D-amino acid formation induced by a chiral field within a human lens protein during aging.

We have previously shown that Asp-151 in alphaA-crystallin from aged human lens are converted to the biologically uncommon D-isomer to a high degree, showing that the formation of D-isomer was not simple racemization, but stereoinvertion. This suggests that alphaA-crystallin has a chiral reaction field which promotes the inversion of L-Asp to D-Asp residues in the native higher order structure of alphaA-crystallin itself. Here, we show that when the aged human alphaA-crystallin, enriched at Asp-151 with the D-isomer (D/L ratio of 5.7), was unfolded by heating at 70 degrees C or 6 M urea, the D-Asp-151 in the unfolded alphaA-crystallin was rapidly racemized (D/L ratio of 2.17 to 1.21). This presumably reflects a relaxation of the chiral field that was initially inducing the stereoinversion from the natural L-isomer to the D-isomer.     

Formation of four isomers at the asp-151 residue of aged human alphaA-crystallin by natural aging

Although proteins are generally composed entirely of l-amino acids, we have previously shown that Asp-151 in alphaA-crystallin from aged human lens is converted to the biologically uncommon d-isomer to a high degree. The formation of d-isomer was not simple racemization, but stereoinvertion. The reaction was also accompanied with isomerization to form beta-Asp (isoaspartate) residue simultaneously; therefore, four isomers of Asp-151, normal l-alpha-Asp and biologically uncommon l-beta-Asp, d-alpha-Asp, and d-beta-Asp, are formed in alphaA-crystallins. In the present study, we measured the ratio of the four isomers of Asp-151 in alphaA-crystallins obtained from total lens proteins of human lenses of newborn and 30-, 60-, and 80-year-olds. The isomers increased with age, and the total amount of three isomers was more than that of normal l-alpha-Asp in the alphaA-crystallin of the human lenses of the 80-year-olds. These drastic changes started at birth, with about 45% of normal l-alpha-Asp lost by 30 years. These modifications of the Asp residue likely affect the three-dimensional packing array of the lens proteins.     

Simultaneous stereoinversion and isomerization at the Asp-4 residue in βB2-crystallin from the aged human eye lenses

The lens proteins are composed of α-, β-, and γ-crystallins that interact with each other to maintain the transparency and refractive power of the lens. Because the lens crystallins are long-lived proteins, they undergo various post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation. In βB2-crystallin, which is the most abundant β-crystallin, the deamidation of asparagine and glutamine residues has been reported. Here, we found that the aspartyl (Asp) residue at position 4 of βB2-crystallin in the lenses of elderly human individuals undergoes a significant degree of inversion and isomerization to the biologically uncommon residue D-β-Asp. Surprisingly, the D/L ratio of β-Asp at position 4 in βB2-crystallin from elderly donors (67-77 year old) was 0.88-3.21. A D/L ratio of amino acids greater than 1.0 is defined as an inversion of configuration from the L- to D-form, rather than a racemization. These extremely high D/L ratios are equivalent to those of Asp-58 and Asp-151 (D/L ratio: 3.1 for Asp-58 and 5.7 for Asp-151) in αA-crystallin from elderly donors (~80 year old) as reported previously. Initially, we identified specific Asp residues in the β-crystallin family of proteins that undergo a high degree of inversion. These results show that the isomerization and inversion of Asp residues occurs both in the α- and β-crystallins of the lens. Inversion of these Asp residues directly affects the higher order structure of the protein. Hence, this modification may change crystallin-crystallin interactions and disrupt the function of crystallins in the lens.     

Comparison of d-aspartic acid contents in alpha A-crystallin from normal and age-matched cataractous human lenses

We have previously shown that biologically uncommon d-beta-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of alpha A-crystallin from aged human lenses. The amounts increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-beta-aspartic acids in alpha A-crystallin and cataract formation, we measured the d/l ratio of beta-Asp-151 of alpha A-crystallin from both cataractous and age-matched normal human lenses. alpha A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the alpha- and beta-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of beta-Asp-151 of alpha A-crystallin from normal lenses is not statistically significant from that of alpha A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.     

Comparison of post-translational modifications of alpha A-crystallin from normal and hereditary cataract rats

Summary. In order to investigate the relationship between lens opacities and the various modifications of lens proteins, we analyzed and compared the properties of lens proteins of 85-day old normal Wistar rats and the hereditary cataract model, ICR/f rats. The present study identified many differences between normal and mutant lens proteins. In the ICR/f mutant rats, the relative amounts of gamma-crystallin decreased and high molecular weight (HMW) protein increased. Racemization and isomerization of Asp-151 of alpha A-crystallin was observed in the mutant ICR/f rats, and Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. These modifications were not found in the age-matched normal rats. These tendencies are consistent with aged and cataractous human lenses.     

Free and protein-bound glutathione in normal and cataractous human lenses

Protein-bound glutathione was identified and measured in normal and cataractous human lenses. In a major group of cataracto us lenses the bound glutathione concentration was higher than normal. Study of normal lenses showed that their glutathione content is age-dependent, decreasing steadily from about 3.5mumol/g of lens at age 20 years to about 1.8mumol/g of lens at age 65 years. Cataract brings further decreases.     

3-Hydroxykynurenine oxidizes alpha-crystallin: potential role in cataractogenesis

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.     

Determination of calcium, sodium, potassium and magnesium concentrations in human senile cataractous lenses

Cataractous lenses have been found to have a distribution of the intracellular ionic environment, the concentrations of potassium and magnesium decreasing and the concentrations of sodium and calcium increasing relative to the cytosol of most cells. This arises as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. These changes have been found to be initiated as a result of normal ageing of the human lens. In this study, total Ca2+, K+, Na+ and Mg2+ contents have been determined in human normal and cataractous lenses using atomic absorption and flame emission spectroscopy. The normal human lens Ca2+ is between 0.15 and 0.5 miromol g(-1) fresh lens weight; in senile cataracts the value increased up to 9.31 micromol g(-1) ( p < 0.0001). The normal levels of Na+, Mg2+ and K+ are 20, 5.5 and 60 micromol g(-1) respectively; these changed to 136.10, 3.60 and 9.33 micro mol g(-1), respectively in cataractous senile human lenses ( p < 0.002, p < 0.002 and p < 0.01). The remarkable differences in these elements may play some role in cataractogenesis.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

Evidence for distinct cholesterol domains in fiber cell membranes from cataractous human lenses

Previous studies in our laboratory have provided direct evidence for the existence of distinct cholesterol domains within the plasma membranes of human ocular lens fiber cells. The fiber cell plasma membrane is unique in that it contains unusually high concentrations of cholesterol, with cholesterol to phospholipid (C/P) mole ratios ranging from 1 to 4. Since membrane cholesterol content is disturbed in the development of cataracts, it was hypothesized that perturbation of cholesterol domain structure occurs in cataracts. In this study, fiber cell plasma membranes were isolated from both normal (control) and cataractous lenses and assayed for cholesterol and phospholipid. Control and cataractous whole lens membranes had C/P mole ratios of 3.1 and 1.7, respectively. Small angle x-ray diffraction approaches were used to directly examine the structural organization of the cataractous lens plasma membrane versus control. Both normal and cataractous oriented membranes yielded meridional diffraction peaks corresponding to a unit cell periodicity of 34.0 A, consistent with the presence of immiscible cholesterol domains. However, comparison of diffraction patterns indicated that cataractous lens membranes contained more pronounced and better defined cholesterol domains than controls, over a broad range of temperature (5-40 degrees C) and relative humidity (52-92%) levels. In addition, diffraction analyses of the sterol-poor regions of cataractous membranes indicated increased membrane rigidity as compared with control membranes. Modification of the membrane lipid environment, such as by oxidative insult, is believed to be one potential mechanism for the formation of highly resolved cholesterol domains despite significantly reduced cholesterol content. The results of this x-ray diffraction study provide evidence for fundamental changes in the lens fiber cell plasma membrane structure in cataracts, including the presence of more prominent and highly ordered, immiscible cholesterol domains.     

Further studies on elastase and trypsin inhibitory activities in mammalian lenses

Elastase and trypsin inhibitory capacities increased significantly on heat treatment of the lens extract for 15 min at 60 degrees C in human infant (mean increase 290 and 335%), human adult (130 and 245%), ovine (90 and 140%), and bovine (70 and 90%) lenses. No increase was observed in human cataractous lenses. Preincubation with target enzymes in the absence of substrate abolished the antitryptic activity in lenses whereas antielastase activity was more resistant. No decrease in antielastase activity in human adult and cataractous lenses was observed on 15-min preincubation whereas about 50% of activity was abolished in human infant lenses. The differences were attributed to the changes in the levels of endogenous proteinases and proenzymes during cataractogenesis and aging.     

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography.     

Determination of rate constants for β-linkage isomerization of three specific aspartyl residues in recombinant human αA-crystallin protein by reversed-phase HPLC

The major soluble eye lens protein, αA-crystallin, has a very long half-life. Thus, many post-translational modifications, including stereoinversion, have been found in its constituent amino acids. We determine the rates of β-linkage isomerization, which is the main reaction through the formation of a succinimide intermediate, of specific Asp residues of recombinant human αA-crystallin protein by simple RP-HPLC method. Kinetic analyses of the β-linkage isomerization were performed on the three Asp residues of αA-crystallin, (58)Asp, (84)Asp, and (151)Asp, because the d/l ratios of both the (58)Asp and (151)Asp residues were higher than 1.0 in the αA-crystallin isolated from aged human eye lens. The β-linkage isomerizations of both the (58)Asp and (84)Asp residues were suppressed in the recombinant protein by approximately 0.4-0.5 times compared to those in the synthetic peptide below 50 °C, whereas the isomerization of the (151)Asp residue occurred at the same rate for the whole protein and synthetic fragmentary peptide. The suppression of (58)Asp isomerization in the recombinant protein relaxed to some extent when the αA-crystallin protein was incubated at a high temperature. The far-UV CD spectra showed that the secondary structure of the protein was partially disordered at temperatures greater than 60 °C in the recombinant αA-crystallin protein. These results suggest that the (58)Asp residue was restrained from forming the succinimide intermediate by the higher order structure of the αA-crystallin protein, and that the structural environment around the (151)Asp residue of the αA-crystallin was similar to that of the synthetic fragmentary peptide with respect to succinimide formation. The difference in the influence of the secondary structure of the αA-crystallin protein inverts the order of the succinimide formations of the (58)Asp and (151)Asp residues in the recombinant protein as compared with the order in the synthetic fragmentary peptides.     

Synchronous scan fluorescence spectroscopy of proteins and human eye lenses

Scanning both the excitation and emission monochromators synchronously while recording the fluorescence spectrum results in a considerable decrease in the apparent band width and shift in the peak position. We demonstrate the potential of this approach in the studies on proteins and their interactions as well as fluorophores in condensed media. We have chosen crystallins, eye lens proteins and human lenses. Synchronous scan spectra of alpha-, beta- and gamma-crystallins are clearly distinguishable and appear to provide specific signatures. The spectrum of the mixed solution could be simulated by the linear combination of components indicating that these proteins might not have any specific interaction in the dilute solutions. Synchronous spectra of the human lenses, both normal and cataractous, show several distinguishable features.     

Covalent change in alpha crystallin during human senile cataractogenesis

The high molecular weight aggregates (HMWA) obtained from normal and cataractous human lens nuclei have been resolved by SDS-polyacrylamide gel electrophoresis, and the alpha crystallin band has been probed with antisera made against the whole alpha crystallin molecule and with antisera made against synthetic peptides of alpha crystallin (alpha A2 147-161 and alpha A2 163-173). Quantitation of these antisera binding demonstrated that the anti-alpha A2 163-173 serum and the anti-alpha whole sera bound equally well to the alpha crystallin band from the HMWA fraction from normal and cataractous lenses. In contrast, the anti-alpha A2 147-161 serum bound little, if at all, to alpha crystallin from normal lenses, while it bound well to alpha crystallin from cataractous lenses. These results demonstrate a covalent alteration in the alpha crystallin molecule, and suggest a possible location of a covalent change that may occur during the cataractogenic process in the aged human lens.     

Proteases and protease inhibitory activities in normal mammalian lenses and human cataractous lenses

Trypsin inhibition (reduction in benzoyl arginine p-nitroanilide hydrolysis), elastase inhibition (reduction in succinyl trialanyl p-nitroanilide hydrolysis), and chymotrypsin inhibition (reduction in acetyl tyrosine ethyl ester hydrolysis) by neutral extracts of mammalian lenses were estimated. The activities were found to be markedly elevated in human cortical cataract lenses compared to normal adult lenses (antielastase 7.21 +/- 3.90 units (mean +/- SD) in cataract compared to 1.46 +/- 0.57 in normals; antitryptic, 0.54 +/- 0.38 and 0.12 +/- 0.04; antichymotryptic, 1.03 +/- 0.61 and 0.297 +/- 0.055). Antielastase activity was distinctly higher in adult normal human lenses compared to infant lenses (0.159 +/- 0.068). Elastase- and trypsin-like activities were detected at low levels in all mammalian lenses. Chymotrypsin-like activity could not be observed in the lenses. The cataractous lenses had lower trypsin- and elastase-like activities compared to normal human lenses (elastase 1.20 +/- 0.643 in normal compared to 0.062 +/- 0.035 in cataract; trypsin, 0.367 +/- 0.154 and 0.069 +/- 0.038). The role of protease: inhibitor complexes in the expression of the individual activities and their role in cataractogenesis are discussed.     

Determination of 19 elements in human eye lenses

Neutron activation analysis was used to determine the concentrations of 19 elements in normal and senile human cataractous lenses. It was found that the concentrations of Ca, Na, Cl, Eu, Sb, and Fe were significantly higher, and those of K, Rb, Cs, Cr, Mn, Co, Sc, and Ce were significantly lower in senile mature cataractous lenses than those in normal human eye lenses. No changes were found for the concentrations of Se, Zn, Mg, S, and Th in the two groups. Positive correlations between Na, Cl, and Ca and K, Rb, and Cs were found, whereas a significantly negative correlation between na, Ca, Cl and K, Rb, Cs were found. The roles of these elements in the evolution of cataract are discussed.     

2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nalpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nalpha-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.     

Conformational changes in human lens proteins in cataract

The reactivity of protein thiol groups in human lens and the susceptibility of the proteins to tryptic digestion were investigated. Both were found to be greater in some cataractous lenses, indicating that lens proteins have unfolded during cataractogenesis. Almost all the tyrosine in the proteins of the normal human lens reacts with tetranitromethane and is therefore probably on the outside of the major lens proteins.     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.