Oxidação de resíduos de triptofano em proteínas: formação de ligação cruzada ditriptofano e implicações patofisiológicas / Oxidation of tryptophan residues in proteins: formation of the ditryptophan cross-link and pathophysiological implications

Apesar de extensa investigação das modificações oxidativas irreversíveis sofridas pelas proteínas in vitro e in vivo, os produtos formados pela oxidação de resíduos de triptofano ainda permanecem apenas parcialmente conhecidos. Recentemente, nosso grupo caracterizou uma ligação cruzada de ditriptofano produzida pela recombinação de radicais hSOD1-triptofanila gerados pelo ataque do radical carbonato produzido durante a atividade peroxidásica da enzima superóxido dismutase humana (hSOD1). Neste trabalho, examinamos se a ligação ditriptofano pode ser formada em outras proteínas, além da hSOD1 e por outros oxidantes, além do radical carbonato. A lisozima da clara do ovo e a beta cristalino bovina foram utilizadas como alvos de oxidação. A lisozima foi utilizada por ser uma enzima pequena (129 aminoácidos) e de estrutura bem conhecida, contendo seis resíduos de Trp. Os resultados mostraram que o radical carbonato, gerado enzimatica ou fotoliticamente, promove a oxidação, dimerização e inativação da lisozima. Os principais produtos de oxidação caracterizados por análise de nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano e N-formilquinurenina juntamente com um dímero de lisozima (lisozima-Trp28-Trp28-lisozima) e um hetero dímero lisozima-hSOD1 (lisozima-Trp28-Trp32-hSOD1), ambos ligados por uma ligação ditriptofano. Também demonstramos que a irradiação da lisozima com luz UVC leva à formação do dímero lisozima-Trp28-Trp28-lisozima. Em consequência, resolvemos tratar a beta cristalino bovina com radical carbonato gerado fotoliticamente ou com luz UVC, e a proteína também sofreu oxidação, dimerização e agregação. Os principais produtos de oxidação caracterizados por nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano, N-formilquinurenina, DOPA e um dímero de beta cristalino (ßB2-Trp151-Trp151-ßB2). A irradiação com luz UVC também levou à formação de um dímero intra-cadeia, caracterizado como ßA2-Trp78-Trp81. Quando a beta cristalino foi irradiada com um simulador de luz solar (UVA e UVB) também foi possível observar um dímero, caracterizado como ßA2-Trp150-Trp150-ßA2. A presença de produtos de oxidação de resíduos de Trp, dentre eles a ligação cruzada ditritpofano, também foi avaliada in vivo, utilizando o cristalino de pacientes que foram submetidos a cirurgia para remoção de catarata. Beta, alfa e gama cristalino foram as principais proteínas identificadas nas frações solúvel e insolúvel do cristalino. A principal modificação pós-traducionais identificada foi deamidação. Um alto conteúdo de resíduos de metionina e triptofano oxidados foram identificados nas proteínas presentes na fração insolúvel. Os principais produtos de oxidação de Trp identificados por nano-ESI-Q-TOF-MS/MS foram quinurenina e N-formilquinurenina. A presença de dímeros covalentes no cristalino com catarata foi confirmada por análises de massas. A completa caracterização desses dímeros (ßB1-Trp127-Trp127-ßB1 e ßB1-Trp193-Trp193-ßB1) confirmou que as cadeias polipeptídicas foram ligadas por uma ligação ditriptofano. Em síntese, nossos dados demonstraram que o radical carbonato e a luz UV podem produzir dímeros de ditriptofano em diferentes proteínas. Também, a presença da ligação cruzada de ditriptofano in vivo (catarata humana) foi pela primeira vez detectada

Despite extensive investigation of irreversible oxidative modifications suffered by proteins in vitro and in vivo, the products formed by oxidation of tryptophan residues remain partially characterized. Our group recently described a ditryptophan cross-link produced by recombination of hSOD1-tryptophanyl radicals generated by attack of the carbonate radical produced during the peroxidase activity of the human superoxide dismutase (hSOD1) enzyme. Here, we examine whether the ditryptophan cross-link can be produced in others proteins besides the hSOD1 and by other oxidants, in addition to the carbonate radical. The egg white lysozyme and bovine beta crystalline were used as targets. Lysozyme was used because it is a small enzyme (129 amino acids) with a well-known structure, containing six Trp residues. The results showed that the carbonate radical, generated enzymatically or photolytically, promotes lysozyme oxidation, inactivation and dimerization. The major oxidation products characterized by nano-ESI-Q-TOF-MS/MS analysis were hydroxy-tryptophan and N-formylkynurenine together with a dimer of lysozyme (lysozyme-Trp28-Trp28-lysozyme) and a hetero dimer hSOD1-lysozyme (lysozyme-Trp28-Trp32-hSOD1), both bound by a ditryptophan cross-link. Also, it was demonstrated that lysozyme irradiation with UVC light leads to the formation of the dimer lysozyme-Trp28-Trp28-lysozyme. In view of these results, we decided to treat beta crystalline bovine with photolytically generated carbonate radical and UVC. Beta crystalline also suffered oxidation, dimerization and aggregation. The major oxidation products characterized were hydroxy-tryptophan, N-formylkynurenine, DOPA and a beta crystalline dimer (ßB2-Trp151-Trp151-ßB2) by nano-ESI-Q-TOF-MS/MS. Irradiation with UVC light also led to the formation of an intra-chain dimer, which was characterized as ßA2-Trp78-Trp81. When beta crystalline was irradiated with a solar simulator (UVA and UVB), it was also possible to observe a dimer which was characterized as ßA2-Trp150-Trp150-ßA2. The presence of oxidized tryptophan products, including the ditryptophan cross-link, was also evaluated in vivo in the lenses of patients submitted to cataract removal. Beta, alpha and gamma crystalline were the main proteins identified in soluble and insoluble fractions of the lenses. The main post translational modification identified was deamidation. A high content of oxidized methionine and tryptophan residues were identified in proteins present in the insoluble fraction. The main tryptophan oxidation products identified by nano-ESI-Q-TOF-MS/MS were kynurenine and N-formylkynurenine. The presence of covalent dimers in the lenses with cataract was demonstrated by mass analysis. Full MS/MS characterization of the dimers ßB1-Trp127-Trp127-ßB1 and ßB1-Trp193-Trp193-ßB1 confirmed that they were linked by a ditryptophan bond. In summary, our data demonstrate that the carbonate radical and UV light can produce ditryptophan dimers in different proteins. Also, the presence of the ditryptophan cross-link was first detected in vivo (human cataract)

Reproducibility of Peripapillary Retinal Nerve Fiber Layer Thickness Measured by Spectral Domain Optical Coherence Tomography in Pseudophakic Eyes

PURPOSE: To assess the reproducibility of circumpapillary retinal nerve fiber layer (cpRNFL) thickness measurement (measurement agreement) and its color-coded classification (classification agreement) by Cirrus spectral domain optical coherence tomography (OCT) in pseudophakic eyes. METHODS: Two-hundred five participants having glaucoma or glaucoma suspected eyes underwent two repeated Cirrus OCT scans to measure cpRNFL thickness (optic disc cube 200 x 200). After classifying participants into three different groups according to their lens status (clear media, cataract, and pseudophakic), values of intra-class coefficient (ICC), coefficient of variance, and test-retest variability were compared between groups for average retinal nerve fiber layer (RNFL) thicknesses and that corresponding to four quadrant maps. Linear weighted kappa coefficients were calculated as indicators of agreement of color code classification in each group. RESULTS: ICC values were all excellent (generally defined as 0.75 to 1.00) for the average and quadrant RNFL thicknesses in all three groups. ICC values of the clear media group tended to be higher than those in the cataract and pseudophakic groups for all quadrants and average thickness. Especially in the superior and nasal quadrants, the ICC value of the cataract group was significantly lower than that of the clear media and pseudophakic groups. For average RNFL thickness, classification agreement (kappa) in three groups did not show a statistically significant difference. For quadrant maps, classification agreement (kappa) in the clear media group was higher than those in the other two groups. CONCLUSIONS: Agreement of cpRNFL measurement and its color code classification between two repeated Cirrus OCT scans in pseudophakic eyes was as good as that in eyes with clear crystalline lens. More studies are required to ascertain the effect of lens status on the reproducibility of Cirrus OCT according to different stages of glaucoma patients.

Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo

The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor-kappaB (NF-kappaB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-one-week-old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-kappaB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal-treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-kappaB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-kappaB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-kappaB-dependent and pro-apoptotic.

Mechanisms of Apoptosis on Human Lens Epithelium after Ultraviolet Light Exposure

PURPOSE: The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes. METHODS: Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway. RESULTS: Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased. CONCLUSIONS: Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.

Trans-differentiation of iris pigmented epithelial cells of Euphlyctis cyanophlyctis tadpoles into lens in vitro.

Meshed pigmented iris epithelium along with neural retina of tadpoles of the frog Euphlyctis cyanophlyctis were found to undergo dedifferentiation and subsequently transdifferentiate into lens in culture medium. During lag period, depigmentation (dedifferentiation) occurred in many cells. When culture became confluent 3-4 weeks after seeding tiny lens like structures differentiated from foci of cultured pigmented iris epithelium cells. The percentage of lens formation was higher in vitamin A treated cases. The culture system appears to be a suitable for investigating the changes occurred during trans-differentiation of pigmented epithelial cells into lens.

Protective Effects of Epigallocatechin Gallate After UV Irradiation of Cultured Human Lens Epithelial Cells

PURPOSE: To evaluate the protective effects of epigallocatechin gallate (EGCG) against UV irradiation of cultured human lens epithelial cells. METHODS: We irradiated cultured human lens epithelial cells with a 30-second pulse from a UV lamp with an irradiance of 0.6 mW/cm2. Five minutes and 1 hour after UV irradiation, we administered 0, 5, 10, 15, 25, 50, or 100 uM EGCG. The cell number was measured with a microscopic counting chamber and cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Compared to untreated cells, the total number of cultured human lens epithelial cells was markedly higher after UV irradiation. In a dose-dependent manner, viability was also higher in EGCG-treated cells. CONCLUSIONS: EGCG increased the cell count and cell viability after UV irradiation of cultured human lens epithelial cells, indicating that EGCG can protect lens epithelium against UV damage.

The Efficacy of an Acrylic Intraocular Lens Surface Modified with Polyethylene Glycol in Posterior Capsular Opacification

To investigate if the surface modification of intraocular lens (IOL) is efficient in the prevention of posterior capsular opacification (PCO), the acrylic surface of intraocular lens (Acrysof(R)) was polymerized with polyethylene glycol (PEG-IOL). The human lens epithelial cells (1x10(4) cells/mL) were inoculated on PEG grafted or unmodified acrylic lenses for the control. The adherent cells on each IOL surface were trypsinized and counted. The every PEG-IOL was implanted in 20 New Zealand rabbits after removal of crystalline lens. The formations of PCO were checked serially through retroilluminated digital photography, and the severity scores were calculated using POCOman(R). The cell adherence patterns on each IOL were examined by scanning electron microscopy. As a result, the mean number of adherent cells of PEG-IOL (3.2+/-1.1x10(3)) tended to be smaller than that of the acrylic controls (3.6+/-1.9x10(3)) without a statistical significance (p=0.73). However, the mean severity of PCO formation in PEG-IOL was significantly lower than that in the control during the third to sixth weeks after surgery. Scanning electron microscopy revealed that the more patch-like cells were found firmly attached to the IOL surface in control than in the PEG-IOL. Conclusively, PEG polymerization to the acrylic IOL would possibly lessen the formation of PCO after cataract removal.

IGF-1 Counteracts TGF-beta-Mediated Enhancement of Fibronectin for in Vitro Human Lens Epithelial Cells

PURPOSE: To determine whether insulin-like growth factor (IGF-1) affects transforming growth factor (TGF-beta)- mediated fibronectin accumulation in human lens epithelial cell line (HLE B-3) cells. MATERIALS AND METHODS: HLE B-3 cells were incubated for 24 hours with TGF-beta (10ng/ mL), IGF-1 (10ng/mL), or both. Expression of the fibronectin gene was determined using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Fibronectin levels were examined using western blot analysis and immunofluorescence staining. RESULTS: Expression of the fibronectin gene was not different between the TGF-beta/IGF-1 treated group and the TGF-beta treated group (p= 0.116). However, western blot analysis demonstrated decreased fibronectin levels in human lens epithelial cells treated with TGF-beta and IGF-1 compared to those treated with TGF-beta only (p < 0.01). Immunofluorescence staining disclosed inhibition of TGF-beta-induced fibronectin in the presence of IGF-1. CONCLUSION: This study suggests that IGF-1 counteracts TGF-beta-mediated fibronectin accumulation in human lens epithelial cells.

Expression of transforming growth factor-beta in cultured normal human lens epithelia cells / 华中科技大学学报（医学）（英德文版）

In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor beta (TGF-beta), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-beta mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-beta immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-beta, and LEC could be affected by TGF-beta through autocrine action.

Transdifferentiation of Cultured Bovine Lens Epithelial Cells into Myofibroblast-like Cells by Serum Modulation

An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT) -related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.

Possible Role of Amyloid beta- (1-40) -BSA Conjugates in Transdifferentiation of Lens Epithelial Cells

We investigated whether amyloid beta (Abeta) aggregates have transforming growth factor beta- like cytokine activity and cause transdifferention of lens epithelial cells, leading to certain types of cataract. In order to mimic Abetaaggregates, Abeta- (1-40) was crosslinked to bovine serum albumin (BSA) with disuccinimidyl suberate according to a previously described procedure. When human lens epithelial B-3 (HLE B-3) cells were treated with the Abeta- (1-40) -BSA conjugates, we observed the translocation of Smad-3, as well as the induced mRNA levels of fibronectin (FN), collagen type I (Col I), smooth muscle actin (SMA) and matrix metalloproteinase-2 (MMP-2). In addition, we investigated the morphology of rat whole lens cultured for 5 days in the presence of Abeta- (1-40) -BSA, and the immunohistochemical localizations of Abeta- (1-40) /amyloid precursor protein (APP) in human clinical tissues beneath the anterior capsules. In rat whole lens cultures, treatment with Abeta- (1-40) -BSA produced a transformed morphology that had multiple layers of lens epithelial cells. To compare the anterior capsules in anterior subcapsular cataracts with those in nuclear cataracts, immunohistochemical studies of Abeta/APP in human clinical tissues revealed that the predominant immunostaining of Abeta occurs in the anterior epithelial plaques, which likely produces the abnormal extracellular matrix. Thus, these findings suggest that Abeta aggregates in vivo are possibly involved in the regulatory process by which lens epithelial cells may transdifferentiate into fibroblast-like cells, as well as help understand the mechanisms which lead to certain types of cataractogenesis.

The effective concentration and exposure time of mitomycin-C for the inhibition of lens epithelial cell proliferation in rabbit eyes

The proliferation of residual lens epithelial cells following cataract surgery is assumed to be a major cause of posterior capsular opacification. To assess the efficacy of mitomycin-C in preventing posterior capsular opacification, we determined the effective concentration and exposure time of mitomycin-C in inhibiting rabbit lens epithelial cell proliferation. The fourth-passaged rabbit lens epithelial cells were maintained for one day and then exposed to mitomycin-C for 1, 2, 3, and 5 minutes, respectively. There were 9 different plating concentrations of mitomycin-C with two-fold serial dilution. The maintenance of the phenotypic properties of lens epithelial cells was confirmed by continuous transcription of lambda-crystalline mRNA determined by reverse transcription-polymerase chain reaction and the polymorphism of the restriction fragment. Cell proliferation was assayed with 3H-thymidine incorporation into DNA. The fourth-passaged cells maintained the expression of lambda-crystalline mRNA, suggesting that they are phenotypically authentic lens epithelial cells. The effective concentrations and exposure time of mitomycin-C were 0.1 mg/ml for 1 minute and 2 minutes, and 0.025 mg/ml for 2 minutes. By these results, we postulated that mitomycin-C at relatively short incubation times could be clinically used for prevention of posterior capsular opacification after cataract surgery.