Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins.

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.

[1]  R. Nagaraj,et al.  The presence of a glucose‐derived Maillard reaction product in the human lens , 1996, FEBS letters.

[2]  F. J. Giblin,et al.  Glutathione: a vital lens antioxidant. , 2000, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[3]  R. Ansari,et al.  Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis. , 2005, Investigative ophthalmology & visual science.

[4]  M. A. Ostrovsky,et al.  Mechanism of Chaperone-like Activity. Suppression of Thermal Aggregation of βL-Crystallin by α-Crystallin† , 2005 .

[5]  F. Giblin,et al.  A protective role for glutathione-dependent reduction of dehydroascorbic acid in lens epithelium. , 1995, Investigative ophthalmology & visual science.

[6]  B. Ortwerth,et al.  LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro. , 2006, Biochimica et biophysica acta.

[7]  H. James,et al.  Spontaneous generation of superoxide anion by human lens proteins and by calf lens proteins ascorbylated in vitro. , 1999, Experimental eye research.

[8]  M. Feather,et al.  The precipitation and cross-linking of lens crystallins by ascorbic acid. , 1988, Experimental eye research.

[9]  B. Ortwerth,et al.  2-Ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) Is a Newly Identified Advanced Glycation End Product in Cataractous and Aged Human Lenses* , 2004, Journal of Biological Chemistry.

[10]  B. Ortwerth,et al.  Studies on the solubilization of the water-insoluble fraction from human lens and cataract. , 1992, Experimental eye research.

[11]  B. Ortwerth,et al.  Studies on the nature of the water-insoluble fraction from aged bovine lenses. , 1989, Experimental eye research.

[12]  E. Abraham,et al.  Evidence of a glycemic threshold for the development of cataracts in diabetic rats. , 1999, Current eye research.

[13]  K. Gilliland,et al.  Ultrastructural characterization and Fourier analysis of fiber cell cytoplasm in the hyperbaric oxygen treated guinea pig lens opacification model. , 2003, Experimental eye research.

[14]  E. Abraham,et al.  Lens protein composition, glycation and high molecular weight aggregation in aging rats. , 1987, Investigative ophthalmology & visual science.

[15]  A. Pirie,et al.  Changes to the proteins of the human lens nucleus in cataract. , 1974, Experimental eye research.

[16]  S P Wolff,et al.  Pro-oxidant activation of ocular reductants. 2. Lens epithelial cell cytotoxicity of a dietary quinone is associated with a stable free radical formed with glutathione in vitro. , 1987, Experimental eye research.

[17]  J. Baynes,et al.  Oxidation of glycated proteins: age-dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins. , 1989, Biochemistry.

[18]  M. Glomb,et al.  Amides are novel protein modifications formed by physiological sugars. , 2002, Journal of Biological Chemistry.

[19]  K. W. Lee,et al.  A systematic approach to evaluate the modification of lens proteins by glycation-induced crosslinking. , 1999, Biochimica et biophysica acta.

[20]  Neer Asherie,et al.  Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin. , 2005, Biochemistry.

[21]  B. Ortwerth,et al.  K2P—A Novel Cross‐Link from Human Lens Protein , 2005, Annals of the New York Academy of Sciences.

[22]  A. Spector,et al.  Age-dependent changes in the molecular size of human lens proteins and their relationship to light scatter. , 1974, Investigative ophthalmology.

[23]  D. Balasubramanian,et al.  Transglutaminase‐mediated cross‐linking of α‐crystallin: structural and functional consequences , 2001, FEBS letters.

[24]  M. Vitek,et al.  Amyloid β Peptides Do Not Form Peptide-derived Free Radicals Spontaneously, but Can Enhance Metal-catalyzed Oxidation of Hydroxylamines to Nitroxides* , 1999, The Journal of Biological Chemistry.

[25]  V. J. Stevens,et al.  Diabetic cataract formation: potential role of glycosylation of lens crystallins. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Truscott,et al.  An impediment to glutathione diffusion in older normal human lenses: a possible precondition for nuclear cataract. , 1998, Experimental eye research.

[27]  B. Ortwerth,et al.  Separation of the yellow chromophores in individual brunescent cataracts. , 2003, Experimental eye research.

[28]  R. Nagaraj,et al.  Protein Cross-linking by the Maillard Reaction , 1996, The Journal of Biological Chemistry.

[29]  P. Jap,et al.  X-ray induced cataract in rabbit lens. , 1975, Experimental eye research.

[30]  L. Chylack,et al.  Studies on human cataracts. III. Structural elements in nuclear cataracts and their contribution to the turbidity. , 1981, Investigative ophthalmology & visual science.

[31]  L. Chylack,et al.  Role of nonenzymatic glycosylation in experimental cataract formation. , 1980, Biochemical and biophysical research communications.

[32]  B. Ortwerth,et al.  Singlet oxygen production correlates with his and Trp destruction in brunescent cataract water-insoluble proteins. , 1998, Experimental eye research.

[33]  N. Robinson,et al.  Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry. , 2006, Molecular vision.

[34]  M. Delaye,et al.  Short-range order of crystallin proteins accounts for eye lens transparency , 1983, Nature.

[35]  M. Obrenovich,et al.  Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model , 2006, Proceedings of the National Academy of Sciences.

[36]  K. K. Sharma,et al.  Effect of cross-linking on the chaperone-like function of alpha crystallin. , 1995, Experimental eye research.

[37]  I. Birlouez-Aragon,et al.  Decrease in vitamin C concentration in human lenses during cataract progression. , 1998, International journal for vitamin and nutrition research. Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung. Journal international de vitaminologie et de nutrition.

[38]  K. W. Lee,et al.  The relative ability of glucose and ascorbate to glycate and crosslink lens proteins in vitro. off. , 1998, Experimental eye research.

[39]  P. Thampi,et al.  α-Crystallin chaperone function in diabetic rat and human lenses , 2004, Molecular and Cellular Biochemistry.

[40]  E. Abraham,et al.  Inhibition of lens crystallin glycation and high molecular weight aggregate formation by aspirin in vitro and in vivo. , 1989, Investigative ophthalmology & visual science.

[41]  B. Ortwerth,et al.  The non-oxidative degradation of ascorbic acid at physiological conditions. , 2000, Biochimica et biophysica acta.

[42]  J. Baynes,et al.  N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. , 1997, The Biochemical journal.

[43]  B. Ortwerth,et al.  Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue. , 2002, Biochimica et biophysica acta.

[44]  K. Uchida,et al.  Argpyrimidine, a blue fluorophore in human lens proteins: high levels in brunescent cataractous lenses. , 2001, Investigative ophthalmology & visual science.

[45]  E. Abraham,et al.  Advanced glycation end products in human senile and diabetic cataractous lenses , 2000, Molecular and Cellular Biochemistry.

[46]  John I. Clark,et al.  Theoretical and experimental basis for the inhibition of cataract , 1999, Progress in Retinal and Eye Research.

[47]  R. Nagaraj,et al.  Protein crosslinking by the Maillard reaction: dicarbonyl-derived imidazolium crosslinks in aging and diabetes. , 1999, Archives of biochemistry and biophysics.

[48]  J. King,et al.  Molecular basis of a progressive juvenile-onset hereditary cataract. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[49]  E. Abraham,et al.  Glycation mediated lens crystallin aggregation and cross-linking by various sugars and sugar phosphates in vitro. , 1993, Experimental eye research.

[50]  K. W. Lee,et al.  Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation. , 2001, Biochimica et biophysica acta.

[51]  L. Takemoto,et al.  Interaction of lens alpha and gamma crystallins during aging of the bovine lens. , 2005, Experimental eye research.

[52]  A. Kamei,et al.  Contribution of glycation to human lens coloration. , 1991, Chemical & pharmaceutical bulletin.

[53]  Michael J Harms,et al.  Laser light‐scattering evidence for an altered association of βB1‐crystallin deamidated in the connecting peptide , 2004, Protein science : a publication of the Protein Society.

[54]  D. Tappan.  A light scattering technique for measuring protein concentration. , 1966, Analytical biochemistry.

[55]  K. Satoh.  Age-related changes in the structural proteins of human lens. , 1972, Experimental eye research.

[56]  G. Benedek,et al.  On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses. , 1973, Experimental eye research.

[57]  A. Spector,et al.  Pro-oxidant activation of ocular reductants. 1. Copper and riboflavin stimulate ascorbate oxidation causing lens epithelial cytotoxicity in vitro. , 1987, Experimental eye research.

[58]  G. Glinsky,et al.  Superoxide free radical generation by Amadori compounds: the role of acyclic forms and metal ions. , 1999, Chemical research in toxicology.

[59]  J. Baynes,et al.  Role of the Maillard Reaction in Aging of Tissue Proteins , 1998, The Journal of Biological Chemistry.

[60]  L. Chylack,et al.  In vivo measurement of the aging rabbit lens using quasielastic light scattering. , 1986, Current eye research.

[61]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[62]  V. Monnier,et al.  Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process. , 1989, The Journal of biological chemistry.

[63]  F. Giblin,et al.  A physiological level of ascorbate inhibits galactose cataract in guinea pigs by decreasing polyol accumulation in the lens epithelium: a dehydroascorbate-linked mechanism. , 1994, Experimental eye research.

[64]  Liang Jj,et al.  Heat-induced conformational change of human lens recombinant alphaA- and alphaB-crystallins. , 2000 .

[65]  J. C. van der Pols.  A possible role for vitamin C in age-related cataract , 1999, Proceedings of the Nutrition Society.

[66]  T. Crawford,et al.  Synthesis of L-ascorbic acid. , 1980, Advances in carbohydrate chemistry and biochemistry.

[67]  V. Monnier,et al.  High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[68]  G. Benedek,et al.  Theory of transparency of the eye. , 1971, Applied optics.

[69]  L T Chylack,et al.  Quantitative detection of the molecular changes associated with early cataractogenesis in the living human lens using quasielastic light scattering. , 1987, Current eye research.