3-Hydroxykynurenine-mediated Modification of Human Lens Proteins
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m501419200
ISSN1083-351X
AutoresMagdalena Staniszewska, Ram H. Nagaraj,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoTryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified Nα-acetyl lysine, Nα-acetyl histidine, Nα-acetyl arginine, and Nα-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and Nα-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-α-((5S)-5-acetamino-5-carboxypentyl amino)-γ-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation. Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified Nα-acetyl lysine, Nα-acetyl histidine, Nα-acetyl arginine, and Nα-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and Nα-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-α-((5S)-5-acetamino-5-carboxypentyl amino)-γ-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation. Crystallins are the major proteins of the lens, and they constitute 90% of the soluble proteins. A number of physicochemical changes occur in lens proteins during aging, and during cataract formation, similar changes occur at an exaggerated rate. The most striking changes in these proteins include yellowing and browning of proteins, intra- and intermolecular cross-linking, and cross-linking with fiber cell membrane proteins (1Bron A.J. Vrensen G.F. Koretz J. Maraini G. Harding J.J. Ophthalmologica. 2000; 214: 86-104Crossref PubMed Scopus (221) Google Scholar, 2Spector A. Isr. J. Med. Sci. 1972; 8: 1577-1582PubMed Google Scholar, 3Cobb B.A. Petrash J.M. J. Biol. Chem. 2000; 275: 6664-6672Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 4de Jong W.W. Mulders J.W. Voorter C.E. Berbers G.A. Hoekman W.A. Bloemendal H. Adv. Exp. Med. Biol. 1988; 231: 95-108PubMed Google Scholar). Several mechanisms have been proposed for such changes, including oxidation (5Spector A. FASEB J. 1995; 9: 1173-1182Crossref PubMed Scopus (798) Google Scholar, 6Reddy V.N. Exp. 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Protein-free tryptophan in the lens undergoes both enzymatic (12Goldstein L.E. Leopold M.C. Huang X. Atwood C.S. Saunders A.J. Hartshorn M. Lim J.T. Faget K.Y. Muffat J.A. Scarpa R.C. Chylack Jr., L.T. Bowden E.F. Tanzi R.E. Bush A.I. Biochemistry. 2000; 39: 7266-7275Crossref PubMed Scopus (163) Google Scholar) and non-enzymatic oxidation (13Zhang H. Andrekopoulos C. Joseph J. Crow J. Kalyanaraman B. Free Radic. Biol. Med. 2004; 36: 1355-1365Crossref PubMed Scopus (63) Google Scholar) to produce reactive kynurenines (Fig. 1). Absorption of ultraviolet light A by the lens is attributed in part to these products. The enzymatic oxidation to N-formylkynurenine (NFK) 1The abbreviations used are: NFK, N-formylkynurenine; MEM, minimal essential medium; AA, anthranilic acid; benzoic acid; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HBA, 4-hydroxybenzoic acid; KYN, d, l-kynurenine; 3OHKYN, 3-hydroxykynurenine; HLE-B3, human lens epithelial cells B3; 3OHKYNG, 3-hydroxykynurenine O-β-d-glucoside; KLH, keyhole limpet hemocyanin; NFDM, nonfat dry milk; PBS, phosphate-buffered saline; PBST, PBS + 0.05% Tween 20; RNase A, ribonuclease A; ROS, reactive oxygen species; WS-HLP, water-soluble human lens proteins; HPLC, high pressure liquid chromatography; t-Boc, tert-butoxycarbonyl; mAb, monoclonal antibody. is initiated by indoleamine 2, 3-dioxygenase, an enzyme that is up-regulated by interferon-γ. The next enzymatic steps produce kynurenine (KYN), 3-hydroxykynurenine (3OHKYN), 3-hydroxyanthranilic acid, quinolinic acid, and nicotinic acid (12Goldstein L.E. Leopold M.C. Huang X. Atwood C.S. Saunders A.J. Hartshorn M. Lim J.T. Faget K.Y. Muffat J.A. Scarpa R.C. Chylack Jr., L.T. Bowden E.F. Tanzi R.E. Bush A.I. Biochemistry. 2000; 39: 7266-7275Crossref PubMed Scopus (163) Google Scholar). The kynurenines within the lens become enzymatically glycosylated to O-β-glucosides. Quantification of these products in the human lens shows that 3-hydroxykynurenine O-β-d-glucoside (3OHKYNG) is the most abundant form followed by 4-(2-amino-3-hydroxyphenyl)-4-oxabutanoic acid O-β-d-glucoside, l-kynurenine, and 3-hydroxykynurenine (14Taylor L.M. Aquilina A.J. Jamie J.F. Truscott R.J. Exp. Eye Res. 2002; 75: 165-175Crossref PubMed Scopus (78) Google Scholar). The kynurenine products produced from protein-free tryptophan readily pass through cell membranes and could thus diffuse through the cortex of the lens. Kynurenines are unstable at physiological pH; they undergo side chain deamination to produce α, β unsaturated ketoalkenes (15Vazquez S. Garner B. Sheil M.M. Truscott R.J. Free Radic. Res. 2000; 32: 11-23Crossref PubMed Scopus (75) Google Scholar). Kynurenine levels decrease in the lens with age (16Bova L.M. Sweeney M.H. Jamie J.F. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 2001; 42: 200-205PubMed Google Scholar) and cataract formation (17Streete I.M. Jamie J.F. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 2004; 45: 4091-4098Crossref PubMed Scopus (45) Google Scholar), which may be due to their reaction with lens proteins. Kynurenines react with nucleophilic amino acids, such as cysteine, histidine, and lysine in lens proteins (18Garner B. Shaw D.C. Lindner R.A. Carver J.A. Truscott R.J. Biochim. Biophys. Acta. 2000; 1476: 265-278Crossref PubMed Scopus (72) Google Scholar, 19Hood B.D. Garner B. Truscott R.J. J. Biol. Chem. 1999; 274: 32547-32550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Aquilina J.A. Carver J.A. Truscott R.J. Exp. Eye Res. 1997; 64: 727-735Crossref PubMed Scopus (71) Google Scholar) and cysteinyl residue in GSH through Michael addition to form covalent adducts. One of the products of the reaction of 3OHKYNG with lens GSH is glutathionyl-3-hydroxykynurenine glucoside (21Garner B. Vazquez S. Griffith R. Lindner R.A. Carver J.A. Truscott R.J. J. Biol. Chem. 1999; 274: 20847-20854Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), which accumulates during lens aging and accumulates to relatively high levels in cataractous lenses (19Hood B.D. Garner B. Truscott R.J. J. Biol. Chem. 1999; 274: 32547-32550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Vazquez et al. (22Vazquez S. Aquilina J.A. Jamie J.F. Sheil M.M. Truscott R.J. J. Biol. Chem. 2002; 277: 4867-4873Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Vazquez S. Parker N.R. Sheil M. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 2004; 45: 879-883Crossref PubMed Scopus (30) Google Scholar) demonstrated modification of histidine and lysine residues of lens proteins by KYN and their accumulation in aging lenses, but these authors also noted that the modified products decrease in senile cataracts. Another recent study confirmed specific histidine modification in αB-crystallin that was incubated with KYN; modification of histidine 83 was thought to affect its chaperone function (24Aquilina J.A. Truscott R.J. Biochem. Biophys. Res. Commun. 2001; 285: 1107-1113Crossref PubMed Scopus (20) Google Scholar). Other crystallins appear to be modified by kynurenines as well (25Aquilina J.A. Carver J.A. Truscott R.J. Biochemistry. 1999; 38: 11455-11464Crossref PubMed Scopus (45) Google Scholar), suggesting that kynurenines are responsible, in part, for protein cross-linking during aging and cataract formation. Kynurenines also play a role in reactive oxygen species (ROS)-mediated crystallin modification. For example, kynurenines such as 3OHKYN that contain a hydroxyl group can generate ROS through transition metal reduction reactions (12Goldstein L.E. Leopold M.C. Huang X. Atwood C.S. Saunders A.J. Hartshorn M. Lim J.T. Faget K.Y. Muffat J.A. Scarpa R.C. Chylack Jr., L.T. Bowden E.F. Tanzi R.E. Bush A.I. Biochemistry. 2000; 39: 7266-7275Crossref PubMed Scopus (163) Google Scholar). Such reactions are implicated in cross-linking of crystallins. Because kynurenine-modified crystallins can generate ROS through photochemical reactions (26Parker N.R. Jamie J.F. Davies M.J. Truscott R.J. Free Radic. Biol. Med. 2004; 37: 1479-1489Crossref PubMed Scopus (87) Google Scholar), weakened defenses against oxidative stress due to age or cataract formation could exacerbate lens protein modifications. Glycation is a firmly established mechanism for protein modification during lens aging and cataract formation (7Cheng R. Feng Q. Argirov O.K. Ortwerth B.J. J. Biol. Chem. 2004; 279: 45441-45449Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 9Nagaraj R.H. Sell D.R. Prabhakaram M. Ortwerth B.J. Monnier V.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10257-10261Crossref PubMed Scopus (253) Google Scholar, 27Lyons T.J. Silvestri G. Dunn J.A. Dyer D.G. Baynes J.W. Diabetes. 1991; 40: 1010-1015Crossref PubMed Scopus (193) Google Scholar, 28Padayatti P.S. Ng A.S. Uchida K. Glomb M.A. Nagaraj R.H. Investig. Ophthalmol. Vis. Sci. 2001; 42: 1299-1304PubMed Google Scholar). In this reaction, sugars, ascorbate, and dicarbonyl compounds react with amino groups of lysine and arginine residues on proteins through the formation of ketoamine adducts on proteins (10Baynes J.W. Exp. Gerontol. 2001; 36: 1527-1537Crossref PubMed Scopus (288) Google Scholar, 29Dyer D.G. Blackledge J.A. Katz B.M. Hull C.J. Adkisson H.D. Thorpe S.R. Lyons T.J. Baynes J.W. Z. Ernahrungswiss. 1991; 30: 29-45Crossref PubMed Scopus (86) Google Scholar). These adducts can produce ROS during their modification to advanced glycation end products (11Brownlee M. Metabolism. 2000; 49: 9-13Abstract Full Text PDF PubMed Scopus (271) Google Scholar, 30Ortwerth B.J. Prabhakaram M. Nagaraj R.H. Linetsky M. Photochem. Photobiol. 1997; 65: 666-672Crossref PubMed Scopus (63) Google Scholar). We reasoned that glycation-derived ROS might induce the oxidation of tryptophan and contribute to lens protein modification by kynurenines. The present study was conducted to understand the role of 3OHKYN in lens protein modification and to determine the effect of glycation on tryptophan oxidation. Using a novel monoclonal antibody raised against 3OHKYN-modified keyhole limpet hemocyanin (KLH), we demonstrate that 3OH-KYN-derived products are present in human cataractous lenses, provide structure of a major antigenic product, and show that glycation products do not influence tryptophan oxidation and kynurenine-mediated modification of proteins. Incubation of Proteins with 3OHKYN—Bovine serum albumin (BSA) or ribonuclease A (RNase A) at 10 mg/ml in 0.1 m sodium phosphate buffer (pH 7.4) were incubated under sterile conditions in the dark with 25 mm 3OHKYN for 3 days at 37 °C. The incubation mixture was stirred after every 24 h, and the pH was adjusted to 7.4. The incubated material was then dialyzed against 4 liters of PBS for 48 h at 4 °C. Production of a Monoclonal Antibody against 3OHKYN-derived Modification—KLH at 10 mg/ml in 0.1 m sodium phosphate buffer (pH 7.4) was incubated with 25 mm 3OHKYN for 3 days followed by dialysis against 4 liters of PBS for 48 h at 4 °C. The antibody was prepared according to the method of Oya et al. (31Oya T. Hattori N. Mizuno Y. Miyata S. Maeda S. Osawa T. Uchida K. J. Biol. Chem. 1999; 274: 18492-18502Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Briefly, mice were initially immunized by intraperitoneal injection with 30 μg of 3OHKYN-modified KLH followed by three booster intraperitoneal injections with 20 μg of the modified protein. After the final booster injection, spleen cells were collected and fused with P3/U1 murine myeloma cells using polyethylene glycol. The hybridomas were cultured in hypoxantine/aminopterin/thymidine selection medium. An enzyme-linked immunosorbent assay (ELISA) was used to screen culture supernatants of hybridomas. Microplate wells were coated by incubation for 16 h at 4 °C with one of the following (1 μg/well) in 0.05 m sodium carbonate buffer (pH 9.7): unmodified BSA, unmodified RNase A, 3OHKYN-modified BSA, or 3OHKYN-modified RNase A. The wells were washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 300 μl of 5% nonfat dry milk (NFDM) in PBST for 2 h at room temperature. The wells were then washed three times with PBST and incubated with 50 μl of hybridoma supernatant for 2 h at room temperature in a humid chamber. Following incubation, the wells were washed three times with PBST and incubated with 50 μl of either goat anti-mouse IgG (1:15,000 dilution in PBST) or goat anti-mouse IgM (1:2,500 dilution in PBST) antibody for 1 h as described above. The wells were finally washed three times with PBST and incubated with 100 μl of 3,3′,5′,5′-tetramethylbenzidine substrate (Sigma). The enzyme reaction was stopped by the addition of 50 μl of 2 n H2SO4, and absorption of the chromophore was measured at 450 nm in a Dynex MRX 5000 Microplate Reader. An IgG antibody producing hybridoma with high specificity against 3OHKYN-modified proteins was expanded. We then purified the antibody from the culture medium on a protein G-Sepharose column (Amersham Biosciences) and stored aliquots at –20 °C. The monoclonal antibody was determined to be of IgG1k subclass. Modification of Human Lens Proteins and Nα-Acetyl Amino Acids by Tryptophan Oxidation Products—Water-soluble proteins (WS-HLP) were extracted from human lenses by homogenizing each lens in 2.0 ml of PBS followed by centrifugation at 20,000 × g for 30 min at 4 °C. The supernatant was dialyzed against 4 liters of PBS for 24 h at 4 °C. Samples of this material (5 mg/ml) were then incubated at 37 °C for 7 days in 0.1 m sodium phosphate buffer (pH 7.4) with one of the following: Trp, 3OHKYN, anthranilic acid (AA) (all from Sigma), KYN (Fluka), or NFK. NFK was synthesized according to Simat and Steinhart (32Simat T.J. Steinhart H. J. Agric Food Chem. 1998; 46: 490-498Crossref PubMed Scopus (191) Google Scholar). We also used benzoic acid and 4-hydroxybenzoic acid (HBA) (Sigma) for incubations to test the specificity of the antibody. Trp and its oxidation products were used at 10 times the molar concentration of lysine in lens proteins. After incubation, the mixtures were dialyzed extensively against PBS for 48 h at 4 °C with a change of dialysis medium after 24 h. Nα-Acetyl lysine, Nα-acetyl arginine, Nα-acetyl histidine, and Nα-acetyl cysteine (all from Sigma) were incubated independently with either tryptophan or tryptophan oxidation products at a 5:1 molar ratio for 7 days at 37 °C as described above. Purification of Antigen from the Reaction Mixture of Nα-Acetyl Lysine and 3OHKYN—Nα-acetyl lysine (59 mm) was incubated with 3OHKYN (9.9 mm) in 0.1 m sodium phosphate buffer (pH 7.4) at 37 °C for 7 days under sterile conditions. A sample (250 μl) of the reaction mixture was loaded on semipreparative reversed phase HPLC on a C18 reversed phase column (VYDAC, 218TP1010, 10 μm, 10 × 25 cm, Separations Group, Hesperia, CA). A linear gradient was established with solvent A (0.1% trifluoroacetic acid in water) and solvent B (50% acetonitrile and 0.1% trifluoroacetic acid in water). The following program was applied: 0–5 min, 0% B; 5–50 min, 40% B; 50–60 min, 100% B; 60–68 min, 100% B; and 68–78 min, 0% B with a flow rate of 2.0 ml/min. We used an online UV detector (Jasco Corp., Tokyo, Japan, Model UV-970) to monitor the column effluent for absorbance at 365 nm. Four major fractions were collected and dried in a Savant SpeedVac concentrator (Savant Instruments, Hicksville, NY) and resuspended in 200 μl of water. Each fraction (17 μl/well) was tested in a competitive ELISA for immunoreactivity against the antibody (ELISA procedure described below). The material eluted at ∼30 min had the strongest reaction with the antibody in this assay. To obtain a higher amount of pure antigen, we modified the HPLC method (described above) so that the reaction mixture of Nα-acetyl lysine and 3OHKYN was first passed through a Sep-Pak Light C18 cartridge (Waters, Milford, MA). After the application of the sample, the cartridge was sequentially eluted with stepwise gradient of acetonitrile in 0.1% trifluoroacetic acid. The eluate from 0.1% trifluoroacetic acid in water was collected, dried in a Savant SpeedVac concentrator, and finally suspended in water. This material was then purified with the same semipreparative reversed phase HPLC that we used previously for the crude mixture. A linear gradient of solvent A (0.1% trifluoroacetic acid in water) and solvent B (50% acetonitrile and 0.1% trifluoroacetic acid in water) was used. The program was slightly altered to improve separation of the compound: 0–5 min, 0% B; 5–50 min, 25% B; 50–80 min, 100% B; and 80–88 min, 0% B. The flow rate was 2.0 ml/min. The antigen peak now eluted at around 36 min. We collected this peak from 10 injections of 500 μl of each and pooled. This pooled sample was dried, suspended in water, and reinjected to the same column under same HPLC conditions. A single homogeneous peak at Rt = ∼36 min was obtained by this procedure. This material was lyophilized, and the product was characterized by spectroscopy. One-dimensional and two-dimensional 1H and 13C NMR spectra were recorded on a Varian Inova 600 MHz spectrometer. Samples were dissolved in D2O for 1H-13C correlation experiments (heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC)). Fast atom bombardment-mass spectroscopy analyses were done in the Mass Spectrometry Facility at Michigan State University, East Lansing, MI in a JEOL HX-110 double focusing mass spectrometer. UV-visible spectra were recorded using Amersham Biosciences Spectra Max 190 spectrometer. Treatment of 3OHKYN with Ninhydrin—The procedure was as described by Miyata and Monnier (33Miyata S. Monnier V. J. Clin. Investig. 1992; 89: 1102-1112Crossref PubMed Scopus (216) Google Scholar). 40 mm 3OHKYN was incubated with 10 mm ninhydrin in 300 μl of ethanol/acetic acid, pH 5.0, for 10 min at 65 °C. 25 mml-lysine was then added and incubated at 65 °C for 10 min. The mixture was dried on a Savant SpeedVac concentrator, dissolved in 200 μl of water, and analyzed by an ELISA outlined below. A control experiment in which l-lysine was used in the place of 3OHKYN was run simultaneously. Preparation of Protein-free Filtrate from Human Lenses—Four normal lenses from donors of 30–50 years of age were each homogenized in 2 ml of water and centrifuged at 20,000 × g for 30 min at 4 °C. The supernatant fraction was filtered through a Centricon YM-10 filter (Millipore, Bedford, MA), and the filtrate was lyophilized and reconstituted in 100 μl of water. Incubation of Proteins with Human Lens Protein-free Filtrate—One half of the protein-free filtrate was digested with β-glucosidase (Sigma) to obtain 3OHKYN from its glucoside form. For this step, we used a 1% enzyme solution in 0.04 m sodium phosphate buffer (pH 5.6) and incubated the material for 2 h at 37 °C. The sample was then filtered through a Centricon YM-10 filter to remove the enzyme. The digested and non-digested protein-free filtrates were incubated for 5 days at 37 °C with 1.0 mg of either RNase A or WS-HLP (see above) in 1.0 ml of 0.1 m sodium phosphate buffer (pH 7.4). The incubated samples were then dialyzed against 2 liters of PBS at 4 °C. Finally, 30 μg of modified protein in 50 μl of PBS/well was tested for antigen using a competitive ELISA (see below). ELISA—Microplate wells were coated with 3OHKYN-modified RNase A in 0.05 m carbonate buffer (pH 9.7) at a concentration of 1 μg/50 μl, incubated at 4 °C overnight, and then washed three times with PBST. Before use in the assay, the wells were blocked for 2 h at room temperature with 300 μl of 5% NFDM in PBST and washed three times with PBST. The monoclonal antibody (1:200 diluted in 1% NFDM/PBS for proteins modified Trp oxidation products and 1:60 diluted for amino acids modified by 3OHKYN) was preincubated with the competitor for 2 h at 37 °C and then dispensed into specified wells and incubated for 1.5 h at room temperature. The washed plates were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega Corp., Madison, WI, diluted 1:15,000) for 1 h at 37 °C as described earlier. The enzyme reaction was assessed by the addition of 100 μl of 3,3′,5′,5′-tetramethylbenzidine (Sigma) followed by the addition of 50 μlof2 n H2SO4 and measurement of chromophore absorbance at 450 nm. Results were expressed as the ratio: B/B0, where B is the absorbance in the presence of competitor, and B0 is the absorbance in the absence of competitor. Western Blotting—WS-HLP from cataractous and normal lenses were digested with proteinase K (2% w/w in PBS) for 30 min at 37 °C, aliquots of the digest corresponding to 20 μg of protein were separated on 18% reducing gels, and the proteins were transferred electrophoretically to Immobilon P membranes (Millipore). Comparable gels were stained with BioSafe Coomassie Blue (Bio-Rad). The membranes were then blocked with 5% NFDM in PBS for 2 h and incubated overnight at 4 °C with a 1:20 dilution of the anti-KLH-3OHKYN (10.3 μg/ml) monoclonal antibody. After washing five times for 10 min with PBST, the membranes were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Promega Corp.) diluted 1:15,000. After repeated washing (five times for 10 min with PBST), the membranes were treated with SuperSignal West Pico chemiluminescent substrate (Pierce) for 5 min and exposed to x-ray film (Pierce, CL-XPosure Film). Proteins modified by Trp oxidation products were similarly subjected to Western blotting, except that 12% gels were used and 2 μg of protein/lane was loaded. Immunostaining—HLE-B3 cells (from Dr. Usha Andley, Washington University, St. Louis, MO) (between passages 13 and 19) were cultured in chamber slides with minimal essential medium (MEM) containing 20% fetal bovine serum, 2 mml-glutamine, and 50 μg/ml gentamycin. The cells were washed twice with PBS and treated with MEM containing 0 (control), 20, 50, 100, or 200 μm 3OHKYN in the absence of fetal bovine serum for 24 h. The treated cells were washed twice with PBS and fixed with 4% paraformaldehyde at –20 °C for 15 min. After washing twice with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS at –20 °C for 5 min and then washed five times with PBS to remove the detergent. Next, the slides were blocked with 3% NFDM/1% BSA for 30 min at room temperature and washed twice for 5 min each time with PBS. The slides were incubated with the anti-KLH-3OHKYN antibody (10.3 μg/ml, diluted in 0.1% BSA) or non-immune mouse IgG diluted to the same concentration for 1 h at room temperature and washed twice for 5 min each time. In some experiments, we preincubated the antibody for 1 h at 37 °C with 3OHKYN-modified RNase A (0.2 mg/ml). All slides were incubated with secondary antibody (anti-mouse IgG) conjugated with Oregon Green (Molecular Probes, Eugene, OR). The secondary antibody was diluted in 0.1% BSA/PBS and applied to the slides for 1 h at room temperature. After this incubation, all slides were washed twice for 5 min with PBS and then developed by incubation for 20 min at room temperature with Texas Red-phalloidin (Molecular Probes) diluted in 0.1% BSA/PBS and washed as described above. Finally, all slides were incubated with 4′, 6-diamidino-2-phenylindole (Molecular Probes, diluted in PBS) for 1 min and washed twice for 5 min with PBS. After mounting, the slides were observed with an Olympus System (Model BX60) fluorescence microscope, and images were acquired with an attached digital camera (Diagnostic Instruments, Inc. Spot RT Slider) connected to a Macintosh computer using Spot RT Slider software, version 3.5.5. Immunohistochemistry—Cataractous and age-matched normal lenses from donors between the ages of 65 and 70 years were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5-μm sections. Following dehydration, heat-induced epitope retrieval was done in 10 mm citrate buffer (pH 6.0), and the sections were treated with 3.0% hydrogen peroxide to block endogenous peroxidases. The sections were blocked with 1.5% normal horse serum and incubated for 2 h either with the anti-KLH-3OHKYN antibody (10.3 μg/ml) diluted in PBS or with non-immune mouse IgG, diluted to the same protein concentration as the primary antibody. For adsorption experiments, the antibody was preincubated overnight at 4 °C with 3OHKYN-modified RNase A in PBS (0.4 mg/ml). After washing, the slides were incubated with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) followed by ABC reagent (Vector Laboratories). They were stained initially with 3,3′-diaminobenzidine, counterstained with hematoxylin, and then permanently mounted. Incubation of Nα-Acetyl Tryptophan with Ribated Lysine-Sepharose-4B—To determine whether glycation could catalyze the oxidation of tryptophan, we incubated Nα-acetyl-tryptophan with ribated lysine-Sepharose (Amersham Biosciences). 1 g of lysine-Sepharose was washed thoroughly with 0.1 m sodium phosphate buffer (pH 7.4) containing 0.1 mm EDTA and 1 mm diethylene triamine pentaacetic acid and then incubated at 37 °C for 2 days with 500 mm ribose in the same buffer. The tubes were bubbled with argon for 15 min and sealed before incubation. The ribose-treated gel was extensively washed with PBS to remove unbound ribose. Finally, 250-mg samples of either modified or unmodified (control) gel were incubated at 37 °C with 5.0 mmNα-acetyl tryptophan in 0.1 m sodium phosphate buffer (pH 7.4) for 3, 7, and 10 days. Aliquots were subjected to C18 reversed phase HPLC using a gradient program consisting of solvents A (water with 0.01 m heptafluorobutyric acid) and B (70% acetonitrile in water and 0.01 m heptafluorobutyric acid). The column was eluted with a linear gradient of B from 5 to 50 min at a flow rate of 1.0 ml/min, and the column effluent was monitored for fluorescence at excitation/emission wavelengths of 290/320 nm. Nα-Acetyl tryptophan eluted at Rt = ∼22 min. We also incubated Nα-acetyl tryptophan during glycation; ribose (6.7 mm), lysine (0.6 mm), arginine (0.6 mm), and Nα-acetyl tryptophan (13.2 mm) were incubated for 5 days at 37 °C in 0.1 m sodium phosphate buffer (pH 7.4). Other incubati
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