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Methylglyoxal-Derived Hydroimidazolone Advanced Glycation End-Products of Human Lens Proteins

¹From the Department of Biological Sciences, University of Essex, Colchester, United Kingdom; ²Departments of Ophthalmology and ³Internal Medicine IV, Friedrich Schiller University, Jena, Germany; and ⁴George Haik Eye Clinic, New Orleans, Louisiana.

	Abstract

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PURPOSE. To determine the concentrations of methylglyoxal-derived advanced glycation end-products (AGEs), the hydroimidazolones MG-H1 and -H2, in soluble human lens proteins and compare them with the concentrations of other methylglyoxal-derived AGEs and pentosidine.

METHODS. Lens protein samples were hydrolyzed enzymatically. AGEs were assayed without derivatization by HPLC with tandem mass spectrometry; the fluorescent AGEs argpyrimidine and pentosidine were assayed by fluorometric detection. MG-H1 and -H2 were resolved and assayed by fluorometric detection after derivatization with 6-aminoquinolyl-N-hydroxysuccimidylcarbamate (AQC).

RESULTS. The methylglyoxal-derived hydroimidazolones MG-H1 and -H2 were detected and quantified in human lens proteins. AGE concentrations (mean ± SEM) were: MG-H1 4609 ± 411 pmol/mg protein, MG-H2 3085 ± 328 pmol/mg protein, argpyrimidine 205 ± 19 pmol/mg protein, and pentosidine 0.693 ± 0.104 pmol/mg protein. The concentration of MG-H1 in human lens protein correlated positively with donor age (correlation coefficient = 0.28, P < 0.05), the concentration of MG-H2 (correlation coefficient = 0.78, P < 0.001) and argpyrimidine (correlation coefficient = 0.42, P < 0.01). The concentrations of AGEs were increased in cataractous lenses in comparison with noncataractous lenses: the increases were MG-H1 85%, MG-H2 122%, argpyrimidine 255%, and pentosidine 183% (P < 0.001). Multiple logistic regression analysis showed a significant link of cataract to donor age (regression coefficient ß = 0.094, P = 0.026) and argpyrimidine (ß = 0.022, P = 0.002).

CONCLUSIONS. Methylglyoxal hydroimidazolones are quantitatively major AGEs of human lens proteins. These substantial modifications of lens proteins may stimulate further glycation, oxidation, and protein aggregation leading to the formation of cataract.

Methylglyoxal reacted reversibly with cysteine residues to form hemithioacetal adducts, and lysine and arginine residues to form glycosylamine residues.⁹ Further reaction with lysine residues occur irreversibly to form N-(1-carboxyethyl)lysine (CEL)¹⁰ and 1,3-di(N-lysino)-4-methyl-imidazolium (MOLD).¹¹ An irreversible reaction of methylglyoxal with arginine residues forms N-(4-carboxy-4,6-dimethyl-5,6-di-hydroxy-1,4,5,6-tetra-hydropyrimidine-2-yl)ornithine (THP),¹² and argpyrimidine,¹³ but the major adduct in proteins modified minimally, as found in vivo, is methylglyoxal-derived hydroimidazolone (MG-H). MG-H is formed as three structural isomers: N-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), 2-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic acid (MG-H2), and 2-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3) (Fig. 1) . MG-H3 is unstable and was not usually quantified.^{14 15}

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Methylglyoxal-derived AGEs.

	Materials and Methods

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Human Lens Samples
Human cataractous lenses were obtained from patients in the Department of Ophthalmology, Friedrich-Schiller University (Jena, Germany) who were undergoing routine cataract surgery. Noncataractous lenses of patients with injury or trauma were used as the control. Further noncataractous lenses were obtained from donor eyes and assessed by one of the authors (GMH) through an arrangement with the Southern Eye Bank, Inc. (New Orleans, LA). Lenses were removed by an aseptic technique with a surgical loop within 8 hours of death. The lenses were examined in toto by transillumination and low-power microscopy. Color, clarity, and capsular integrity were determined. Medical history was noted. The lenses were stored at -84°C. Samples were dispatched on dry ice to the University of Essex for AGE analysis. Decapsulated individual lenses were homogenized on ice in 5 mL of phosphate-buffered saline (PBS) followed by centrifugation at 100,000g for 1 hour at 4°C. The supernatants containing the water-soluble lens proteins were stored at -80°C before analysis.

Sixteen lenses without cataract were from donors of mean age 56 ± 10 years; 3 were women and 13 were men. Thirty-nine samples with cataract were from donors with a mean age of 71 ± 14 years, which was significantly higher than that of the noncataractous donors (P < 0.001); 20 were women and 19 were men. Twenty-six subjects had diabetes mellitus and 29 were nondiabetic, with mean ages of 66 ± 16 and 67 ± 12 years, respectively (P > 0.05). Cataract was graded by color of the lens protein (grades 1 to 3). Five subjects had grade 1 (light yellow lens, mean age, 64 ± 20 years), 37 had grade 2 (yellow-light brown, mean age, 65 ± 14 years), and 13 had grade 3 (dark yellow-brown, mean age, 74 ± 13 years), a significantly higher age than subjects with grade 2 color; P < 0.05). This research followed the tenets of the World Medical Association Declaration of Helsinki on ethical principles for medical research involving human subjects.

Delipidification, Washing, and Enzymatic Hydrolysis of Lens Proteins
Lens protein samples (100–500 µg; 2 mg/mL) were extracted with a 2 x 1-volume of water-saturated diethyl ether to remove lipids, and the residual ether was removed by centrifugal evaporation. An aliquot of protein sample (500 µg) was then diluted to 500 µL with water and concentrated to approximately 50 µL by ultrafiltration (12-kDa cutoff membrane). This washing procedure was repeated a further two times. The protein concentration was then determined by the Bradford method.¹⁶ Protein samples were hydrolyzed enzymatically under nitrogen, as described.¹⁴ This hydrolysate was used in the assay of AGEs by the LC-MS/MS and fluorescence techniques.

Assay of AGEs by the LC-MS/MS, Intrinsic Fluorescence, and AQC-Derivatization Chromatographic Methods
The concentration of MG-H, arginine, and lysine in soluble lens protein hydrolysates was determined by LC-MS/MS using the stable isotope-substituted standards for internal standardization with reference to calibration curves of authentic standards. [Guanidino-¹⁵N₂]-L-arginine, [¹³C₆]-L-lysine, (all 98% isotopic purity) were purchased from Cambridge Isotope Laboratories (Andover, MA). [Guanidino-¹⁵N₂]-MG-H1 was prepared from [guanidino-¹⁵N₂]-L-arginine after conversion to the N-t-BOC derivative.^{14 17} Samples were assayed by LC-MS/MS using a separation module (model 2690) with a triple quadrupole mass spectrometric detector (Quattro Ultima; Waters-Micromass, Manchester, UK). Two 5 µm columns (Hypercarb; Thermo Hypersil, Ltd., Runcorn, UK) in series were used: 2.1 x 50 mm (column 1) and 2.1 x 250 mm (column 2). The mobile phase was 26 mM ammonium formate (pH 3.8), with a two-step gradient of acetonitrile (17–25 minutes, 0%–31% acetonitrile; 25–30 minutes, 31% acetonitrile). The flow rate was 0.2 mL/min. The flow was diverted to bypass column 2 at 20 minutes to facilitate elution of MG-H. Flow from the column during the interval of 4 to 30 minutes was directed to the MS/MS detector. Amino acids and AGEs were detected by electrospray-positive ionization-mass spectrometric multiple-reaction monitoring (MRM), with which the strongest fragment ion response, formed from a specific parent ion, was detected. The ionization source temperature was 120°C and the desolvation gas temperature, 350°C. The cone gas and desolvation gas flow rates were 150 and 550 L/h, respectively. The capillary voltage was 3.55 kV and the cone voltage, 80 V. Argon gas (2.7 x 10^-3 mbar) was in the collision cell. Programed molecular ion and fragment ion masses and collision energies were optimized to ±0.1 Da and ±1 eV for MRM detection of analytes. Amounts of internal standard used were: 10 nmol for amino acids and 50 pmol for MG-H1. The retention times, MRM transitions (molecular ion > fragment ion masses), collision energy, and fragment losses for analytes and calibration standards were, respectively: lysine 5.0 minutes, 147.1 > 84.3 Da, 15 eV, H₂CO₂+NH₃, [¹³C₆]-lysine; arginine 10.9 minutes, 175.2 > 70.3 Da, H₂CO₂+NH₂C(NH)NH₂, 15 eV, [¹⁵N₂]-arginine; MG-H 23.6 and 24.0 minutes (two epimers), 229.2 > 114.3 Da, 14 eV, NH₂CH(CO₂H)CH₂CHCH₂ and [¹⁵N₂]-MG-H1. The concentration of the fluorescent AGEs argpyrimidine and pentosidine was determined by HPLC with fluorometric detection (model 2475 fluorometric detector; Waters). The column was a 5-µm particle size, with dimensions of 50 x 2.1 mm (Hypercarb; Waters). The mobile phase was 0.1% trifluoroacetic acid in 10% acetonitrile with a linear gradient to 50% acetonitrile at 15 minutes and isocratic 50% acetonitrile thereafter. The limits of detection for argpyrimidine and pentosidine were 400 fmol and 20 fmol, respectively. The recoveries of MG-H1, argpyrimidine, and pentosidine in enzymatic hydrolysis were 83%, 84%, and 101% and the recoveries of arginine and lysine compared with acid hydrolysis were 94%.

MG-H structural isomers coeluted in LC-MS/MS analysis, and therefore the AQC derivatization technique that resolved MG-H isomers^{14 15} enabled the analysis of MG-H1 and -H2. Aliquots of hydrolysate (50 µL, equivalent to 50 µg protein) were derivatized by AQC and analyzed by HPLC with fluorometric detection, as described.^{14 15}

The formation of AGEs in lens proteins by glycation with methylglyoxal in vitro was investigated by incubation of lens protein under conditions similar to those used to prepare human serum albumin modified minimally by methylglyoxal.¹⁵ Lens protein (6 mg/mL) was incubated with and without methylglyoxal (500 µM) in sodium phosphate buffer (100 mM [pH 7.4] and at 37°C) for 24 hours. The samples were then washed by ultrafiltration and analyzed for AGEs, as described earlier.

	Results

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Methylglyoxal-Derived Hydroimidazolones, Argpyrimidine, and Pentosidine in Human Lens Proteins
Methylglyoxal-derived AGEs in enzymatic hydrolysates of lens proteins were determined without derivatization by LC-MS/MS with stable isotope-substituted internal standardization. In the volatile buffer eluent method used, the epimer pairs (LD- and LL-stereoisomers) of the structural isomers MG-H1, -H2, and -H3 were partially resolved in the LC-MS/MS chromatogram, but the structural isomers coeluted. There were therefore two peaks reflecting the LD- and LL-epimers coeluting of all three structural isomers (Fig. 2a . The detector response to each isomer was not significantly different, and therefore MG-H determination by this method reflects the sum of all isomers. The internal standard [¹⁵N₂]MG-H1 coeluted with the MG-H isomers (Fig. 2b) . The MG-H/[¹⁵N₂]MG-H1 detector response ratio was calibrated with authentic MG-H1 (1–100 pmol). There were high levels of MG-H in human lens proteins (8 nmol/mg protein).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Detection of methylglyoxal-derived hydroimidazolone in human lens protein by HPLC with tandem mass spectrometry. (a) MG-H; (b) [15N2]MG-H1 (50 pmol) in human lens protein (50 µg equivalent).

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Analysis of methylglyoxal-derived hydroimidazolones in human lens proteins by the chromatographic AQC method. (a) Methylglyoxal-derived hydroimidazolones (500 pmol MG-H1, MG-H2, and the IS -aminobutyric acid) and amino acid standards (10 nmol) by the chromatographic AQC method. (b) Noncataractous lens; (c) cataractous lens; and (d) the noncataractous lens protein in (a) reanalyzed after incubation with 500 µM methylglyoxal for 24 hours at pH 7.4 and 37°C in vitro.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Correlation analysis of methylglyoxal-derived AGEs in human lens proteins. (a) MG-H1 and donor age (correlation coefficient = 0.28, P < 0.05; nondiabetic subjects only), with the regression equation MG-H1 (pmol/mg protein) = 70 x donor age (years) + 705; (b) MG-H2 and MG-H1 (correlation coefficient = 0.78, P < 0.001), with the regression equation MG-H2 (pmol/mg protein) = 0.47 x MG-H1 (pmol/mg protein) + 674; and (c) argpyrimidine and MG-H1 (correlation coefficient = 0.42, P < 0.01) with the regression equation argpyrimidine (pmol/mg protein) = 0.021 x MG-H1 (pmol/mg protein) + 105.

	Discussion

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The concentrations of MG-H1 and -H2 in lens proteins are similar in the extent of amino acid modification (1%–2% arginine) to that of fructosamine (1%–2% lysine).¹⁸ This suggests that MG-H1 and -H2 are major glycation adducts quantitatively. Recently, we have found similar high concentrations of MG-H1 in protein extracts of human blood cells and tissues of laboratory rats.¹⁹ Argpyrimidine and pentosidine were present at much lower concentrations (0.1% arginine and 0.0002% arginine in cataractous lenses, respectively). Other methylglyoxal-derived AGEs are also found in lens proteins at low concentrations: CEL 0.1%–0.4% lysine and MOLD 0.1%–0.8% lysine.^{10 11 20} Hence, MG-H is the major methylglyoxal-derived AGE in the lens. The concentration of MG-H1 increased with donor age in the current study, as did the concentrations of CEL and MOLD reported previously.¹⁰ The predicted increases in MG-H1, CEL, and MOLD over the subject age interval of 20 to 75 years are 183%, 92%, and 275%, respectively.

MG-H1, MG-H2, and argpyrimidine are relatively short-lived AGEs with half-lives of only 1 to 2 weeks under physiological conditions.¹⁴ The levels of these analytes in long-lived proteins therefore may reflect the fluctuation of methylglyoxal concentration in the 2 to 4 weeks before lens extraction. The concentrations of these AGEs in long-lived lens proteins reflect the steady state concentration of AGE achieved as a balance of the rates of AGE formation and degradation. When significant correlations of these AGE analytes are found with donor age, factors influencing the steady state AGE concentration that change slowly over many years are likely to produce the correlation. These factors are GSH concentration, glyceraldehyde-3-phosphate dehydrogenase activity, and glyoxalase I activity.^{4 8 21} The activity of glyoxalase I and the concentration of triosephosphates are important variables controlling methylglyoxal concentration and related glycation in cultured rat lens.²²

The association of protein glycation with cataract was further supported in this work by the finding of increased concentrations of MG-H1, argpyrimidine, and pentosidine in soluble protein extracts of cataractous lenses in comparison with noncataractous lens proteins. We found concentrations of argpyrimidine and pentosidine in lens proteins similar to those reported previously.^{23 24} The increased glycation of proteins in cataractous lenses—particularly the higher extent of glycation by MG-H—may induce protein conformational changes that stimulate further glycation and oxidation and trigger protein aggregation leading to cataract. To study the association of variables with cataract formation, a multiple logistic regression model was computed. This indicated that cataract was associated significantly with donor age and argpyrimidine concentration. The formation of argpyrimidine is favored by high concentrations of methylglyoxal¹⁵ and oxidative processes.¹³ Argpyrimidine may also be degraded by oxidative processes.¹⁴ The link of argpyrimidine to cataract in the multiple logistic regression model suggests that the formation of argpyrimidine is either involved in the development of cataract or is a surrogate indicator of other critical factors in cataractogenesis. The lack of significant increases in AGE concentrations in diabetic lenses may have been due to masking of diabetes-associated changes by the effects of donor age and oxidative stress.

We conclude that MG-H is a major AGE in human lens proteins quantitatively. The modification of lens crystallins by methylglyoxal led to a decrease in arginine residues and loss of positive charge.²⁵ The formation of MG-H1 and -H2 are likely adducts producing this effect with up to 2% of total arginine modified. There are 10 to 20 arginine residues in the human crystallin isoforms. There is therefore an expectation that 10% to 20% of crystallin molecules have a MG-H modification. Loss of a single arginine residue may promote cataract formation, as found in the R58H mutation in -D-crystallin.^{26 27} Changes in lens protein charge due to MG-H formation, although short lived, may induce protein refolding and stimulate long-lived irreversible modifications, such as oxidation and proteolysis, that are major protein modifications implicated in cataractogenesis.²⁸ Further studies are needed to investigate the involvement of hydroimidazolone AGEs in cataract formation.

	Acknowledgements

 
The authors thank the Southern Eye Bank Inc., New Orleans, Louisiana, for assistance with the research and Sinan Battah for assistance in the preparation of [¹⁵N₂]MG-H1.

	Footnotes

 
Supported by a contract from the Institute for Pharmaceutical Discovery (IPD), Branford, Connecticut; and the Wellcome Trust, United Kingdom (PJT, NA, GMH).

Submitted for publication June 7, 2003; revised July 14, 2003; accepted July 24, 2003.

Disclosure: N. Ahmed, None; P.J. Thornalley, None; J. Dawczynski, None; S. Franke, None; J. Strobel, None; G. Stein, None; G.M. Haik, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Paul J. Thornalley, Department of Biological Sciences, University of Essex, Central Campus, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK; thorp{at}essex.ac.uk.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Phillips SA, Thornalley PJ. The formation of methylglyoxal from triose phosphates: investigation using a specific assay for methylglyoxal. Eur J Biochem. 1993;212:101–105.[Medline][Order article via Infotrieve]
2. Thornalley PJ, Langborg A, Minhas HS. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem J. 1999;344:109–116.
3. Yoshino K, Saito S, Sano M, Matsuura T, Hagiwara M, Tomita I. Formation of methylglyoxal in reduced nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation of rat liver microsomes. Jpn J Toxicol Environm Health. 1996;42:236–240.
4. Haik GM, Jr, Lo TWC, Thornalley PJ. Methylglyoxal concentration and glyoxalase activities in human lens. Exp Eye Res. 1994;59:497–500.[CrossRef][Medline][Order article via Infotrieve]
5. McLellan AC, Thornalley PJ, Benn J, Sonksen PH. The glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin Sci. 1994;87:21–29.[Medline][Order article via Infotrieve]
6. Thornalley PJ. The glyoxalase system in health and disease. Mol Asp Med. 1993;14:287–371.
7. Phillips SA, Thornalley PJ. Glyoxalase activity in the lens. Med Sci Res. 1992;20:91–92.
8. Kamei A. Glutathione levels of the human crystalline lens in aging and its antioxidant effect against oxidation of lens proteins. Biol Pharm Bull. 1993;16:870–875.[Medline][Order article via Infotrieve]
9. Lo TWC, Westwood ME, McLellan AC, Selwood T, Thornalley PJ. Binding and modification of proteins by methylglyoxal under physiological conditions: a kinetic and mechanistic study with N-acetylarginine, N-acetylcysteine, N-acetyl-lysine, and bovine serum albumin. J Biol Chem. 1994;269:32299–32305.[Abstract/Free Full Text]
10. Ahmed MU, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW. N^e-(Carboxyethyl)lysine, a product of chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J. 1997;324:565–570.
11. Brinkmann F, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW. Role of the Maillard reaction in aging of tissue proteins: advanced glycation end product-dependent increase in imidazolium cross-links in human lens proteins. J Biol Chem. 1998;273:18714–18719.[Abstract/Free Full Text]
12. Oya T, Hattori N, Mizuno Y, et al. Methylglyoxal modification of protein: chemical and immunochemical characterization of methylglyoxal-arginine adducts. J Biol Chem. 2000;274:18492–18502.
13. Shipanova IN, Glomb MA, Nagaraj RH. Protein modification by methylglyoxal: chemical nature and synthetic mechanism of a major fluorescent adduct. Arch Biochem Biophys. 1997;344:29–36.[CrossRef][Medline][Order article via Infotrieve]
14. Ahmed N, Argirov OK, Minhas HS, Cordeiro CA, Thornalley PJ. Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatisation by aminoquinolyl-N-hydroxysuccimidyl-carbamate and application to N-carboxymethyl-lysine- and N-(1-carboxyethyl)lysine-modified albumin. Biochem J. 2002;364:1–14.[Medline][Order article via Infotrieve]
15. Ahmed N, Thornalley PJ. Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatisation with aminoquinolyl-N-hydroxysuccimidyl-carbamate and intrinsic fluorescence. Biochem J. 2002;364:15–24.[Medline][Order article via Infotrieve]
16. Bradford MM. A rapid and sensitive method of quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[CrossRef][Medline][Order article via Infotrieve]
17. Meldal M, Kindtler W. Synthesis of a proposed antigenic hexapeptide from Escherichia-coli k88 protein fimbriae. Acta Chem Scand Ser B. 1986;40:235–241.[Medline][Order article via Infotrieve]
18. Lyons TJ, Silvestri G, Dunn JA, Dyer DG, Baynes JW. Role of glycation in modification of lens crystallins in diabetic and nondiabetic senile cataracts. Diabetes. 1991;40:1010–1015.[Abstract]
19. Babaei-Jadidi R, Karachalias N, Ahmed N, Battah S, Thornalley PJ. Prevention of incipient diabetic nephropathy by high dose thiamine and Benfotiamine. Diabetes. 2003;52:2110–2120.[Abstract/Free Full Text]
20. Chellan P, Nagaraj RH. Protein crosslinking by the Maillard reaction: dicarbonyl-derived imidazolium crosslinks in aging and diabetes. Arch Biochem Biophys. 1999;368:98–104.[CrossRef][Medline][Order article via Infotrieve]
21. Hyslop PA, Hinshaw DB, Halsey WA, Shraufstatter IU. Mechanisms of oxidant-mediated cell injury: the glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J Biol Chem. 1988;263:1665–1675.[Abstract/Free Full Text]
22. Shamsi FA, Sharkey E, Creighton DJ, Nagaraj RH. Maillard reaction in lens proteins: methylglyoxal-mediated modifications in rat lens. Exp Eye Res. 2000;70:369–380.[CrossRef][Medline][Order article via Infotrieve]
23. Wilker SC, Chellan P, Arnold BM, Nagaraj RH. Chromatographic quantification of argpyrimidine, a methylglyoxal-derived product in tissue proteins. Anal Biochem. 2001;290:353–358.[CrossRef][Medline][Order article via Infotrieve]
24. Padayatti PS, Ng AS, Uchida K, Glomb MA, Nagaraj RH. Argpyrimidine, a blue fluorophore in human lens proteins: high levels in brunescent cataractous lenses. Invest Ophthalmol Vis Sci. 2001;42:1299–1304.[Abstract/Free Full Text]
25. Riley ML, Harding JJ. The reaction of methylglyoxal with human and bovine lens proteins. Biochim Biophys Acta. 1995;1270:36–43.[Medline][Order article via Infotrieve]
26. Pande A, Jayanti J, Asherie N, et al. Crystal cataracts: human genetic cataract caused by protein crystallization. Proc Natl Acad Sci USA. 2001;98:6116–6120.[Abstract/Free Full Text]
27. Basak A, Bateman O, Slingsby C, et al. High-resolution X-ray crystal structures of human D crystallin (1.25Å) and the R58H mutant (1.15Å) associated with aculeiform cataract. J Mol Biol. 2003;328:1137–1147.[CrossRef][Medline][Order article via Infotrieve]
28. Hanson SRA, Hasan A, Smith DL, Smith JB. The major in vivo modification of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage. Exp Eye Res. 2000;71:195–207.[CrossRef][Medline][Order article via Infotrieve]

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