Lens Soluble Fraction Research Articles

Effect of chronic hyperglycemia on crystallin levels in rat lens

Crystallins are the major structural proteins in the vertebrate eye lens that contribute to lens transparency. Although cataract, including diabetic cataract, is thought to be a result of the accumulation of crystallins with various modifications, the effect of hyperglycemia on status of crystallin levels has not been investigated. This study evaluated the effect of chronic hyperglycemia on crystallin levels in diabetic cataractous rat lens. Diabetes was induced in rats by injecting streptozotocin and maintained on hyperglycemia for a period of 10weeks. At the end, levels of α-, β-, γ-crystallins and phosphoforms of αB-crystallins (αBC) were analyzed by immunoblotting. Further, solubility of crystallins and phosphoforms of αBC was analyzed by detergent soluble assay. Chronic diabetes significantly decreased the protein levels of α-, β- and αA-crystallins (αAC) in both soluble and insoluble fraction of lens. Whereas γ-crystallin levels were decreased and αBC levels were increased in lens soluble fraction with no change in insoluble fraction in diabetic rat lens. Although, diabetes activated the p38MAPK signaling cascade by increasing the p-p38MAPK in lens, the phosphoforms of αBC were decreased in soluble fraction with a concomitant increase in insoluble fraction of diabetic lens when compared to the controls. Moreover, diabetes strongly enhances the degradation of crystallinsand phosphoforms of αBC in lens. Taken together, the decreased levels of crystallins and insolubilization of phosphoforms of αBC under chronic hyperglycemia could be one of the underlying factors in the development of diabetic cataract.

The critical role of the central hydrophobic core (residues 71–77) of amyloid-forming αA66-80 peptide in α-crystallin aggregation: a systematic proline replacement study

Age-related cataract formation is marked by the progressive aggregation of lens proteins. The formation of protein aggregates in the aging lens has been shown to correlate with the progressive accumulation of a range of post-translational crystallin modifications, including oxidation, deamidation, racemization, methylation, acetylation, N- and C-terminal truncations and low molecular weight (LMW) crystallin fragments. We found that an αA-crystallin-derived peptide, αA66-80 (1.8 kDa), is a prominent LMW peptide concentrated in water-insoluble fractions of the aging lens. The peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from lens-soluble fractions. It binds at multiple sites and forms a hydrophobically driven non-covalent complex with α-crystallin to induce α-crystallin aggregation. To define the specific role of the αA66-80 peptide in age-related protein aggregation and cataract formation, it is important to understand the mechanisms by which this peptide acts. We used scanning proline mutagenesis to identify which particular sequences of the peptide drive it to form amyloid-like fibrils and induce α-crystallin aggregation. The secondary structure and the aggregate morphology of the peptides were determined using circular dichroism and transmission electron microscopy, respectively. Peptides were also tested for their ability to induce α-crystallin aggregation. We found that proline replacement of any residue in the sequence FVIFLDV, which corresponds to residues 71–77, led to an absence of both fibril formation and α-crystallin aggregation. The apparently critical role of 71–77 residues in αA66-80 explains their significance in the self-assembly processes of the peptide and further provide insights into the mechanism of peptide-induced aggregation. Our findings may have applications in the design of peptide aggregation inhibitors.

Isolation of ascorbate free radical reductase from rabbit lens soluble fraction

Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8·4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of p I 7·0–8·6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b 5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.

Ascorbate Free Radical Reductase Activity in Vertebrate Lenses of Certain Species

Purpose: To clarify the function of ascorbate free radical (AFR) reductase in the antioxidation system of different vertebrate lenses. Methods: The soluble and insoluble fractions were prepared from bullfrog, guinea pig, rat, rabbit, swine, and bovine lenses, and membrane-bound enzymes in the insoluble fraction were extracted by 0.3% Triton X-100. Ascorbate free radical reductase and diaphorase activities in each fraction were determined. Results: Ascorbate free radical reductase activity in the lens soluble fraction was the highest in the bullfrog. That in the guinea pig and rabbit was at the next level. There was only a little activity in rat and swine lenses, and none was detected in the bovine lenses. However, a large species difference in AFR reductase activity was not observed in the 0.3% Triton X-100 extracts. Diaphorase activity was three to nine higher than AFR reductase activity in the soluble fractions of bullfrog, guinea pig, and rabbit. In the 0.3% Triton X-100 extracts of all animal species used, it was very high, 108 to 311 times the AFR reductase activity. Conclusion: These results indicate that the lens soluble and membrane-bound AFR reductase in the different animals may be individual enzyme molecules and have different antioxidative functions. Because the lenses of bullfrog, guinea pig, and rabbit are known to contain a near-ultraviolet (UV) light-absorbing compound, reduced pyridine nucleotide, at a high concentration, the soluble AFR reductase activity is expected to be high in the vertebrate lenses with a near-UV light filter, to enhance the antiphoto-oxidation capacity of ascorbate.

Ascorbate Free Radical Reductase Activity in Vertebrate Lenses of Some Species

Purpose: To clarify the function of ascorbate free radical (AFR) reductase in the lens antioxidation mechanism, we investigated the difference among species in AFR reductase activity in different vertebrate lenses. Materials and Methods: Soluble and insoluble fractions were prepared from the lenses of frogs, guinea pigs, rats, rabbits, pigs, and calves. AFR reductase and diaphorase activity of each fraction was determined. Results: AFR reductase activity in the lens soluble fraction was the highest in frogs. That of guinea pigs and rabbits was at the next level; there was only a little activity in rats and pigs, and none was detected in calves. Membrane-bound AFR reductase in the lens insoluble fraction was extracted by 0.3% Triton X-100. The membrane-bound enzyme activity was almost at the same level in frogs, rats, rabbits, and calves, and a little higher in guinea pigs and pigs. However, such species-specificity of AFR reductase activity as in the soluble fraction was not observed in 0.3% Triton X-100 extracts. Diaphorase activity was 3 to 9 times as much as AFR reductase activity in the soluble fractions of frogs, guinea pigs, and rabbits, but in 0.3% Triton X-100 extracts of all vertebrate species used, it was very high, 108 to 311 times the AFR reductase activity. Conclusion: These results suggest that the lens soluble and membrane-bound AFR reductases are individual enzyme molecules and have different anti-oxidative functions. The lenses of frogs, guinea pigs, and rabbits contain a near-ultraviolet (UV) light absorbing compound, reduced pyridine nucleotide at a high concentration. Therefore, the soluble AFR reductase activity may be high in the vertebrate lenses with a near-UV light filter, and enhance the antiphotoxidation of ascorbic acid.

Acetyl- [formula omitted] -Carnitine Decreases Glycation of Lens Proteins: in vitro Studies

Although the role of carnitine system in the ocular tissues is not clearly understood, earlier studies showed that lenticular levels of l -carnitine were the highest among ocular tissues and there was a dramatic depletion of lenticular l -carnitine and acetyl- l -carnitine in streptozotocin-diabetic rats. As protein glycation has been implicated in the development of several diabetic complications including cataracts, this study was initiated to show the possible effects of l -carnitine and acetyl- l -carnitine on the glycation and advanced glycation (AGEs) of lens proteins. Calf lens soluble fraction (crystallins) was incubated with 50 m m glucose (containing14C glucose) with or without 5–50 m ml -carnitine, 5–50 m m acetyl- l -carnitine and 5–50 mm acetyl salicylic acid, for 15 days. The results show that while l -carnitine did not have any effect on in vitro glycation of lens crystallins, acetyl- l -carnitine and acetyl salicylic acid decreased crystallin glycation by 42% and 63%, respectively—this decrease was concentration dependent. Glycated crystallins were separated on HPLC which showed that the rate of glycation is in the following order: α>β>γ. Interestingly, acetyl- l -carnitine inhibited glycation of α crystallin more than other crystallins. In vitro incubations with [3H-acetyl] acetyl- l -carnitine showed that acetyl- l -carnitine acetylates lens crystallins (non-enzymatically) and α crystallin is the major acetylated protein. Furthermore, there was a 70% reduction in anti-AGE antibody reactivity when 50 m m acetyl- l -carnitine was included in the incubation of lens crystallins and 10 m m erythrose, suggesting that inhibition of glycation by acetyl- l -carnitine also affected the generation of AGEs. This in vitro study shows, for the first time, that acetyl- l -carnitine could acetylate potential glycation sites of lens crystallins, and protect them from glycation-mediated protein damage.

Inhibition of Cataracts in Moderately Diabetic Rats by Aminoguanidine

The effect of aminoguanidine (AG), an inhibitor of advanced glycation, on the development of cataracts was studied in diabetic rats. Rats were made diabetic with streptozotocin, and based on the level of plasma glucose they were grouped as moderately (<350mgdl -1plasma glucose) and severely (>350mgdl -1plasma glucose) diabetic. One half of the animals in each group received AG (25mgkg -1body weight each day), intraperitoneally, starting from the day of streptozotocin injection. Progression of lens opacification was recorded using Fundus and Scheimpflug photography at regular time intervals. On the ninetieth day all the rats were killed and the levels of advanced glycation end products (AGE) was determined by measuring the non-tryptophan fluorescence of the lens soluble and insoluble fractions. Densitometric analysis of Scheimpflug images showed that in diabetic rats lens opacification progressed in a biphasic manner, an initial slow progression for the first 60 days, followed by a steep increase during next 30 days. Moderately and severely diabetic rats developed lens opacities more or less at the same time. AGE fluorescence in the lens soluble fractions increased three-fold and seven-fold in the moderately and severely diabetic rats, respectively; whereas in insoluble fractions there was a 30% and three-fold increase in the moderately and severely diabetic rats, respectively. Although AG treatment inhibited the AGE fluorescence of lens soluble and insoluble fractions by about 56% and 75% in moderately diabetic and by 19% and 52% severely diabetic rats, respectively, the development of cataracts was delayed only in the moderately diabetic rats. These results thus suggest that the effect of AG is indeed inhibition of the formation of AGEs. However, in the severely diabetic rats the beneficial effect of AG is overwhelmed by the excessive accumulation of AGEs.

Glycation Mediated Lens Crystallin Aggregation and Cross-linking by Various Sugars and Sugar Phosphates In Vitro

Glycation of lens crystallins results in protein conformational changes, oxidation, browning and aggregation. Though glucose is the major sugar, other sugars and sugar phosphates generated as intermediates of metabolic pathways are present in the lens, albeit at low concentrations. In this study we incubated bovine lens soluble fraction with various sugars and sugar phosphates (5 mM for 10 days). The reactivity was in the order trioses > tetroses > penroses > hexoses. High molecular weight (HMW) aggregates were also formed at a comparable rate. Increased levels of fluorescence were associated with the HMW aggregates with fast reacting sugars. The phosphorylated derivatives were only slightly more reactive than their respective sugars. Interestingly, fructose-1,6-diphosphate was more reactive and cross-linked more readily than fructose-6-phosphate. Gel electrophoresis under reducing and nonreducing conditions showed formation of disulfide linked protein aggregates with slow reacting sugars such as glucose and non-disulfide covalent linked protein aggregates with fast reacting sugars such as erythrose. In contrast, if 0·1 M DTT was present in erythrose incubations (a fast reacting sugar), the HMW aggregate formation was significantly reduced. In order to show the reactivity among the slow reacting hexoses, we incubated lens proteins with 1 M hexoses for 30 days and the results showed that galactose was more reactive and showed higher cross-linking than fructose and glucose. These results thus indicate that relatively low levels of some sugars and sugar phosphates in the lens could be compensated by enhanced lens protein cross-linking and the combined effect could be rather significant with respect to cataractogenesis.

In vitro glycation and acetylation (by aspirin) of rat crystallins

In vitro studies with rat lens crystallins were conducted to explore the mechanism by which aspirin (ASA-acetylsalicylic acid) could inhibit cataractogenesis. The purpose of the present study is to show whether γ-crystallin is the primary target for glycation by glucose and acetylation by ASA. Lens soluble fractions from one and seven month old Sprague-Dawley rats were incubated with 5 mM [ 14C]glucose with and without 10 mM ASA. α, β, and γ-crystallins were separated by molecular sieve HPLC and specific activities of each crystallin determined. In vitro acetylation was also studied by measuring protein bound [ 14C]acetyl groups after incubation with [ 14C]acetyl ASA. There was 2 to 4-fold faster glycation of γ-crystallin than all other crystallins from 1-month-old rats and ASA inhibited glycation of γ-crystallin four times more than that of α and β-crystallins, thus showing preferential glycation of γ-crystallin and its selective inhibition by ASA. [ 14C]acetyl incorporation showed increased acetylation of γ-crystallin in one month old rats, whereas in older lenses acetylation of other crystallins predominated. Treatment with 10 mM ASA showed 35% decrease in free -NH 2 groups but protein thiols remained unchanged.

Differential glycation of rat α-, β- and γ-crystallins

Crystallin glycation seems to play an important role in the development of diabetic cataract. In order to understand the role of glycation in cataractogenesis, levels of glycation of different crystallins were determined by in vitro glycation of rat lens soluble fraction with 50 m m glucose or glucose-6-phosphate (G6P) for up to 5 days and in streptozotocin-diabetic rats during various stages of cataract development. All samples were reduced with [ 3H]NaBH 4 and the tritium incorporation was taken as a measure of glycation. Proteins were routinely separated by molecular sieve HPLC. In vitro studies with glucose showed that γ-crystallin was readily glycated and reached a plateau by 3 days, while α- and β-crystallins were glycated slowly initially up to 3 days followed by a steep increase as seen on the fifth day. Incubation with 50 m m G6P resulted in an approximately two fold increase in glycation compared to glucose of all crystallins. In the diabetic animals also γ-crystallin glycation increased approximately twofold within 15 days after the onset of diabetes and an additional threefold within the next 45 days followed by a slight decrease during the following 90–120 days. Increase in glycation, on the contrary, was very slow up to 30 days for α-crystallin and up to 60 days for β-crystallin, followed by a steep increase during the remainder of the experimental period. The high molecular weight (HMW) aggregates had higher levels of glycation than other proteins; the insoluble HMW aggregates contained higher levels of glycation than the soluble HMW aggregates. Protein conformational changes may be involved in facilitating the glycation of polymeric α-and β-crystallins with increasing duration of glycation, while the monomeric γ-crystallin is readily glycated even from the beginning.