysine 389. This produced a C-terminally truncated protein, similar to that previously reported as a minor product during purification of the recombinant protein. Initial studies here, show that the Lys389 mutant retains enzymatic activity. Purification of this truncated protein, which lacks a presumably mobile terminal region, may increase the likelihood of crystallisation of human IDO.
... However, in the literature, a few studies on trypto- phan metabolism progression with the age are reported. Truscott and Elderfield  found that serum kynurenine, the product of the action of TDO and IDO enzymes, increased in subjects from the 20- 30-year-old group to the 70-80-year-old group. In contrast, Bova et al.  reported that kynurenine concentration decreased linearly as a function of age in human lenses. ...
... αB-crystallin/sHSP has been implicated as a key player in defense of cells against oxidative stress damage. It has been shown to defend cells against oxidative stress-induced loss of protein function [31,32], disruption of cytoskeletal assembly [33,34] and apoptotic induction [28,353637383940414243 . It has been shown to preserve cellular viability upon oxidative stress exposure in multiple cells and tissues including glial cells , heart , retina  and kidney . ...
... Sample purification and desulfonation using Helix pomatia sulfatase was performed according to Minchinton, Sang, Burke and Truscott (1982), Hennig, Verkerk, Bonnema and Dekker (2012), and Kusznierewicz, Iori, Piekarska, Namiesnik and Bartoszek (2013) with slight modifications. In brief, Econo-Pac chromatography columns (Bio- Rad, Hercules, CA, USA) were loaded with approximately 3 mL of DEAE Sephadex A-25. ...
... Calcium homeostasis has been recognized to be critical for lens structural integrity and transparency, [19,20] with elevated levels of lens calcium and the subsequent stimulation of calpain activity reported to cause cataract or lens opacification. [21,22,23,24,25,26] Furthermore, intracellular calcium acting together with calmodulin has been reported to regulate the activities of the aquaporin-0 water channel and connexin gap junctions, as well as the stability of crystallin proteins which are required for lens transparency and architecture. [27,28,29,30,31,32,33,34,35,36,37] Therefore, a tightly regulated calcium influx is critical for homeostasis of lens physiology and function. ...
... The lens coloration is formed by an interaction of crystalline proteins with short-wavelength-filtering compounds, functioning as a low-frequency pass cut-off filter with undisturbed transmission at lower wavelengths. In humans, these compounds are kynurenine (KN) related, including kynurenine ; 3-hydroxykynurenine (3OHKN); 3-hydroxykynurenine O-b-D-glucoside (3OHKG); 4-(2-aminophenyl)-4-oxobutanoic acid (AHA); and glutathionyl-kynurenine (GSH-KN; ), and all derived from tryptophan [98,99] . Interestingly, the biochemical pathway leading to 3OHKG production in the eye was also found in some (but not all) teleost fishes , leading to the conclusion that this photochemical protection mechanism evolved relatively early in vertebrate life (within the gnathostomes) and might have been permissive for the evolutionary radiation of terrestrial vertebrates. ...
... In seeming contradiction, there have been frequent reports of human antibodies circu- lating to sites of papain and pepsin IgG hinge cleavage.  Circulating blood platelets proved to be an instructive target system for antibody-mediated clearance of papain and pepsin- mediated antibody fragments. The platelet receptor α IIb β 3 is essential for normal hemostasis but also responsible for platelet thrombosis in diseased arteries. ...
... 30, An interesting enzyme, indoleamine 2,3-dioxygenase or IDO, has also been described in the epithelial cells of the murine epididymis (Figure 1a). 39,40 Contrary to other organs where it is induced by inflammation, IDO is constitutively expressed in the epididymis, 41 suggesting a crucial role for this enzyme. Even if it is generally considered for its tolerogenic properties, IDO is a well-described antimicrobial effector through its catabolic activity which deprives the environment of tryptophan. ...
... Strictosidinic acid (20) was also isolated from Ophiorrhiza tomentosa , O. filistipula , O. kuroiwai , O. liukiuensis  and O. pumila . Nonato et al., 1995 isolated harman (47), lyalosidic acid (12) and palicoside (15) from Ophiorrhiza acuminata leaves . Kitajima et al., 1997a ...
Reference: Secondary metabolites from Ophiorrhiza
... The general domain arrangement along the longitudinal axis is consistent with that of the disulfide-linked dimer (Fig. 1b, top), thus verifying the conformation of the biological assembly. There are, however, two protuberances in the transverse plane of the SAXS envelope that correlate with the three- dimensional space that would be sampled by an unstructured and flexible N-terminal extension (Fig. 1b, bottom), encompassing the first four N-terminal residues (Fig. 1c, black, and e, black boldface text) . A similar protuberance is noted in the ab initio SAXS envelope of βB2-crystallin dimer due to flexible terminal extensions [35,39]. ...
... The total soluble protein content in the supernatant was determined by the method of Lowry et al. (1951). Glucosinolates were extracted and analyzed according to the method of Tholen et al. (1989). Defatted seed sample (400 mg) was homogenized with 3 ml hot distilled water followed by the addition of 0.5M barium sulphate (0.5 ml) and 0.5M lead acetate (0.5 ml). ...