Post-translationally modified human lens crystallin fragments show aggregation in vitro.

BACKGROUND: Crystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties. METHODS: Four crystallin fragments-containing fractions (Fraction I [∼3.5 kDa species], Fraction II [∼3.5-7 kDa species], Fraction III [∼7-10 kDa species] and Fraction IV [>10-18 kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50-70 year old-donors. The crystallin fragments of the Fractions I-IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I-IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins. RESULTS: Crystallin fragments in Fractions I-IV were derived from α-, ß- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in Mr from 5.5×105 to 1.0×108 Da, with diameters of 10-28 nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins. CONCLUSION: The crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development. GENERAL SIGNIFICANCE: Crystallin fragments play a major role in human cataract development.

Downregulation of ß-actin and its regulatory gene HuR affect cell migration of human corneal fibroblasts.

PURPOSE: In an earlier study, we showed that human antigen R (HuR) and ß-actin expression levels were downregulated in fibroblasts isolated from human keratoconus stroma compared to normal corneal stroma. To further extend the finding, we determined whether HuR expression affects ß-actin gene expression and in turn affects corneal fibroblast migration and wound healing. METHODS: Stromal keratocytes from normal human corneas were cultured in the presence of serum. Cells were transfected with siRNA specific for ß-actin or HuR. SiRNAs specific for GAPDH or a scrambled sequence were used as positive and negative controls (siCTR) for transfection, respectively. The effects of gene silencing were analyzed at the transcriptional and translational levels. Specific proteins were immunohistochemically localized using confocal imaging. The effects of gene silencing on cell migration and cell proliferation were analyzed using a modified Boyden chamber and with a wound healing assay, respectively. RESULTS: Reverse-transcription PCR (RT-PCR) and western blot analyses showed that when the HuR gene was silenced, ß-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the ß-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the ß-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced. CONCLUSIONS: The results show that downregulation of the HuR gene results in decreased ß-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased ß-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.

Structural and functional roles of deamidation of N146 and/or truncation of NH2- or COOH-termini in human αB-crystallin.

PURPOSE: The purpose of the study was to determine the relative effects of deamidation and/or truncation on the structural and functional properties of αB-crystallin. METHODS: Using wild-type (WT) αB-crystallin and the αB deamidated mutant (i.e., αB N146D), we generated NH(2)-terminal domain deleted (residues no. 1-66; αB-NT), deamidated plus NH(2)-terminal domain deleted (αB N146D-NT), COOH-terminal extension deleted (residues no. 151-175; αB-CT), and deamidated plus COOH-terminal extension deleted (αB N146D-CT) mutants. All of the proteins were purified and their structural and functional (chaperone activity with insulin as target protein) properties were determined and compared to WT αB-crystallin. RESULTS: The desired deletions in the αB-crystallin mutants were confirmed by DNA sequencing and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. The homomers of αB-CT and its deamidated form (αB N146D-CT) became water insoluble, whereas the αB N146D, αB-NT, and αB N146D-NT species remained water-soluble. CD spectroscopic studies revealed that the mutants with deletion of NH(2)- or COOH-termini or deamidation showed increased ß-sheet and decreased α-helical contents with the exception of αB N146D-CT, which showed a substantial increase in α-helix and decrease in ß-sheet content. Results of intrinsic Trp fluorescence suggested little change in Trp microenvironment of αB N146D relative to WT αB, but substantial alterations on deletion of COOH-terminal extension or a combination of this deletion plus deamidation. Hydrophobic binding studies using the hydrophobic probe 8-anilino-1-naphthalene sulfonate (ANS) showed that, relative to WT αB structure, the N146 deamidation, COOH-terminal extension deletion or a combination of this deamidation and deletion resulted in a relatively compact structure whereas the NH(2)-terminal domain deletion and a combination of this deletion plus deamidation resulted in a relaxed structure. All the αB mutants showed higher molecular mass ranging between 1.2×10(6) to 5.4×10(6) Da, relative to WT αB which had a molecular mass of 5.8×10(5) Da. Chaperone activity across all αB species decreased in the following order: WTαB > αB N146D-CT > αB N146D-NT > αB-NT > αB-CT > αB N146D. Specifically, substantial losses in chaperone activity (only 10% to 20% protection) were seen in αB N146D, αB-NT, and αB-CT. However, in the species with the combination of deamidation plus NH(2)- or COOH-terminal deletion, the percent protection was about 24% in αB N146D-NT and about 40% in αB N146D-CT. CONCLUSIONS: Although all mutants formed oligomers even after deamidation, on deletion of either NH(2)-terminal domain or COOH-terminal extension or a combination of these deletions and deamidation, their structural properties were substantially altered. The results suggested that the NH(2)-terminal domain is relatively more important than the COOH-terminal extension for the chaperone function of αB. The non-deamidated N146 residue, NH(2)-terminal domain and COOH-terminal extension are also of critical importance to the maintenance of αB-crystallin chaperone activity.

Structural and functional properties of NH(2)-terminal domain, core domain, and COOH-terminal extension of αA- and αB-crystallins.

PURPOSE: The purpose of the present study was to determine the biophysical and chaperone properties of the NH(2)-terminal domain, core domain and COOH-terminal extension of human αA- and αB-crystallins and correlate these properties to those of wild type (WT) αA- and αB-crystallins. METHODS: WT αA- and αB-crystallins cloned into pET 100D TOPO vector, were used as templates to generate different constructs encoding specific regions (NH(2)-terminal domain [NTD], core domain [CD], and COOH-terminal extension, [CTE]). The specific regions amplified by PCR using plasmid DNA from WT αA and WT αB were: αA NTD (residues 1-63), αA CD (residues 64-142), αA CTE (residues 143-173), αB NTD (residues 1-66), αB CD (residues 67-146), and αB CTE (residues 147-175). Resultant blunt-end PCR products were ligated to a pET 100 Directional TOPO vector. DNA sequencing results confirmed the desired constructs. Positive clones were transformed into the BL21 Star (DE3) expression cell line. Protein expression and solubility were confirmed by SDS-PAGE and western blot analysis using a monoclonal antibody against a 6× His-tag epitope. Proteins were purified using Ni(2+)-affinity column chromatography, under native or denaturing conditions, and used for biophysical and chaperone function analyses. RESULTS: A total of five constructs were successfully generated: αA NTD, αA CD, αB NTD, αB CD, and αB CTE. SDS-PAGE and western blot analyses showed that αA CD and αB CD were present in both the soluble and insoluble fractions, whereas mutant preparations with NTD alone became insoluble and the mutant with CTE alone became soluble. All purified constructs showed alterations in biophysical properties and chaperone function compared to WT α-crystallins. αA NTD and αB CTE exhibited the most notable changes in secondary structural content. Also, αA NTD and all αB-crystallin constructs showed altered surface hydrophobicity compared to their respective WT α-crystallins. CONCLUSIONS: Although the individual α-crystallin regions (i.e., NH(2)-terminal domain, core domain, and COOH-terminal extension) exhibited varied biophysical properties, each region alone retained some level of chaperone function. The NH(2)-terminal domains of αA and αB each showed the maximum chaperone activity of the three regions with respect to their WT crystallins.

Differential epithelial and stromal protein profiles in keratoconus and normal human corneas.

The purpose of the study was to identify epithelial and stromal proteins that exhibit up- or down-regulation in keratoconus (KC) vs. normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluted the specificity of the proteome of each tissue, we selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, i.e., a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS)(2)] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS)(2) method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset. Relative to normal corneal epithelial proteins, six KC epithelial proteins (lamin-A/C, keratin type I cytoskeletal 14, tubulin beta chain, heat shock cognate 71 kDa protein, keratin type I cytoskeletal 16 and protein S100-A4) exhibited up-regulation and five proteins (transketolase, pyruvate kinase, 14-3-3 sigma isoform, phosphoglycerate kinase 1, and NADPH dehydrogenase (quinone) 1) showed down-regulation. A similar relative analysis showed that three KC stromal proteins (decorin, vimentin and keratocan) were up-regulated and five stromal proteins (TGF-betaig h3 (Bigh3), serotransferrin, MAM domain-containing protein 2 and isoforms 2C2A of collagen alpha-2[VI] chain) were down-regulated. The 2D-DIGE-mass spectrometry followed by Decyder software analysis showed that relative to normal corneas, the KC corneal epithelium exhibited up-regulation of four proteins (serum albumin, keratin 5, L-lactate dehydrogenase and annexin A8) and down-regulation of four proteins (FTH1 [Ferritin heavy chain protein 1], calpain small subunit 1, heat shock protein beta 1 and annexin A2). A similar relative analysis of stroma by this method also showed up-regulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in KC corneas. Together, the results suggested that the Nano-ESI-LC-MS(MS)(2) method was superior than the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up- or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling.

A serine-type protease activity of human lens ßA3-crystallin is responsible for its autodegradation.

PURPOSE: The purpose of the study was to determine whether the autodegradation of human ßA3-crystallin is due to its intrinsic protease activity. METHODS: Recombinant His-tagged human ßA3-crystallin was expressed in E. coli and purified by a Ni+2-affinity column chromatographic method. To determine protease activity, the purified crystallin was incubated for 24 h with either sodium deoxycholate, Triton X-100, or CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate} and with benzoyl DL-arginine p-nitroanilide (BAPNA), a colorimetric protease substrate. The autodegradation of the crystallin at 0 h and 24 h on incubation at 37 °C with and without detergents (CHAPS/Triton X-100) was also determined by sodium dodecylsulfate-PAGE (SDS-PAGE) method. To examine whether the autodegradation of the crystallin was due to its protease activity, the crystallin was incubated with inhibitors of serine-, metallo- and cysteine-proteases. The binding of the intact ßA3-crystallin and its autodegradation products to FFCK [5-carboxyfluorescenyl-1-phenylalaninyl-chloromethyl ketone], an analog of TPCK [1-Chloro-3-tosylamido-4-phenyl-2-butanone, a chymotrypsin-type serine protease inhibitor] was also determined by their incubation followed by SDS-PAGE and scanning for fluorescence using a Typhoon 9400 scanner. RESULTS: ßA3-crystallin protease activity showed activation in the presence of CHAPS but not in presence of Triton X-100. Upon incubation of ßA3-crystallin for 24 h with CHAPS or sodium deoxycholate and BAPNA as a substrate, a time-dependent increase in the Arg-bond hydrolyzing activity was observed. SDS-PAGE analysis exhibited autodegradation products with Mr of 22, 27 and 30 kDa, which on partial NH2-terminal sequencing showed cleavage of Lys17-Met18, Gln4-Ala5 and Thr-Gly (in the NH2-terminal His-tag region) bonds, respectively. Almost no autodegradation of the ßA3-crystallin occurred during its incubation alone or with CHAPS plus serine protease inhibitors (phenylmethylsulfonyl fluoride [PMSF], approtinin, and chymostatin). In contrast, the autodegradation occurred in the presence of metallo-protease inhibitors (EDTA and EGTA) and cysteine protease inhibitors (E-64, N-methylmaleimide and iodoacetamide). The ßA3-crystallin also exhibited binding to FFCK, suggesting existence of a chymotrypsin-type active site in the ßA3-crystallin protease. CONCLUSIONS: The results suggested that a serine-type protease activity of ßA3-crystalllin was responsible for its autodegradation. The specific bonds cleaved during autodegradation (Gln4-Ala5 and Lys17-Met18), were localized in the NH2-terminal arm of ßA3-crystallin.

Identification of crystallin modifications in the human lens cortex and nucleus using laser capture microdissection and CyDye labeling.

PURPOSE: With aging, lens crystallins undergo post-translational modifications (PTMs) and these modifications are believed to play a major role in age-related cataract development. The purpose of the present study was to determine the protein profiles of crystallins and their PTMs in the cortical and nuclear regions within an aging human lens to gain a better understanding about changes in crystallins as fiber cells migrate from cortical to nuclear region. METHODS: Laser capture microdissection (LCM) was used to select and capture cells from cortical and nuclear regions of 12 mum, optimum cutting temperature (OCT) compound-embedded frozen lens sections from a 69-year-old human lens. Proteins were extracted and then analyzed by 2-D difference gel electrophoresis (2-D DIGE) with sulfonated indocyanine dye (CyDye) labeling. Crystallin identities and their PTMs were then determined by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) and Electrospray Ionization Quadripole Linear Ion-Trap Liquid Chromatography (ESI-QTRAP LC-MS/MS) mass spectrometry. RESULTS: Crystallin fragments (M(r) <20 kDa) were present in both cortical and nuclear regions, while high molecular weight (HMW) aggregates (M(r) > 35 kDa) were mostly localized in the nuclear region. HMW complexes contained a relatively large number of truncated and modified beta-crystallins, compared to alpha- and gamma-crystallins, and two lens-specific intermediate filaments, CP49 (phakinin) and filensin. Modified alpha-crystallins were in low abundance in the nuclear region compared to the cortical region. Several PTMs, including deamidation, oxidation, phosphorylation, ethylation, methylation, acetylation, and carbamylation, were identified in virtually all crystallins and CP49. The data provide the first report of human lens crystallin profiling by a combination of LCM, 2D-DIGE, and mass spectrometric analysis. CONCLUSIONS: The results suggested that as the fiber cells migrate from cortical region to the nuclear region, the crystallin degradation begins in the cortical region and continues in the nuclear region. However, a greater number of the HMW complexes exist mainly in the nuclear region.

Truncated human betaB1-crystallin shows altered structural properties and interaction with human betaA3-crystallin.

The purpose of the study was to determine the effects of truncation of various regions of betaB1-crystallin on its structural properties and stability of heterooligomers formed by wild-type (WT) betaB1 or its deletion mutants with WT betaA3-crystallin. For these analyses, seven deletion mutants of betaB1-crystallin were generated with the following sequential deletions of either N-terminal arm [betaB1(59-252)], N-terminal arm + motif I [betaB1(99-252)], N-terminal arm + motif I + motif II [betaB1(144-252)], N-terminal arm + motif I + motif II + connecting peptide [betaB1(149-252)], C-terminal extension [betaB1(1-234)], C-terminal extension plus motif IV [betaB1(1-190)], or C-terminal extension + motif III + motif IV [betaB1(1-148)]. The betaB1-crystallin became water insoluble on the deletion of C-terminal extension and subsequent deletions of the C-terminal domain (C-terminal extension plus motifs III and IV) while it remained partially soluble on the deletion of the N-terminal domain (N-terminal arm plus motifs I and II). However, circular dichroism spectral analysis showed that the deletion of the N-terminal domain but not the C-terminal domain exhibited relatively greater structural changes in the crystallin. The deletion of the C-terminal domain resulted in a greater exposure and disturbance in the microenvironment of Trp-100, Trp-123, and Trp-126 (localized in the motif II), suggesting a relatively greater role of the C-terminal domain than the N-terminal domain in the structural stability of the crystallin. The deletion of the N-terminal extension in betaB1 resulted in maximum exposure of hydrophobic patches and compact structure and in a maximum loss of subunit exchange with WT betaA3-crystallin compared to deletion of either the C-terminal extension, the N-terminal domain, or the C-terminal domain. The thermal stability results of the heterooligomer of betaB1- plus betaA3-crystallins suggested that oligomers lose their stability on deletion of the C-terminal domain. Together, the results suggested that the N-terminal arm of betaB1-crystallin plays a major role in interaction with betaA3-crystallin during heterooligomer formation, and the solubility of betaB1-crystallin per se and that of the heterooligomer with betaA3-crystallin are dependent on the intact C-terminal domain of betaB1-crystallin.

Isolation and characterization of betaA3-crystallin associated proteinase from alpha-crystallin fraction of human lenses.

PURPOSE: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses. METHODS: An inactive, Arg-bond hydrolyzing proteinase in the alpha-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions). RESULTS: An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The three protein bands were identified as betaA3-, betaB1-, and betaB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (alpha-crystallin, beta(H)-crystallin, and membrane fractions). CONCLUSIONS: A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses.

The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate-PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M(r) between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of alpha-, beta-, and gamma-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of alpha-, beta-, and gamma-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.

Child sexual abuse and sexually transmitted infections: review of joint genitourinary medicine and paediatric examination practice.

Joint examination by doctors with complementary skills and screening for sexually transmitted infections (STIs) are recommended in children who may have been sexually abused or have been found to have an STI. Our study showed that criminal proceedings were more likely to be brought in cases with physical signs of sexual abuse. It could be difficult to prove whether sexual abuse had taken place or not with microbiological evidence alone, in the absence of other evidence. Significance of viral STIs in the context of sexual abuse should be evaluated carefully. The review of our practice re-enforced the importance of joint examination of children with suspected STIs.

UV-A-induced structural and functional changes in human lens deamidated alphaB-crystallin.

PURPOSE: To determine comparative effects of ultraviolet (UV)-A irradiation on structural and functional properties of wild type (WT) alphaB-crystallin and its three deamidated mutant proteins (alphaB-Asn78Asp, alphaB-Asn146Asp, and alphaB-Asn78/146Asp). METHODS: Three deamidated mutants previously generated from recombinant WT alphaB-crystallin, using a site-specific mutagenesis procedure as previously described [32], were used. The WT alphaB-crystallin and its three deamidated species were exposed to UV-A light (320-400 nm) at intensities of 20 or 50 J/cm(2). The UV-A-unexposed and UV-A-exposed preparations were examined for their chaperone activity, and their activities were correlated with the UV-A-induced structural changes. The structural properties studied included dimerization and degradation, intrinsic tryptophan (Trp) fluorescence, ANS (8-anilino-1-naphthalenesulfate)-binding, far ultraviolet circular dichroism (UV-CD) spectral analysis, molecular sizes by dynamic light scattering, and oxidation of Trp and methionine (Met) residues. RESULTS: The WT alphaB-crystallin and its three deamidated mutant proteins showed enhanced dimerization to 40 kDa species and partial degradation with increasing doses during UV-A-exposure. Compared to the deamidation of asparagines (Asn) 78 residue to aspartic acid (Asp) or both Asn78 and Asn146 residues to Asp, the deamidation of Asn146 residue to Asp resulted in a greater loss of chaperone activity. The UV-A-induced loss of chaperone activity due to structural changes was studied. The ANS-binding data suggested that the alphaB-Asn146Asp mutant protein had a relatively compact structure and an increase in surface hydrophobic patches compared to WT and two other deamidated proteins. Similarly, UV-A-exposure altered the Trp microenvironment in the deamidated mutant proteins compared to the WT alphaB-crystallin. Far-UV CD spectral analyses showed almost no changes among WT and deamidated species on UV-A-exposure except that the alphaB-Asn146Asp mutant protein showed maximum changes in the random coil structure relative to WT alphaB-crystallin and two other deamidated proteins. The UV-A-exposure also resulted in the aggregation of WT and the three deamidated mutant proteins with species of greater mass compared to the non-UV-A exposed species. Among the four spots recovered after two-dimensional (2D)-gel electrophoresis from WT and the three deamidated species, the Met and Trp residues of alphaB-Asn146Asp mutant showed maximum oxidation after UV-A exposure, which might account for its greater loss in chaperone activity compared to WT alphaB-crystallin and two other deamidated species. CONCLUSIONS: After UV-A-exposure, the deamidated alphaB-Asn146Asp mutant protein showed a complete loss of chaperone activity compared to WT alphaB and alphaB-Asn78Asp and alphaB-Asn78/146Asp deamidated species. Apparently, this loss of chaperone activity was due to oxidative changes leading to its greater structural alteration compared to other alphaB-species.

Proteomic analysis of water insoluble proteins from normal and cataractous human lenses.

PURPOSE: The purpose of the study was to compare and analyze the composition of crystallin species that exist in the water insoluble-urea soluble (WI-US) and water insoluble-urea insoluble (WI-UI) protein fractions of a human cataractous lens and an age-matched normal lens. METHODS: The water soluble (WS) and water insoluble (WI) protein fractions from a 68-year-old normal lens and a 61-year-old cataractous lens were isolated, and the WI proteins were further solubilized in urea to separate WI-US and WI-UI protein fractions. The WI-US and WI-UI protein fractions from normal and cataractous lenses were individually analyzed by two-dimensional (2D) gel electrophoresis. The protein spots were excised from 2D gels, digested with trypsin, and analyzed by the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) method. The tryptic peptides from individual spots were further analyzed by the electrospray tandem mass spectrometry (ES-MS/MS) method to determine their amino acid sequences. RESULTS: The comparative 2D gel electrophoretic analyses of WI-US proteins of normal and cataractous lenses showed that the majority of species in a normal lens (68 years old) and a cataractous lens (61 years old) had M(r) between 20 to 30 kDa. The ES-MS/MS analyses showed that the individual WI-US protein spots from normal and cataractous lenses contained mostly either alphaA- or alphaB-crystallin with beta-crystallins, or alpha- and beta-crystallins with filensin as well as vimentin. Similar sequence analyses of tryptic fragments of 2D gel spots of WI-UI proteins revealed that the normal lens showed either individual alphaA- and alphaB-crystallins, a mixture of betaA3/A1-, betaB1-, and betaB2-crystallins and filensin, betaA4-, betaB1-, betaB2-, betaS-crystallins and filensin, or alphaA-, alphaB1-, filensin, and vimentin or alphaB-, betaA3-, betaA4-, betaB1-, betaB2-, and betaS-crystallins. In contrast, the WI-UI proteins from a cataractous lens showed three intact crystallins (alphaB-, gammaS-, and betaB2-crystallins), and three spots containing a mixture of beta-crystallins (the first containing betaB1- and betaB2-crystallins, the second gammaS-, betaB1-, and betaB2-crystallins, and the third betaA3-, betaA4-, and betaB1-crystallins). CONCLUSIONS: The compositions of WI-US and WI-UI proteins, isolated from one normal and one cataractous lens, were different. The absence of alphaA- but not of alphaB-crystallin and preferential insolubilization mostly of beta-crystallins in the WI-US protein fraction from the cataractous lens but not in the normal lens was observed. Similarly, in contrast to the normal lens, the WI-UI proteins of the cataractous lens contained alphaB-crystallin while alphaA-crystallin was absent, which suggested a major role of alphaB-crystallin in the insolubilization process of crystallins.

Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure.

The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.

BetaB2-crystallin undergoes extensive truncation during aging in human lenses.

Based on the present literature, it is unclear whether betaB2-crystallin undergoes age-related truncation in human lenses. To answer this question, the purpose of this study was to determine in vivo truncation of betaB2-crystallin in human lenses during aging by examining its fragments in the beta(H)-crystallin fraction. The WS-protein fraction was isolated from lenses of desired ages and separated by a size-exclusion Agarose A 1.5m column to recover alpha-, beta(H)-, beta(L)-, and gamma-crystallin fractions. The beta(H)-crystallin fractions, isolated from lenses of 24- and 70-year-old donors, were utilized for two-dimensional (2D) gel electrophoresis (isoelectric focusing in the first dimension followed by SDS-PAGE in the second dimension). The partial N-terminal sequences of the desired fragments (Molecular weights [M(r)]<18-19kDa) from a 2D-gel of WS-proteins from lenses of a 70-year-old donor were determined. More than 37 crystallin fragments with M(r) between 4 and 19kDa were observed on a 2D-gel. Nine fragments in beta(H)-crystallin fraction were from betaB2-crystallin but additional single fragments of alphaA-, gammas-, betaA4, and of either gammaB-, gammaC- or gammaD-crystallins were also observed. Seven cleavage sites in the betaB2-crystallin were identified, which included two sites at Q(7)-A(8) and A(8)-G(9) bonds in the N-terminal extension, two sites at E(46)-K(47) and G(49)-S(50) bonds in the motif 1, one site at S(94) -S(95) in the motif 2, and two sites at N(115)-F(116) and Q(135)-Y(136) in motif 3. No fragments with cleavage in the motif 4 and C-terminal extension of betaB2-crystallin were seen. Apparently, three betaB2-crystallin fragments with only N-terminal cleavage and five with both N- and C-terminal cleavages were observed. Additional fragments with cleavage sites at Q(54)-Y(55) in alphaA-crystallin, at E(112)-N(113) in betaA4-crystallin, at G(4)-T(5) in gammas-crystallin, at M(69)-G(70) in either gammaB-, gammaC- or gammaD-crystallins (three have identical sequences at the cleaved bond), and at G(1)-K(2) in gammaB or gammaC (both have identical sequences at the cleavage site) were observed.Conclusions. The results showed that betaB2-crystallin undergoes age-related truncation producing fragments with M(r) between 4 and 19kDa that existed in the beta(H)-crystallin oligomer. The beta(H)-crystallin fraction also contained single fragments of alpha-, betaA4-, gammas-, and other gamma-crystallins.

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface.

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.

Characterization of a sodium deoxycholate-activatable proteinase activity associated with betaA3/A1-crystallin of human lenses.

A human lens proteinase was purified by a five-step procedure that included two consecutive size-exclusion agarose A 1.5 m chromatographies, a preparative non-denaturing gel-electrophoretic separation, HPLC on a size-exclusion column (TSK G-3000 PW(XL)) followed by preparative isoelectric focusing. A 2300-fold purified enzyme showed a major band of 22 kDa during SDS-PAGE, a pH optimum of 7.8, pI between 4.5 and 5.0, a loss of activity above 45 degrees C and a serine type nature. The partial N-terminal sequence of the enzyme, i.e. P-M-P-G-S-L-G-P-W, matched with the sequence of human lens betaA3/A1-crystallin starting at residue No. 23. Based on the Western blot results of the enzyme with five different site-specific polyclonal antibodies raised against betaA3/A1-crystallin, it was concluded that the 22 kDa crystallin enzyme had a cleaved N-terminus but an intact C-terminus. The betaA3/A1-crystallin, isolated from human lenses, also exhibited proteinase activity following detergent activation and size-exclusion chromatography. The mouse recombinant betaA3/A1-crystallin proteinase was purified by the above five-step procedure, from a homogenate of Sf-9 cells transfected with baculovirus containing the full length coding sequence of betaA3/A1-crystallin. The mouse 22 kDa species also exhibited proteinase activity and immunoreactivity with anti-betaA3/A1-C-terminal antibody. Together, the data suggest that a truncated species of betaA3/A1-crystallin exhibits proteinase activity.

cAMP-dependent phosphorylation of betaig-h3 protein in human corneal endothelial cells.

PURPOSE: To determine the identity of a major membrane/ extracellular matrix (ECM)-associated 66-kDa protein in human corneal endothelial cells and its phosphorylation in vivo. METHODS: A membrane/ECM-associated 66 kDa protein was purified from human corneal endothelial cells of 50-80 year-old donors by a three-step procedure that included preparative SDS-PAGE, preparative isoelectric focusing (IEF) and HPLC using a C-18 column. The phosphorylation of the 66-kDa protein was determined by both endogenous kinases and exogenous protein kinase A in the presence of endogenous or added cAMP, or added Walsh inhibitor (a specific inhibitor of protein kinase A). The phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. The phosphoamino acids were identified following hydrolysis of the purified phosphorylated 66-kDa protein and thin layer chromatography with standard phosphoamino acids. RESULTS: Following purification, the 66-kDa protein showed a single band on SDS-PAGE, a single species on two dimensional (2D)-gel electrophoresis and a single peak during C-18 HPLC. The partial N-terminal sequence of the 66-kDa protein matched with that of the 68-kDa beta ig-h3 protein (minus the signal peptide) of lung adenocarcinoma cells. Furthermore, on Western blot analysis, the 66-kDa protein immunoreacted with anti-beta ig-h3 past signal peptide (residue nos.24-32) antibody but not with the anti-beta ig-3 signal peptide (residue nos.1-9) antibody. During the phosphorylation of endothelial proteins by endogenous kinases or added protein kinase A in the presence of endogenous or added cAMP, the 66 kDa protein was the major substrate with its Ser residues phosphorylated in both cytosolic- and membrane/ECM-fractions. CONCLUSIONS: The human corneal endothelial 66-kDa protein is identical to the 68-kDa beta ig-h3 protein (minus signal peptide) from lung adenocarcinoma cells. The corneal protein exists in a phosphorylated state in vivo with its Ser residues phosphorylated by a cAMP-dependent protein kinase A.

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.

Age-related degradation of betaA3/A1-crystallin in human lenses.

The aim of this study was to determine age-related degradation of betaA3/A1-crystallin in human lenses. The betaA3/A1-crystallin fragments were identified by Western blot analysis using two site-specific anti-betaA3/A1-crystallin antibodies. The first antibody was raised against a N-terminal region (residues 35-66), and the second to the C-terminal (residues 203-214) region of the crystallin. During the analyses, either preparative SDS-PAGE-separated fragments from betaH-crystallin fraction or water-soluble (WS) protein fractions from lenses of different aged donors were used. In lenses from 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-betaA3/A1-N-terminal antibody, suggesting their intact N-terminus but cleaved C-terminus. A similar analysis with the anti-betaA3/A1-C-terminal antibody identified 15-, 18-, 19-, and 20-kDa species and also five species between 4 and 11 kDa that had intact C-terminus but cleaved N-terminus. In lenses from a 5-year-old donor only two crystallin species, a major 15-kDa and a minor 18-kDa species, showed an intact N-terminus and cleaved C-terminus, whereas, eight species with Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-terminus. Upon two-dimensional gel electrophoresis of a betaH-crystallin fraction from the lenses of a 70-year-old donor, a degradation profile almost similar to the crystallin mentioned above was observed. However, the existence of multiple spots with identical Mr's of truncated betaA3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modifications. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-terminal sequence of N-F-Q-G, suggesting cleavage at the E39-N40 bond during their production. Together, the data suggest that the majority of age-related cleavages in betaA3/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E39-N40 bond generating some of these fragments.