Bovine filensin possesses primary and secondary structure similarity to intermediate filament proteins

The cDNA coding for calf filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755- amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di- arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the filensin rod, which is the shortest among IF proteins, can be subdivided into three subdomains (coils 1a, 1b, and 2). A 29 amino acid truncation in the coil 2 region accounts for the smaller size of this domain. The filensin tail contains 6 1/2 tandem repeats which match analogous motifs of mammalian neurofilament M and H proteins. We suggest that filensin is a novel IF protein which does not conform to any of the previously described classes. Purified filensin fails to form regular filaments in vitro (Merdes, A., M. Brunkener, H. Horstmann, and S. D. Georgatos. 1991. J. Cell Biol. 115:397-410), probably due to the missing segment in the coil 2 region. Participation of filensin in a filamentous network in vivo may be facilitated by an assembly partner.     

The 47-kD lens-specific protein phakinin is a tailless intermediate filament protein and an assembly partner of filensin

In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lens- specific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek phi alpha kappa omicron sigma = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3% identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long alpha-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakinin lacks a COOH-terminal tail domain and it therefore represents the second known example of a naturally tailless IF protein. Confocal microscopy on frozen lens sections reveals that phakinin colocalizes with filensin and is distributed along the periphery of the lens fiber cells. Quantitative immunoblotting with whole lens fiber cell preparations and fractions of washed lens membranes suggest that the natural stoichiometry of phakinin to filensin is approximately 3:1. Under in vitro conditions, phakinin self- assembles into metastable filamentous structures which tend to aggregate into thick bundles. However, mixing of phakinin and filensin at an optimal ratio of 3:1 yields stable 10-nm filaments which have a smooth surface and are ultrastructurally indistinguishable from "mainstream" IFs. Immunolabeling with specific antibodies shows that these filaments represent phakinin/filensin heteropolymers. Despite its homology to the cytokeratins, phakinin does not coassemble with acidic (type I), or basic (type II) cytokeratins. From these data we conclude that filensin and phakinin are obligate heteropolymers which constitute a new membrane-associated, lens-specific filament system related to, but distinct from the known classes of IFs.     

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.     

Tropomodulin binds to filensin intermediate filaments

Tropomodulin (Tmod) is an actin filament pointed end capping protein found in the membrane skeleton of lens fiber cells. We demonstrate that Tmod4 is able to bind the lens-specific intermediate filament protein, filensin, in either co-sedimentation or solid phase binding assays in a saturable fashion, but with low affinity and stoichiometry. Furthermore, Tmod4 does not bind the 53 kDa rod domain of filensin, nor to CP49, the obligate assembly partner of filensin. Finally, the binding of filensin to Tmod4 does not inhibit the actin capping activity of Tmod4 in vitro, suggesting that the two functions are not mutually exclusive.     

Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and β-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and β/γ-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.     

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.     

Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers

Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.     

Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens- epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host- specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.     

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry,Transparency, and Mechanical Stiffness of the Mouse Lens

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.     

Overexpression of the vimentin gene in transgenic mice inhibits normal lens cell differentiation

To investigate the role of the intermediate filament protein vimentin in the normal differentiation and morphogenesis of the eye lens fiber cells, we generated transgenic mice bearing multiple copies of the chicken vimentin gene. In most cases, the vimentin transgene was overexpressed in the lenses of these animals, reaching up to 10 times the endogenous levels. This high expression of vimentin interfered very strongly with the normal differentiation of the lens fibers. The normal fiber cell denucleation and elongation processes were impaired and the animals developed pronounced cataracts, followed by extensive lens degeneration. The age of appearance and extent of these abnormalities in the different transgenic lines were directly related to the vimentin level. Electron microscopic analysis revealed that the accumulated transgenic protein forms normal intermediate filaments.     

Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.     

Seeing is believing! The optical properties of the eye lens are dependent upon a functional intermediate filament cytoskeleton

Beaded filaments are the major cytoskeletal element of the eye lens and they are essential to the optical properties of the eye lens. They were discovered in 1972 by Harry Maisel and Margaret Perry and have since been found to comprise two novel intermediate filament proteins, CP49 and filensin. These proteins possess unique structure features and unusual assembly characteristics, which distinguish them from canonical IF proteins. Whilst CP49 is completely tailless, filensin has a rather short rod domain and extremely large C-terminal tail domain. In vitro, CP49 and filensin do not form IFs on their own. In vitro studies suggest that CP49 and filensin have a distinct coassembly mechanism. Whilst CP49 self-assembles into thick bundles of filaments, filensin only forms short fibrils, but when combined together they form filaments. The generation of gene knockouts by the targeted deletion of Bfsp1 and Bfsp2 that encode filensin and CP49, respectively, have been made to explore the function of beaded filaments in the lens. Our results suggest that the lens-specific beaded filaments are the key cytoskeletal element in organising and maintaining lens fibre cell architecture and are a key factor in determining the optical properties of the lens. We have also found that some common mouse strains contain a natural mutation in Bfsp2 that will effectively generate a CP49 knockout. This finding has important implications for lens research involving other gene knockouts maintained on a 129 background. It has also been observed that mutations in Bfsp2 are the genetic basis of inherited human cataract. Collectively, these data demonstrate that beaded filaments are fundamental to lens function.     

Identification of a novel intermediate filament-linked N-cadherin/gamma-catenin complex involved in the establishment of the cytoarchitecture of differentiated lens fiber cells

Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell-cell junctions. Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, strengthened by association with cytoskeletal networks during development. Both β- and γ-catenins are reported to link classical cadherins to the actin cytoskeleton, but only γ-catenin binds to the desmosomal cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we provide the first biochemical evidence that, in vivo, γ-catenin also mediates interactions between classical cadherins and the intermediate filament cytoskeleton, linked through desmoplakin. In the developing lens, which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a differentiation-state specific manner. This newly identified junctional complex was tissue specific but not unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through γ-catenin or β-catenin we developed an innovative “double” immunoprecipitation technique. This approach made possible, for the first time, the separation of N-cadherin/γ-catenin from N-cadherin/β-catenin complexes and the identification of multiple members of each of these isolated protein complexes. The study revealed that vimentin was associated exclusively with N-cadherin/γ-catenin junctions. Assembly of this novel class of cadherin junctions was coincident with establishment of the unique cytoarchitecture of lens fiber cells. In addition, γ-catenin had a distinctive localization to the vertices of these hexagonally shaped differentiating lens fiber cells, a region devoid of actin; while β-catenin co-localized with actin at lateral cell interfaces. We believe this novel vimentin-linked N-cadherin/γ-catenin junction provides the tensile strength necessary to establish and maintain structural integrity in tissues that lack desmosomes.     

The beaded intermediate filaments and their potential functions in eye lens

The elongated fiber cells of the eye lens contain a unique cytoskeletal system, the beaded chain filaments (BFs). The BFs had been morphologically identified more than two decades ago, but the precise identity of their subunit molecules remained unknown. Recently, use of recombinant DNA approaches, refined morphological and immunochemical studies and experiments with mutant mice have allowed the molecular dissection of these structures and provided clues about their potential functins. The BFs represent a highly specialized network of intermediate filaments (IFs) juxtaposed to the plasma membrane. They are obligate heteropolymers composed of two lens-specific polypeptides, filensin and phakinin. In this review we discuss the properties, molecular interactions and in situ arrangement of these two proteins, and comment on their potential roles during lens development.     

Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.     

A protein antigenically related to nuclear lamin B mediates the association of intermediate filaments with desmosomes

Desmosomes are specialized domains of epithelial cell plasma membranes engaged in the anchoring of intermediate filaments (IF). So far, the desmosomal component(s) responsible for this binding has not been unambiguously identified. In the present work, we have examined bovine muzzle epidermis desmosomes for the presence of protein(s) structurally and functionally related to lamin B, the major receptor for IF in the nuclear envelope (Georgatos, S. D., and G. Blobel. 1987. J. Cell Biol. 105:105-115). By using polyclonal antibodies to lamin B in immunoblotting experiments, we find that a desmosomal protein of 140-kD shares epitope(s) with lamin B. Immunoelectron microscopic and urea extraction experiments show that this protein is a peripheral protein localized at the cytoplasmic side of the desmosomes (desmosomal plaques). Furthermore, this protein binds vimentin in an in vitro assay. Since this binding is inhibited by lamin B antibodies, the epitopes common to the 140-kD protein and to lamin B may be responsible for anchoring of intermediate filaments to desmosomes. These data suggest that lamin B-related proteins (see also Cartaud, A., J. C. Courvalin, M. A. Ludosky, and J. Cartaud. 1989. J. Cell Biol. 109:1745-1752) together with lamin B, provide cells with several nucleation sites, which can account for the multiplicity of IF organization in tissues.     

Differentiation-dependent modification and subcellular distribution of aquaporin-0 suggests multiple functional roles in the rat lens

Using immunohistochemistry and mass spectrometry, differentiation-dependent changes in the subcellular distribution and processing of aquaporin-0 (AQP0) have been mapped in the rat lens. Sections labelled with C-terminal tail AQP0 antibodies yielded two concentric rings of labelling with minimal signal in the lens core. The rings were separated by a transient zone of decreased labelling located prior to the transition of differentiating fiber (DF) cells into mature denucleated fiber (MF) cells. Mass spectrometry showed that the loss of core labelling was due to AQP0 cleavage, while the transient loss of labelling was more likely caused by masking of the antibody epitope. AQP0 subcellular distribution changed with radial distance into the lens. In peripheral DF cells, AQP0 was found throughout both broad and narrow side membranes. In deeper-lying DF cells, AQP0 aggregated into plaque-like structures located on the broad sides. This shift occurred prior to the transient loss of AQP0 signal, and coincided with formation of broad-side membrane invaginations between adjacent fiber cells to which filensin, a known binding partner of AQP0, was also localized. After nuclei loss, AQP0 was once again distributed throughout MF cell membranes. In the absence of protein synthesis, the observed subcellular redistribution of AQP0 in DF and subsequent cleavage of AQP0 in MF are suggestive of a switch in the function of AQP0 from a water channel to a junctional protein.     

A Slow Maturation of a Cysteine Protease with a Granulin Domain in the Vacuoles of Senescing Arabidopsis Leaves

Arabidopsis RD21 is a cysteine protease of the papain family. Unlike other members of the papain family, RD21 has a C-terminal extension sequence composed of two domains, a 2-kD proline-rich domain and a 10-kD domain homologous to animal epithelin/granulin family proteins. The RD21 protein was accumulated as 38- and 33-kD proteins in Arabidopsis leaves. An immunoblot showed that the 38-kD protein had the granulin domain, whereas the 33-kD protein did not. A pulse-chase experiment with Bright-Yellow 2 transformant cells expressing RD21 showed that RD21 was synthesized as a 57-kD precursor and was then slowly processed to make the 33-kD mature protein via the 38-kD intermediate. After a 12-h chase, the 38-kD intermediate was still detected in the cells. These results indicate that the N-terminal propeptide was first removed from the 57-kD precursor, and the C-terminal granulin domain was then slowly removed to yield the 33-kD mature protein. Subcellular fractionation of the Bright-Yellow 2 transformant showed that the intermediate and mature forms of RD21 were localized in the vacuoles. Under the acidic conditions of the vacuolar interior, the intermediate was found to be easily aggregated. The intermediate and the mature protein were accumulated in association with leaf senescence. Taken together, these results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.     

Detection of gender differences in rat lens proteins using 2-D-DIGE

The glass-like transparency of the human eye lens is achieved by the tight packing of abundant crystallin proteins. However, the precise role of the accessory non-crystallin proteins is not well understood. We have carried out 2-DE mapping of these proteins in rat lens. This showed the presence of the high molecular weight filamentous structural proteins spectrin, filensin, tubulin, vimentin, actin and phakinin as well as several forms of potential crystallin oligomers comprised of alphaA, betaB1, betaA1 and betaA4 chains. Other proteins that were present include, heat shock protein 71, WD repeat protein 1, and several enzymes including alpha-enolase, pyruvate kinase, transketolase and aldose reductase. 2-D-DIGE analysis revealed several expression differences between the lens proteomes of male and female rats. Female rat lenses contained lower levels of aldose reductase, increased proteolyic fragments of the structural proteins filensin, vimentin and phakinin and higher levels of potential alphaA, betaB1 and betaA1 crystallin oligomers. Taken together these findings suggest that there are potential differences in oxidative stress regulation between male and female rat lenses, which may have implications on susceptibility to cataract formation. Future studies aimed at elucidating pre-cataractic changes in the non-crystallin proteins described here may facilitate identification of novel markers involved in cataractogenesis.