Modifications of human betaA1/betaA3-crystallins include S-methylation, glutathiolation, and truncation

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human gamma-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that betaA1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of betaA1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found.     

Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure

A major component of human nuclear cataracts is water-insoluble, high molecular weight protein. A significant component of this protein is disulfide bonded gamma S-crystallin that can be reduced to monomers by dithiothreitol. Analysis of this reduced gamma S-crystallin showed that deamidation of glutamine and asparagine residues is a principal modification. Deamidation is one of the modifications of lens crystallins associated with aging and cataractogenesis. One proposed hypothesis of cataractogenesis is that it develops in response to altered surface charges that cause conformational changes, which, in turn, permit formation of disulfide bonds and crystallin insolubility. This report, showing deamidation among the disulfide bonded gamma S-crystallins from cataractous lenses, supports this hypothesis.     

A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2

Juvenile-onset cataracts are distinguished from congenital cataracts by the initial clarity of the lens at birth and the gradual development of lens opacity in the second and third decades of life. Genomewide linkage analysis in a multigenerational pedigree, segregating for autosomal dominant juvenile-onset cataracts, identified a locus in chromosome region 3q21.2-q22.3. Because of the proximity of the gene coding for lens beaded filament structural protein-2 (BFSP2) to this locus, we screened for mutations in the coding sequence of BFSP2. We observed a unique C-->T transition, one that was not observed in 200 normal chromosomes. We predicted that this led to a nonconservative R287W substitution in exon 4 that cosegregated with cataracts. This mutation alters an evolutionarily conserved arginine residue in the central rod domain of the intermediate filament. On consideration of the proposed function of BFSP2 in the lens cytoskeleton, it is likely that this alteration is the cause of cataracts in the members of the family we studied. This is the first example of a mutation in a noncrystallin structural gene that leads to a juvenile-onset, progressive cataract.     

Autosomal recessive juvenile onset cataract associated with mutation in BFSP1

A genome wide scan in a consanguineous family of Indian origin with autosomal recessive developmental cataracts was performed by two-point linkage analysis with 382 microsatellite markers. It showed linkage to markers on chromosome 20q, between D20S852 and D20S912, with a maximum lod score of 5.4 obtained with D20S860. This region encompasses the beaded filament structural protein 1 (BFSP1) gene. Direct sequencing revealed a 3343 bp deletion including exon 6 (c.736-1384_c.957-66 del) predicted to result in a shift of the open reading frame. This mutation was absent in 50 control individuals from south India. This is the first report of a mutation in the BFSP1 gene associated with human inherited cataracts. This further increases the genetic heterogeneity of inherited cataracts and provides clues as to the importance of BFSP1 in the cell biology of intermediate filaments and their role in the eye lens.     

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu²⁺-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.     

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.     

Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.     

Evidence of Highly Conserved β-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens*

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H₂O₂, whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.Aging lens crystallins accumulate post-synthetic modifications that can be broadly classified into three categories, namely (1) protein backbone changes, such as racemization and truncation (1–3), (2) conversion of one amino acid into another, such as deamidation of asparagine into aspartate or deguanidination of arginine into ornithine, deamination of lysine into allysine and 2-aminoadipic acid (4–6), and (3) amino acid residue damage from reactive carbonyls and reactive oxygen species (7,8). Carbonyl damage results from the Maillard Reaction by glucose, methylglyoxal, or oxidation products of ascorbate, tryptophan or lipids which form adducts and crosslinks with nucleophilic group of lysine, arginine and cysteine. Examples include carboxymethyl-lysine, pentosidine, methylglyoxal hydroimidazolones, HNE-cysteine adducts and kynurenine (7,9–12). Oxidative damage results from reactive oxygen species that directly damage amino acid residues, e.g. oxidizing tryptophan into N-formyl kynurenine and kynurenine, methionine into its sulfoxide, and cysteine into cysteine disulfides or cysteic acid (13–15).Because of their relevance to age-related cataract, the impact of each of these modifications on crystallin structure and stability is the subject of intense investigation. Importantly, Benedek proposed that high molecular weight (HMW)¹ crystallin aggregates the size of 50 million daltons are needed in order for lens opacification to be visible(16,17). Crystallin aggregation conceivably occurs by one of several mechanisms that include conformational changes as a consequence amino acid mutations (18) or physical-chemical protein modifications. Of the latter, one mechanism that is dominant in several types of cataract involves oxidation of cysteines into protein disulfides (18) and formation of HMW aggregates that scatter light (19).In order to mimic the oxidative process and formation of protein disulfides linked to low concentrations of glutathione (GSH) in the nucleus of the human lens, we recently created the LEGSKO mouse in which lenticular GSH was lowered by knocking out the γ-glutamyl cysteine ligase subunit Gclc (20). These mice develop full-blown nuclear cataract by about 9 months and represent an important model for the development of drugs that might block or reverse the oxidation of crystallin sulfhydryls and presumably protein aggregation. However, this assumption in part depends on whether the sites of disulfide bond formation are similar in mouse and human age-related cataract. To test this hypothesis we performed the first comparative analysis of the cataract prone LEGSKO mouse and human aging and cataractous lens crystallin disulfidome, and compared the results with the disulfidome from mouse lens homogenate oxidized in vitro with H₂O₂ as a model of crystallin aggregation and opacification.     

Insights into the beaded filament of the eye lens

Filensin (BFSP1) and CP49 (BFSP2) represent two members of the IF protein superfamily that are thus far exclusively expressed in the eye lens. Mutations in both proteins cause lens cataract and careful consideration of the detail of these cataract phenotypes alerts us to several interesting features concerning the function of filensin (BFSP1) and CP49 (BFSP2) in the lens. With the first filensin (BFSP1) mutation now having been reported to cause a recessive cataract phenotype, there is the suggestion that the mutation could predispose heterozygote carriers to the early onset of age-related nuclear cataract. In the case of CP49 (BFSP2), there are now three unrelated families who have been identified with a common E233 Delta mutation. Very interestingly this is linked to myopia in one family. Despite the apparent phenotypic differences of the filensin (BFSP1) and CP49 (BFSP2) mutations, the data are still consistent with the beaded filament proteins being essential for lens function and specifically contributing to the optical properties of the lens. The fact that none of the mutations thus far reported affect either the conserved LNDR or TYRKLLEGE motifs that flank the central rod domain supports the view that this pair of IF proteins have unusual structural features and a distinctive assembly mechanism. The multiple sequence divergences suggest these proteins have been adapted to the specific functional requirements of lens fibre cells, a function that can be traced from squid to man.     

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products: IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION*

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm², 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.     

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.     

Structural and functional characterization of CcmG from Pseudomonas aeruginosa,a key component of the bacterial cytochrome c maturation apparatus

The cytochrome c maturation process is carried out in the bacterial periplasm, where some specialized thiol‐disulfide oxidoreductases work in close synergy for the correct reduction of oxidized apocytochrome before covalent heme attachment. We present a structural and functional characterization of the soluble periplasmic domain of CcmG from the opportunistic pathogen P. aeruginosa (Pa‐CcmG), a component of the protein machinery involved in cyt c maturation in gram‐negative bacteria. X‐ray crystallography reveals that Pa‐CcmG is a TRX‐like protein; high‐resolution crystal structures show that the oxidized and the reduced forms of the enzyme are identical except for the active‐site disulfide. The standard redox potential was calculated to be E^0′ = ?0.213 V at pH 7.0; the pK_a of the active site thiols were pK_a = 6.13 ± 0.05 for the N‐terminal Cys74 and pK_a = 10.5 ± 0.17 for the C‐terminal Cys77. Experiments were carried out to characterize and isolate the mixed disulfide complex between Pa‐CcmG and Pa‐CcmH (the other redox active component of System I in P. aeruginosa). Our data indicate that the target disulfide of this TRX‐like protein is not the intramolecular disulfide of oxidized Pa‐CcmH, but the intermolecular disulfide formed between Cys28 of Pa‐CcmH and DTNB used for the in vitro experiments. This observation suggests that, in vivo, the physiological substrate of Pa‐CcmG may be the mixed‐disulfide complex between Pa‐CcmH and apo‐cyt. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Racemization at the Asp 58 residue in αA‐crystallin from the lens of high myopic cataract patients

Post‐translational modifications in lens proteins are key causal factors in cataract. As the most abundant post‐translational modification in the lens, racemization may be closely related to the pathogenesis of cataract. Racemization of αA‐crystallin, a crucial structural and heat shock protein in the human lens, could significantly influence its structure and function. In previous studies, elevated racemization from l ‐Asp 58 to d ‐isoAsp58 in αA‐crystallin has been found in age‐related cataract (ARC) lenses compared to normal aged human lenses. However, the role of racemization in high myopic cataract (HMC), which is characterized by an early onset of nuclear cataract, remains unknown. In the current study, apparently different from ARC, significantly increased racemization from l ‐Asp 58 to d ‐Asp 58 in αA‐crystallin was identified in HMC lenses. The average racemization rates for each Asp isoform were calculated in ARC and HMC group. In ARC patients, the conversion of l ‐Asp 58 to d ‐isoAsp 58, up to 31.89%, accounted for the main proportion in racemization, which was in accordance with the previous studies. However, in HMC lenses, the conversion of l ‐Asp 58 to d ‐Asp 58, as high as 35.44%, accounted for the largest proportion of racemization in αA‐crystallin. The different trend in the conversion of αA‐crystallin by racemization, especially the elevated level of d ‐Asp 58 in HMC lenses, might prompt early cataractogenesis and a possible explanation of distinct phenotypes of cataract in HMC.     

The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.     

In vitro translation of cytoskeletal beaded-chain filament proteins from chicken lens mRNA

The beaded-chain filament is a unique cytoskeletal structure that appears in the elongating fiber cells during the differentiation of lens epithelial cells to form the mature fiber cells. This beaded-chain structure is made up of two proteins of molecular weight 95 kDa and 49 kDa. As a prerequisite for cloning the cDNAs of these proteins, newborn chicken lens total poly(A+) mRNA was translated in vitro, using a rabbit reticulocyte lysate system and [35S]-L-methionine. The labelled translation products were analyzed by one-and two dimensional gel electrophoresis followed by autoradiography. Immunoprobing of the translation products on Western blots using specific polyclonal antibodies identified the above proteins, and demonstrated the presence and expression of specific mRNAs in the neonatal chick lens, that code for the in vitro synthesis of these two cytoskeletal proteins. These mRNAs are low abundant mRNAs as compared to the crystallin mRNAs.     

The role of glycation and autoxidation on crystallin aggregation

SUMMARY

Incubation of α-crystallin with glucose and CuSO₄ resulted in crystallin changes similar to those observed in cataracts. Examination of the reaction mixtures by polyacrylamide slab gel electrophoresis showed progressive crystallin aggregation through non-disulfide covalent bonds and parallel increases in ultraviolet absorbance and non-tryptophan fluorescence. Both glucose and copper were required; iron was less effective. The reaction can be accelerated by increasing glucose concentration or by utilizing ribose which has a higher percentage of free aldehyde groups than glucose. These observations are consistent with a mechanism involving crystallin glycation. The reaction is mediated by hydrogen peroxide and transition metals since it is inhibited by catalase and by chelating agents. These results, in turn, are consistent with copper-catalyzed autoxidation of glucose and of glycated crystallin. This reaction generates superoxide free radical which dismutates to yield hydrogen peroxide. The latter, in turn, generates hydroxyl radicals in presence of transition metal ions (Fenton reaction). Hydroxyl radical attack leads to cross-linking which is enhanced in glycated proteins. Under hyperglycemic conditions, such as in diabetes mellitus, high levels of glucose occur in insulin-independent tissues such as the lens. Elevated cupremia and oxidative stress are also known to occur in diabetic patients. There-fore, our findings are consistent with crystallin glycation and superimposed oxyradical generation during diabetic cataractogenesis.     

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Crystallins of the octopus lens. Recruitment from detoxification enzymes.

The eye lens crystallins of the octopus Octopus dofleini were identified by sequencing abundant proteins and cDNAs. As in squid, the octopus crystallins have subunit molecular masses of 25-30 kDa, are related to mammalian glutathione S-transferases (GST), and are encoded in at least six genes. The coding regions and deduced amino acid sequences of four octopus lens cDNAs are 75-80% identical, while their non-coding regions are entirely different. Deduced amino acid sequences show 52-57% similarity with squid GST-like crystallins, but only 20-25% similarity with mammalian GST. These data suggest that the octopus and squid lens GST-like crystallin gene families expanded after divergence of these species. Northern blot hybridization indicated that the four octopus GST-like crystallin genes examined are lens-specific. Lens extracts showed about 40 times less GST activity using 1-chloro-2,4-dinitrobenzene as substrate than liver extracts of the octopus, indicating that the major GST-like crystallins are specialized for a lens structural role. A prominent 59-kDa crystallin polypeptide, previously observed in octopus but not squid and called omega-crystallin (Chiou, S.-H. (1988) FEBS Lett. 241, 261-264), has been identified as an aldehyde dehydrogenase. Since cytoplasmic aldehyde dehydrogenase is a major protein in elephant shrew lenses (eta-crystallin; Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) the octopus aldehyde dehydrogenase crystallin provides the first example of a similar enzyme-crystallin in vertebrates and invertebrates. The use of detoxification stress proteins (GST and aldehyde dehydrogenase) as cephalopod crystallins indicates a common strategy for recruitment of enzyme-crystallins during the convergent evolution of vertebrate and invertebrate lenses. For historical reasons we propose that the octopus GST-like crystallins, like those of the squid, are called S-crystallins.     

A role for lengsin, a recruited enzyme, in terminal differentiation in the vertebrate lens

Lengsin is an eye lens-specific member of the glutamine synthetase (GS) superfamily. Lengsin has no GS activity, suggesting that it has a structural rather than catalytic role in lens. In situ hybridization and immunofluorescence showed that lengsin is expressed in terminally differentiating secondary lens fiber cells. Yeast two-hybrid (Y2H) and recombinant protein experiments showed that full-length lengsin can bind the 2B filament region of vimentin. In affinity chromatography, lengsin also bound the equivalent region of CP49 (BFSP2; phakinin), a related intermediate filament protein specific to the lens. Both the vimentin and CP49 2B fragments bound lengsin in surface plasmon resonance spectroscopy with fast association and slow dissociation kinetics. Lengsin expression correlates with a transition zone in maturing lens fiber cells in which cytoskeleton is reorganized. Lengsin and lens intermediate filament proteins co-localize at the plasma membrane in maturing fiber cells. This suggests that lengsin may act as a component of the cytoskeleton itself or as a chaperone for the reorganization of intermediate filament proteins during terminal differentiation in the lens.