Downregulation of ß-actin gene and human antigen R in human keratoconus.

PURPOSE: The purpose of this study was to determine the expression levels and regulation of ß-actin in the stroma of keratoconus (KC) and normal corneas. METHODS: A total of 15 different human corneas from both KC and normal individuals were used for this study. Additionally, 3 Fuch's dystrophic corneas were also used. The ß-actin gene expression was analyzed at the transcriptional and translational levels in the epithelium and stroma of the KC and normal corneas. The human antigen R (HuR) gene expression was analyzed by real-time PCR in the stroma of five KC and five normal corneas. The keratocytes from three normal and three KC corneas were cultured in the presence of serum, and the expression levels of ß-actin and human antigen R (HuR) were analyzed by using confocal imaging in both normal and KC fibroblasts. RESULTS: The expression of the ß-actin gene was downregulated in the stroma of the six KC corneas but not in the stroma of six normal and Fuchs' dystrophic corneas. Immunofluorescence detection of ß-actin showed that it was absent in the KC fibroblast. The real-time PCR analysis of the HuR gene showed a relative 4.7-fold lower expression in KC corneas relative to the normal corneas, which was further confirmed by the immunofluorescence detection of HuR in fibroblasts of KC corneas. CONCLUSIONS: Although ubiquitous ß-actins are essential for cell survival during early embryogenesis, the effects on various stages of development are not well understood. Our results show that ß-actin is downregulated in the corneal stroma of patients with KC, which may be related to reduced levels of a stabilizing factor (HuR) for ß-actin mRNA. We propose that loss of ß-actin in the corneal stroma might be a triggering factor in the development of KC.

High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function.

MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)-MS using nanoLC (nano-liquid chromatography)-ESI (electrospray ionization)-MS or direct-infusion ESI-MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas.

PURPOSE: The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus (KC) corneas compared to normal corneas. METHODS: Two-dimensional (2-D) gel electrophoretic profiles of epithelial cell proteins from normal and keratoconus corneas were compared, and the selected protein spots that showed either up- or downregulation were identified. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, two proteins, alpha-enolase and beta-actin, were further analyzed by immunohistochemical and western blot methods, using respective antibodies. To determine the presence of mRNA of the two proteins in the epithelial cells, RT-PCR studies were performed. RESULTS: On comparison of the 2-D gel electrophoretic protein profiles, two protein spots were identified in normal corneas that were either absent or present at lower levels in keratoconus corneas. The two spots were determined to be alpha-enolase (48 kDa) and beta-actin (42 kDa) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and ES-MS/MS mass spectrometric methods. Immunohistochemical analysis revealed that alpha-enolase and beta-actin were present at extremely low levels in the epithelial superficial and wing cells of the keratoconus corneas compared to these cells of normal corneas. 2-D gel electrophoresis followed by western blot analysis revealed relatively greater degradation of the two proteins in the keratoconus corneas compared to normal corneas. RT-PCR analysis showed the mRNA expression of the two proteins in the epithelial cells of both normal and keratoconus corneas. CONCLUSIONS: The results showed relatively low or negligible levels of alpha-enolase and beta-actin in the wing and superficial epithelial cells of keratoconus corneas compared to normal corneas. This was attributed to relatively greater degradation of the two proteins in keratoconus corneas compared to normal corneas.

Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.

PURPOSE: The aim of the study was to comparatively analyze crystallin fragments in the water soluble high molecular weight (WS-HMW) and in the water insoluble (WI) protein fractions of human cataractous (with nuclear opacity) and age matched normal lenses to determine the identity of crystallin species that show cataract specific changes such as truncation and post-translational modifications. Because these changes were cataract specific and not aging specific, the results were expected to provide information regarding potential mechanisms of age related cataract development. METHODS: The WS-alpha-crystallin, WS-HMW protein, and WI protein fractions were isolated from normal lenses of different ages and from cataractous lenses. The three fractions were subjected to two dimensional (2D) gel electrophoresis (IEF in the first dimension and SDS-PAGE in the second dimension). Individual spots from 2D gels were trypsin digested and the tryptic fragments were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. RESULTS: The 2D protein profiles of WS-alpha-crystallin fractions of normal human lenses showed an age related increase in the number of crystallin fragments. In young normal lenses, the WS-alpha-crystallin fragments were mostly C-terminally truncated, but in older lenses these were both N- and C-terminally truncated. The WS-HMW protein fraction from normal lenses contained mainly fragments of alphaA- and alphaB-crystallin, whereas additional fragments of betaB1- and betaA3-crystallin were present in this fraction from cataractous lenses. Similarly, the WI proteins in normal lenses contained fragments of alphaA- and alphaB-crystallin, but cataractous lenses contained additional fragments of betaA3- and betaB1-crystallin. The modifications identified in the WS-HMW and WI crystallin species of cataractous lenses were truncation, oxidation of Trp residues, and deamidation of Asn to Asp residues. CONCLUSIONS: The results show that the components of WS-HMW and WI protein fractions of cataractous lenses differed from normal lenses. Selective insolubilization of fragments of betaA3/A1- and betaB1-crystallin occurred during cataract development compared to normal lenses. Further, the crystallin species of cataractous lenses showed increased truncation, deamidation of Asn to Asp residues, and oxidation of Trp residue.

Crosslinking of human lens 9 kDa gammaD-crystallin fragment in vitro and in vivo.

PURPOSE: [corrected] The aims of this study were to determine in vitro crosslinking of a 9 kDa gammaD-crystallin fragment alone and with alpha-, beta-, or gamma-crystallins, the existence of covalent multimers of the polypeptide in vivo, and posttranslational modifications in the three isoforms of the polypeptide. METHODS: A mixture of crystallin fragments (3-14 kDa), a 9 kDa gammaD-crystallin polypeptide or the polypeptide and individual alpha-, beta-, or gamma-crystallins, were incubated at 37 degrees C for a desired length of time and the crosslinked species were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion Agarose A 1.5 gel chromatography, and western blot analysis. In addition, the existence of covalent multimers of the 9 kDa polypeptide in human lens water soluble (WS) and water insoluble (WI) protein fractions of normal and cataractous human lenses was determined by western blot analyses. The posttranslationally modified amino acids of three isofroms of the polypeptide were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and ES-MS/MS mass spectrometric analyses. RESULTS: Following incubation of a mixture of the crystallin fragments or the 9 kDa polypeptide, covalently crosslinked species held via non-disulfide bonding were seen on SDS-PAGE analysis. The polypeptide also exhibited crosslinking with individual alpha-, beta-, and gamma-crystallins. After western blot analysis with site specific anti-9 kDa antibodies, both WS and WI protein fractions from normal and cataractous lenses showed immunoreactive 27 and 45 kDa multimers. The mass spectrometric analysis of the three isoforms of the polypeptide (with identical molecular weight but different charges) showed oxidized methionine and tryptophan residues, with the latter residue containing two oxygens. CONCLUSIONS: The data suggest that a 9 kDa gammaD-crystallin fragment demonstrated crosslinking properties, which might be due to oxidation of its methionine and tryptophan residues.

Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses.

PURPOSE: The aims of this study were to characterize lens crystallin fragments having a molecular mass of <10 kDa, isolated by solubilization in trichloroacetic acid, in order to identify cleavage sites in the parent crystallins for their origin and determine post-translational modifications in the fragments. METHODS: The water-soluble (WS) and water-insoluble (WI) protein fractions were isolated from normal human lenses of 60 to 80 year old donors and from age-matched cataractous lenses. Both WS and WI protein fractions were treated with TCA at 60 degrees C for 2 h and the TCA-soluble fractions were recovered following centrifugation. The preparations were dialyzed against H2O to remove TCA, concentrated by lyophilization and subjected to two dimensional gel electrophoresis (2D-GE). The spots from 2D-gels were analyzed by western blot analysis, partial N-terminal sequencing, or excised for mass spectrometric analysis. RESULTS: SDS-PAGE analysis showed that TCA solubilized polypeptides having a molecular mass of <10 kDa from both WS and WI protein fractions of normal and cataractous lenses. Following 2D-GE of TCA-solubilized species from normal lenses, 8 and 5 polypeptides from the WS and WI protein fractions, respectively, were observed. Using similar 2D-GE analysis of TCA solubilized species from cataractous lenses, 9 and 5 polypeptides from WS and WI protein fractions, respectively, were seen. Partial N-terminal sequence analysis showed that the majority of the polypeptides from both WS and WI protein fractions of normal and cataractous lenses were derived from alphaB-crystallin following cleavage at the D129-P130 bond. Western blot and partial N-terminal sequence analyses identified three additional 4-kDa alphaA-crystallin fragments with cleavage at the D136-G137 bond in the WS proteins from normal lenses. MALDI-TOF mass spectrometric analysis showed that all TCA soluble polypeptides from cataractous lenses, except one from normal lenses, contained residue number 130 to 175 from alphaB-crystallin. No further truncation occurred at the C-terminal region of the alphaB-crystallin polypeptides. Following comparison of the isotopic distribution in MALDI-TOF profiles of a tryptic fragment having a mass of 2,014 among the alphaB-crystallin polypeptides, a gain of one single Dalton was observed. This suggested deamidation of the N146 residue in alphaB-crystallin fragments. CONCLUSIONS: The results show that the N146 residue in human alphaB-crystallin undergoes in vivo deamidation and several fragments containing this modification exist in both WS and WI protein fractions of normal and cataractous human lenses.