Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0086543 (cataract)
 29,165 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of these experiments was to determine if truncation and deamidation alter the structure of a human lens protein, beta B1-crystallin. Recombinant wild type and a deamidated form of recombinant beta B1 were expressed in Escherichia coli. Wild type beta B1 was also enzymatically cleaved to generate a physiologically-relevant truncated beta B1. Purity and size of the expressed proteins were confirmed by SDS-PAGE and electrospray ionization mass spectrometry. Size exclusion chromatography and light scattering were used to determine aggregation states of beta B1. Protein conformations were predicted from sedimentation velocity analysis. Molecular weights of 49,000 and 54,000 Da were obtained for wild type beta B1 by sedimentation equilibrium and light scattering, respectively. A sedimentation coefficient of 2.7 S was determined for wild type beta B1. Molecular weights of 54,000 and 60,000 Da were determined for deamidated beta B1 by sedimentation equilibrium and light scattering, respectively. However, deamidated beta B1 eluted earlier than wild type beta B1 on size exclusion chromatography, with an estimated molecular weight between 78,000 and 116,000 Da. Loss of the extensions of beta B1 caused abnormal association of the protein with the stationary phase during size exclusion chromatography. Wild type beta B1 was predicted to form a dimer with an elongated structure. The earlier elution of the deamidated beta B1 dimer on size exclusion chromatography suggested the dimer was less compact. Truncation caused abnormal column interactions suggesting an altered conformation. These changes are important because truncation and deamidation occur extensively in aging human lenses and may be important for senile cataract formation.
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PMID:Deamidation of human beta B1 alters the elongated structure of the dimer. 1118 Sep 77

An autosomal dominant congenital cataract in humans is associated with mutation of Arg-116 to Cys in alphaA-crystallin (alphaA-R116C). The chaperone activity and biophysical properties of reconstituted alpha-crystallin from different proportions of wild-type alphaB-crystallin (alphaB-wt) and alphaA-R116C-crystallin were studied by gel permeation chromatography, SDS-polyacrylamide gel electrophoresis, and fluorescence and circular dichroism spectroscopy and compared with those of reconstituted alpha-crystallin from alphaB-wt and wild-type alphaA-crystallin (alphaA-wt). The reconstituted alpha-crystallin containing alphaA-R116C and alphaB-wt had a higher molecular mass, a higher thermal sensitivity to exposition of Trp side chains, fewer available hydrophobic surfaces, and lower chaperone activity than the alpha-crystallin containing alphaA-wt and alphaB-wt. The secondary structure exhibited very small changes, whereas the tertiary structure was distinctly different for alpha-crystallin formed from alphaA-R116C and alphaB-wt. Most importantly, subunit exchange studies by fluorescence resonance energy transfer showed that alphaA-R116C forms heteroaggregates faster than alphaA-wt with alphaB-wt, and the reconstituted alpha-crystallins were true heteroaggregates of two interacting subunits. These findings suggest that the molecular basis for the congenital cataract with the alphaA-R116C mutation is the formation of highly oligomerized heteroaggregates of alpha-crystallin with modified structure. However, contrary to the earlier conclusions based on the studies of homoaggregates, the loss in chaperone activity of the heteroaggregates having alphaA-R116C does not appear to be large enough to become the main factor in initiating cataract development in the affected individuals.
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PMID:The alphaA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with alphaB-crystallin. 1177 29

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.
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PMID:Heat shock proteins of adult and embryonic human ocular lenses. 1178 56

Eye tissues contain splice variants of muscle-preferred p94 (calpain 3), such as lens-specific Lp82 and Lp85, retina-specific Rt88, and cornea-specific Cn94. The purpose of the present experiment was to analyze the activation and regulation of the best characterized p94 splice variant, Lp82. Recombinant rat Lp82 (rLp82) was expressed using the baculovirus system, purified with Ni-NTA affinity and DEAE-ion exchange chromatographies, and characterized by SDS-PAGE, casein zymography, and immunoblotting. After incubation with calcium, rLp82 autolyzed into two major fragments at approximately 60 and 22 kDa. Sequencing of the autolytic fragments showed loss of three amino acids from the N terminus and cleavage near the IS2 region. Also, Lp82 and calpain 2 were found to hydrolyze each other. Calpastatin inhibited calpain 2 activity, but not Lp82. Homology modeling suggested that the lack of inhibition of Lp82 by calpastatin was due to molecular clashes at the unique AX1 region of Lp82. Lp82 also hydrolyzed calpastatin. These results suggested that Lp82 might regulate other calpain activities and cause hydrolysis of substrates such as crystallins during lens cataract formation.
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PMID:Characterization and regulation of lens-specific calpain Lp82. 1190

BACKGROUND: Development of steroid cataract is a likely outcome following prolonged exposure to glucocorticoids. It has been suggested that formation of steroid-protein adducts is a key event in this lens opacification. In order to explore this possibility, we have monitored the reaction of bovine lens proteins with glucocorticoids and examined the effects of adduct formation on their structures. METHODS: Bovine lens proteins were incubated with high (10(-4) M) and low (10(-8) M) concentrations of dexamethasone or prednisolone for up to 56 days at 37 degrees Celsius. Changes in molecular size and solubility of the crystallins and their polypeptide subunits were examined using gel permeation chromatography and SDS gel electrophoresis. Conformational changes were assessed with the aid of tryptophan fluorescence spectroscopy and oxidation was monitored by measuring protein sulphydryl content. RESULTS: Covalent incorporation of glucocorticoids was observed for all crystallins with relative reactivities for alpha-: beta-: gamma-crystallin of 20: 5: 1. The maximum incorporated was one steroid molecule per 40 to 50 subunits of alpha-crystallin. The proportions and sizes of the soluble crystallins and their subunits were unchanged. Protein sulphydryl contents decreased by eight to 10 per cent more than controls but no intermolecular disulphide bonds were detected. There were no alterations in tryptophan microenvironments. CONCLUSIONS: Steroids form adducts with lens proteins, in particular alpha-crystallin, but it appears unlikely that this reaction is responsible for steroid cataract formation.
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PMID:Steroid adduct formation with lens crystallins. 1248 87

Among polyamines (putrescine, spermidine, and spermine), spermine specifically induces cataract in an organ cultured lens. Spermine uptake nearly paralleled the cataract formation. When polyamines were added to lens soluble proteins, spermine specifically induced turbidity. When lens soluble proteins were separated by gel chromatography, heavy-molecular-weight protein (HMW, high molecular form of alpha-crystallin) and proteins between betaH- and betaL-crystallin fractions reacted with spermine and aggregated. SDS-polyacrylamide gel electrophoresis of the aggregated proteins showed that 43-kDa lens protein was commonly observed in both aggregates. Spermine-affinity chromatography of the total soluble proteins showed the binding of HMW protein to the gel and the chromatogram of the second turbidity peak in the gel chromatography showed the binding of 43-kDa protein. These results indicated that 43-kDa protein, which is present as a subunit in HMW and also in free form, binds spermine and induces turbidity of lens soluble proteins and produces cataract in a cultured lens.
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PMID:Spermine induces cataract and 43-kDa protein that binds spermine possibly participates in the cataract formation. 1252 9

Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions. Fresh intact bovine lenses were incubated with [14C]glucose in a conventional tissue-culture medium with added antibiotics. After 3 and 6 days of incubation, the water-soluble proteins were separated by size-exclusion chromatography. Glycated proteins from the water-soluble fractions were separated by using a sugar affinity column (Affi-Gel 601). Then the radioactive fractions were identified on SDS/polyacrylamide gels. In addition, the whole bovine lenses were incubated with 10 mM fructose and glucose for 3 and 6 days. The glycated proteins from the water-soluble fractions in parallel with the radioactive fractions were separated by affinity chromatography, and were identified further by amino-acid sequencing. A progressive uptake of radioactive label showed that the majority of proteins incorporating both glucose and fructose were water-soluble fractions. Chromatography and SDS/polyacrylamide gel results showed that alpha- and gamma-crystallin and some proteins of a mean molecular mass of 36-37 kDa incorporated sugars early during incubation. After 6 days of incubation, more crystallins were glycated compared with 3 days, in particular beta-crystallin. Affinity-chromatography results indicated that proteins with subunit masses of 36 kDa and 20 kDa were possibly radiolabelled at an early stage. The purified glycated proteins following incubation with both glucose and fructose, which corresponded to 20 kDa and 36 kDa bands on SDS/polyacrylamide gels, were sequenced by Edman degradation. N-terminal sequences of both 20 kDa bands were Gly-Lys-Ile-Thr, characteristic of gamma-crystallins, but the N-termini of both 36 kDa bands were blocked. Further sequencing after digestion of 36 kDa bands with trypsin and running on HPLC revealed that the glucose sample gave the peptide sequences as Gly-Glu-Tyr-Pro-Asp-Tyr-Gln-Gln and Tyr-Glu-Leu-Pro-Asn-Tyr-Arg, which match with bovine gammaIIIb-crystallin. The peptide sequence Tyr-Glu-Leu-Pro-Asn-Tyr-Arg is only present in the published sequence of bovine gammaIIIb-crystallin and not in any other type of gamma-crystallin. The fructose sample gave the peptide sequences Ile-Thr-Phe-Tyr-Glu-Asp-Arg, Arg-Gly-Asp-Tyr-Pro-Asp-Tyr-Gln-Gln-Trp, Gln-Tyr-Leu-Leu-Arg and Val-Val-Asp-Leu-Tyr, which all matched with bovine gammaIIIa-crystallin. The sequence Val-Val-Asp-Leu-Tyr only appears in the sequence of bovine gammaIIIa-crystallin. gammaIII-Crystallin is the most susceptible lens protein to glycation. The primary target of glucose is gammaIIIb-crystallin, whereas that of fructose is gammaIIIa-crystallin. The early glycation of gammaIII-crystallin by glucose and fructose could result in structural alterations, leading to aggregation of crystallin and eventually cataract formation.
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PMID:Gamma III-crystallin is the primary target of glycation in the bovine lens incubated under physiological conditions. 1280 41

Leptospirosis is a widespread zoonotic disease that affects all mammals in different parts of the world. Though there are many commercial kits available for the diagnosis of systemic leptospirosis, the nature of the antigen has not been described. Therefore, identification of a specific antigen is important. Since ocular involvement in leptospirosis has been reported, there is a need to identify and characterize the leptospiral antigen for diagnosis of uveitis associated with past leptospiral infection (leptospiral uveitis) and for confirming the clinical diagnosis. Seven-day-old culture of Leptospira biflexa serovar Patoc was used for preparing the antigen. The present study included serum samples from 81 patients with clinical criteria for leptospiral uveitis, 15 cataract controls and 15 non-leptospiral uveitis controls. Serum samples were assayed by ELISA using our antigenic preparation and by a microscopic agglutination test (MAT) using 19 serovars. The antigen prepared had 280 micro g LPS ml(-1) and no detectable amount of protein. Silver-staining of SDS-PAGE for protein and LPS, dot blot and Western blot analysis and proteinase K and periodate treatment showed that LPS (13-21 kDa and 28 kDa) in our preparation was the relevant antigen for serodiagnosis. IgG antibodies showed reactivity in both leptospiral uveitis patients and controls. However, on the basis of IgM response to LPS, 48 % of the leptospiral uveitis patients were significantly positive compared with controls; 58 % of leptospiral uveitis patients and none of the controls were positive for MAT. When MAT and IgM ELISA results were considered together, 77 % were significantly positive. LPS is identified as a candidate antigen for serodiagnosis of leptospiral uveitis and has sensitivity and specificity of 48 and 90 %, respectively, in ELISA for IgM antibodies. Confirmation of clinical diagnosis with a specific laboratory test would help to initiate the most appropriate treatment for leptospiral uveitis.
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PMID:Identification and evaluation of LPS antigen for serodiagnosis of uveitis associated with leptospirosis. 1286 60

In an attempt to model the processes of free radical-mediated cataractogenesis, we investigated the oxidative modification of rat eye lens proteins by peroxyl radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) under aerobic conditions. When incubated with AAPH, the soluble eye lens proteins precipitated in a time-dependent manner. The insolubilisation was accompanied by the accumulation of protein free carbonyls and the diminution of sulfhydryls, yet the processes were shifted in time. The SDS-PAGE analysis of the AAPH-treated proteins revealed the presence of high molecular weight cross-links and, to a lesser extent, fragments. The aggregation and cross-linking of proteins along with the generation of free carbonyls was significantly inhibited by the chain-breaking antioxidants stobadine and Trolox. On the other hand, the AAPH-initiated sulfhydryl consumption was much less sensitive to the antioxidants studied. The results point to a complex mechanism of peroxyl-radical-mediated modification of eye lens proteins with implications for cataract development and they indicate a potentially protective role of antioxidants.
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PMID:Oxidative modification of rat eye lens proteins by peroxyl radicals in vitro: protection by the chain-breaking antioxidants stobadine and Trolox. 1595 60

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K(50%act)=20 microM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on alphaA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.
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PMID:Biochemical properties of lens-specific calpain Lp85. 1605 32


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