Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract.

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.

[1]  D. Borchman,et al.  Age dependence and distribution of green and blue fluorophores in human lens homogenates. , 1992, Investigative ophthalmology & visual science.

[2]  J. Wollensak,et al.  Non-tryptophan fluorescence of crystallins from normal and cataractous human lenses. , 1987, Investigative ophthalmology & visual science.

[3]  C. Slingsby,et al.  Rapid separation of bovine -crystallin subunits B1, B2, B3, A3 and A4 , 1990 .

[4]  U. Steinbrecher,et al.  Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. , 1987, The Journal of biological chemistry.

[5]  Joseph S. Wall,et al.  Application of a Hammett-Taft Relation to Kinetics of Alkylation of Amino Acid and Peptide Model Compounds with Acrylonitrile2 , 1964 .

[6]  H. Esterbauer,et al.  Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. , 1986, Biochimica et biophysica acta.

[7]  RUTH VAN HEYNINGEN,et al.  Fluorescent Glucoside in the Human Lens , 1971, Nature.

[8]  S. García-Castiñeiras,et al.  Loss of free amino groups in the water-insoluble fraction of nuclear senile cataracts. , 1983, Investigative ophthalmology & visual science.

[9]  D. Shaw,et al.  Amino acid sequence of bovine gamma E (IVa) lens crystallin , 1997, Protein science : a publication of the Protein Society.

[10]  R. Truscott,et al.  Human Lens Coloration and Aging , 1999, The Journal of Biological Chemistry.

[11]  K. K. Sharma,et al.  Identification of 1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic Acid Binding Sequences in α-Crystallin* , 1998, The Journal of Biological Chemistry.

[12]  S. Moore,et al.  Alkylation and identification of the histidine residues at the active site of ribonuclease. , 1963, The Journal of biological chemistry.

[13]  B. Witkop,et al.  The photoreduction of kynurenic acid to kynurenine yellow and the occurrence of 3-hydroxy-L-kynurenine in butterflies. , 1967, Journal of the American Chemical Society.

[14]  R. Truscott,et al.  Changes in human lens proteins during nuclear cataract formation. , 1977, Experimental eye research.

[15]  S. Pyne,et al.  Reactive metabolite hypothesis for human senile cataract. , 1991, Lens and eye toxicity research.

[16]  J. Carver,et al.  Identification of Glutathionyl-3-hydroxykynurenine Glucoside as a Novel Fluorophore Associated with Aging of the Human Lens* , 1999, The Journal of Biological Chemistry.

[17]  R. Dean,et al.  The Hydroxyl Radical in Lens Nuclear Cataractogenesis* , 1998, The Journal of Biological Chemistry.

[18]  R. Truscott,et al.  The modification of proteins by 3-hydroxykynurenine. , 1993, Experimental eye research.

[19]  J. Dillon,et al.  MODEL STUDIES ON THE PHOTOCHEMICAL PRODUCTION OF LENTICULAR FLUOROPHORES , 1994, Photochemistry and photobiology.

[20]  E. Stadtman,et al.  Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. Lou,et al.  Free cysteine levels in normal human lenses. , 1997, Experimental eye research.

[22]  J. Carver,et al.  Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine. , 1999, Biochemistry.

[23]  T. Blundell,et al.  Evolutionary and functional relationships between the basic and acidic beta-crystallins. , 1988, Experimental eye research.

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

[25]  P. Jocelyn.  Spectrophotometric assay of thiols. , 1987, Methods in enzymology.

[26]  Jean B. Smith,et al.  Deamidation and Disulfide Bonding in Human Lens γ-Crystallins , 1998 .

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

[28]  K. Schey,et al.  Identification of tryptophan oxidation products in bovine α‐crystallin , 1998, Protein science : a publication of the Protein Society.

[29]  A. Butenandt,et al.  Über Ommochrome, IV. Mitteilung: Konstitution des Xanthommatins , 1954 .

[30]  D. Moss,et al.  X-ray diffraction and structure of crystallins , 1997, Progress in Retinal and Eye Research.

[31]  Joseph S. Wall,et al.  Additive Linear Free-Energy Relationships in Reaction Kinetics of Amino Groups with α,β-Unsaturated Compounds1,2 , 1966 .

[32]  B. Ortwerth,et al.  Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction. , 1988, Biochimica et biophysica acta.

[33]  M. Skonieczna,et al.  The Photochemical Attachment of the O‐Glucoside of 3‐Hydroxykynurenine to α‐Crystallin: A Model for Lenticular Aging , 1999, Photochemistry and photobiology.

[34]  B. Derham,et al.  α-Crystallin as a molecular chaperone , 1999, Progress in Retinal and Eye Research.

[35]  Sidney Lerman,et al.  Spectroscopic Evaluation and Classification of the Normal, Aging, and Cataractous Lens. (With 1 color plate) , 1976 .

[36]  J. Carver,et al.  A new UV‐filter compound in human lenses , 1994, FEBS letters.

[37]  R. Truscott,et al.  Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions , 2000, Free radical research.

[38]  E. Berman,et al.  Biochemistry of the Eye , 1991, Perspectives in Vision Research.

[39]  J. Carver,et al.  Oxidation products of 3-hydroxykynurenine bind to lens proteins: relevance for nuclear cataract. , 1997, Experimental eye research.

[40]  A. Pirie.  Color and solubility of the proteins of human cataracts. , 1968, Investigative ophthalmology.

[41]  R. Truscott,et al.  UV filters in human lenses: tryptophan catabolism. , 1993, Experimental eye research.