A Rat Eye Lens Model of Cataract Formation.

This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.

Purification and Functional Characterization of the C-Terminal Domain of the ß-Actin-Binding Protein AIM1 In Vitro.

The protein absent in melanoma 1 (AIM1) is a member of the ßγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to ß-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the ß-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and ß-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and ß-actin; we also obtain the crystal of the first two ßγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the ßγ-crystallin domain repeats that bound to ß-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function.

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine ßL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates.

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 Major Chromophore Arising from Glucose Degradation and Oxidative Stress Occurrence during Lens Proteins Glycation Induced by Glucose.

Glucose autoxidation has been proposed as a key reaction associated with deleterious effects induced by hyperglycemia in the eye lens. Little is known about chromophores generated during glucose autoxidation. In this study, we analyzed the effect of oxidative and dicarbonyl stress in the generation of a major chromophore arising from glucose degradation (GDC) and its association with oxidative damage in lens proteins. Glucose (5 mM) was incubated with H2O2 (0.5-5 mM), Cu2+ (5-50 µM), glyoxal (0.5-5 mM) or methylglyoxal (0.5-5 mM) at pH 7.4, 5% O2, 37 °C, from 0 to 30 days. GDC concentration increased with incubation time, as well as when incubated in the presence of H2O2 and/or Cu2+, which were effective even at the lowest concentrations. Dicarbonylic compounds did not increase the levels of GDC during incubations. ¹H, 13C and FT-IR spectra from the purified fraction containing the chromophore (detected by UV/vis spectroscopy) showed oxidation products of glucose, including gluconic acid. Lens proteins solutions (10 mg/mL) incubated with glucose (30 mM) presented increased levels of carboxymethyl-lysine and hydrogen peroxide that were associated with GDC increase. Our results suggest a possible use of GDC as a marker of autoxidative reactions occurring during lens proteins glycation induced by glucose.

Simultaneous separation of taxon-specific crystallins from Mule duck and characterization of their enzymatic activities and structures.

Methods to obtain pure proteins in large amounts are indispensible in protein research. We report here a large-scale/simultaneous isolation of taxon-specific crystallins (ɛ- and δ-crystallin) from the eye lenses of Mule duck. We also investigate the compositions, enzymatic activities, and structures of these purified taxon-specific proteins. A relatively mild method of ion-exchange chromatography was developed to fractionate ɛ-crystallin and δ-crystallin in large amount, ca. ∼6.60mg/g-lens and ∼41.0mg/g-lens, respectively. Both crystallins were identified by electrophoresis, HPLC, and MALDI-TOF-MS. ɛ-Crystallin, with native composition of Mr 142kDa, consisted of two subunits of 35kDa and 36kDa, while δ-Crystallin, with native molecular mass of 200kDa, comprised single subunit of Mr ∼50kDa. Both ɛ- and δ-crystallin were tetramers. The former showed lactate dehydrogenase (LDH) activity, while the latter appeared slightly active in an argininosuccinate lyase (ASL) assay. Raman spectroscopic results indicated that the secondary structures of ɛ- and δ-crystallin were predominantly α-helix as evidenced by the vibrational stretching of amide III over 1260cm-1 and amide I at 1255cm-1, in greatly contrast to the anti-parallel ß-sheet of α- and ß-crystallin as demonstrated by amide III at 1238cm-1 and amide I at 1672cm-1. The microenvironments of aromatic amino acids and the status of thiol groups also vary in different crystallins. The compositions, enzyme activities, and structures of the ɛ- and δ-crystalline of Mule duck are different from those of Muscovy duck (Cairina moschata) or Kaiya duck (Anas Platyrhynchos var. domestica), which reflect faithfully species specificity.

Identification of Sequence Similarities among Isomerization Hotspots in Crystallin Proteins.

The eye lens crystallins represent an ideal target for studying the effects of aging on protein structure. Herein we examine separately the water-soluble (WS) and water-insoluble (WI) crystallin fractions and identify sites of isomerization and epimerization. Both collision-induced dissociation and radical-directed dissociation are needed for detection of these non-mass-shifting post-translational modifications. Isomerization levels differ significantly between the WS and the WI fractions from sheep, pig, and cow eye lenses. Residues that are most susceptible to isomerization are identified site-specifically and are found to reside in structurally disordered regions. However, isomerization in structured domains, although less common, often yields more dramatic effects on solubility. Numerous isomerization hotspots were also identified and occur in regions with aspartic acid and serine repeats. For example, 128KMEIVDDDVPSLW140 in ßB3 crystallin contains three sequential aspartic acid residues and is isomerized heavily in the WI fractions, while it is not modified at all in the WS fractions. Potential causes for enhanced isomerization at sites with acidic residue repeats are presented. The importance of acidic residue repeats extends beyond the lens, as they are found in many other long-lived proteins associated with disease.

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level.

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of "free" Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their "free" counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

Extraction, purification and characterization of the crystallin protein of cataractous eye lens nucleus.

The purpose of this study is to separate and identify the crystallin protein present in the nucleus of a human cataractous eye lens. Cataractous lenses were collected from different eye hospitals from patients of different etiologies with ages between 40 and 80 years. Lens nucleus proteins were extracted into four fractions on the basis of their solubility in different media by applying a reported method. These fractions were buffer-soluble proteins (PS), urea-soluble proteins (PU), yellow fraction proteins (PY) and insoluble proteins (PI). All three soluble fractions were subjected to HPLC and GPC analysis. Both HPLC and GPC analysis showed that each fraction contains α-, ß- and γ-crystallins, a major class of protein present in the lenses of vertebrates. Various chromatographic parameters including precision, accuracy and linearity have been evaluated. Studies of water-insoluble crystallins using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) have demonstrated extreme homogeneity with evidence of major components with molecular masses of 18-70 kDa, similar to the crystallin of the water-soluble portion. The method was found to be suitable for the analysis of various isomers of crystallin protein present in human cataractous eye lens nuclei. The detailed results of the GPC are discussed. This study provides the first HPLC and GPC analysis of a human cataractous eye lens nucleus.

The impact of calcium ion on structure and aggregation propensity of peroxynitrite-modified lens crystallins: new insights into the pathogenesis of cataract disorders.

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has been indicated in the pathogenesis of various ocular disorders. The PON induces mobilization of intra cellular calcium which plays an important function in structure and activity of lens proteins. Moreover, the amount of calcium increases to the pathogenic level in the cataractous lenses. The aim of this study was to assess the impact of calcium ion on structure and aggregation of PON-modified lens crystallins, using spectroscopic techniques and gel mobility shift assay. The PON modification of lens proteins was confirmed with detection of the significantly increased quantity of carbonyl group, dityrosine, nitrotyrosine and nitrotryptophan. Moreover, the modified proteins indicated high levels of solvent exposed hydrophobic surfaces and markedly elevated proteolytic instability which can be explained with their structural alteration upon this type of modification. The results of UV-vis absorption studies suggest that PON-modified lens crystallins are highly sensitive to aggregation in the presence of both physiological and pathological ranges of calcium ion. Also, the results of thioflavin T fluorescence study indicated absence of any ordered aggregate entity in the calcium-induced aggregate samples. The results of gel mobility shift assay demonstrated the importance of calcium ion in the induction of disulfide and dityrosine covalent cross-linking and formation of the oligomeric structure with relatively larger sizes in the PON-modified crystallins compared to the non-modified protein counterparts. Overall, this study may suggest that a simultaneous raise of calcium ion and PON in the eye ball is an important risk factor for development of cataract diseases.

Application of solid-phase extraction for the concentration of chromophores, fluorophores, and photosensitizers from lens protein digests.

Solid-phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid-phase extraction was capable of concentrating the advanced glycation end-products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.

Lactate dehydrogenase like crystallin: a potentially protective shield for indian spiny-tailed lizard (Uromastyx hardwickii) lens against environmental stress?

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyx hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure-function relationship of these proteins.

Amyloid fibrils from readily available sources: milk casein and lens crystallin proteins.

Amyloid fibrils are a highly ordered and robust aggregated form of protein structure in which the protein components are arranged in long fibrillar arrays comprised of ß-sheet. Because of these properties, along with their biocompatibility, amyloid fibrils have attracted much research attention as bionanomaterials, for example as template structures (in some cases following modification) that can be used as biosensors, encapsulators, and biomimetic materials. To use amyloid fibrils for such a range of applications will require them to be obtained relatively easily in large quantities. In this chapter, we describe methods for isolating crystallin and casein proteins from readily available sources that contain abundant protein, i.e., the eye lens and milk, respectively, and the subsequent conversion of these proteins into amyloid fibrils.

Proteomic analysis of the aqueous humor in patients with wet age-related macular degeneration.

PURPOSE: A number of studies have shown that the levels of some proteins in the aqueous humor (AH) are altered and correlate with the mechanisms or prognosis of many eye diseases. To identify the possible mechanisms that lead to the development of wet age-related macular degeneration (AMD), a proteomic analysis of the AH composition from wet AMD patients was performed and compared with that from non-AMD cataract patients. EXPERIMENTAL DESIGN: Six wet AMD and six non-AMD cataract patients were enrolled. A proteomic approach which included two-dimensional electrophoresis coupled with MS and bioinformatics methods were used to identify AH proteins with altered expression in wet AMD compared with non-AMD patients. An ELISA was used to validate the proteomic results. RESULTS: We separated 78 protein spots and identified 68 that were differently expressed in the wet AMD group and controls. Numerous proteins identified in this study are implicated in inflammation, apoptosis, angiogenesis, and oxidative stress. CONCLUSIONS AND CLINICAL RELEVANCE: The AH protein composition was significantly different between wet AMD and non-AMD patients. The proteins identified in this study may be potential biomarkers of wet AMD development and might play a role in the mechanisms of wet AMD.

Polymorphism and higher order structures of protein nanofibers from crude mixtures of fish lens crystallins: toward useful materials.

Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to individual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers; here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.

Structural and functional specificity of small heat shock protein HspB1 and HspB4, two cellular partners of HspB5: role of the in vitro hetero-complex formation in chaperone activity.

The ubiquitous small heat shock proteins are essential elements in cellular protection, through a molecular chaperone activity. Among them, human small heat shock protein HspB1, HspB4 and HspB5 are involved in oncogenesis, anti-apoptotic activity and lens transparency. Therefore, these proteins are potential therapeutic targets in many diseases. Their general chaperone activity is related to their dynamic and multiple oligomeric structures, which are still poorly understood. The tissue selective distribution of HspB1 and HspB4, two cellular partners of HspB5, suggests that these two proteins might have evolved to play distinct physiological functions. Moreover, hetero-complex formation seems to be favoured in vivo, yet the functional specificity of the HspB1-HspB5 and HspB4-HspB5 hetero-complexes compared to the homo-oligomers remains unclear in the stress response pathway. A powerful approach combining biochemistry, biophysics and bioinformatics, allowed us to compare the different assemblies, with a special emphasis on the structural data, subunit exchange properties, activity and sequence evolution. We showed that they all exhibit different properties, from structural organization in physiological versus stress conditions, to chaperone-like activity, whatever the level of sequence conservation. Subunit exchange kinetics leading to HspB1-HspB5 or HspB4-HspB5 hetero-complex formation is also different between these two complexes: HspB5 exchanges more rapidly subunits with HspB1 than with HspB4. The relative sequence conservation in the sHSP superfamily does hide important structural heterogeneity and flexibility, which confer an enlarged range of different surface necessary to efficiently form complexes with various stress-induced cellular targets. Our data suggest that the formation of hetero-complexes could be an original evolutionary strategy to gain new cellular functions.

Conformational heterogeneity and dynamics in a ßγ-crystallin from Hahella chejuensis.

Most of the ßγ-crystallins are structural proteins with high intrinsic stability, which gets enhanced by Ca(2+)-binding in microbial members. Functions of most of these proteins are yet to be known. However, a few of them were reported to be involved in Ca(2+)-dependent and stress-related functions. Hahellin, a microbial homolog, is a natively unfolded protein that acquires a well-folded structure upon Ca(2+) binding. Although the structure of ßγ-crystallin domains is well understood, the dynamical features are yet to be explored. We have investigated for the first time the equilibrium dynamics, conformational heterogeneity and associated low-lying free-energy states of hahellin in its Ca(2+)-bound form using NMR spectroscopy to understand the dynamics of a ßγ-crystallin domain. Hahellin shows large conformational heterogeneity with nearly 40% of the residues, some of which are part of Ca(2+)-binding loops, accessing alternative states. Further, out of the two Greek key motifs, which together constitute the ßγ-crystallin domain, the second Greek key motif is floppy as compared to its relatively rigid counterpart. Taken together, we believe that these characteristics might be of importance to understand the stability and functions of ßγ-crystallin domains.

Lens proteome map and alpha-crystallin profile of the catfish Rita rita.

Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. alphaA-crystallins, member of the alpha-crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1- and 2-D immunoblot analysis with anti-alphaA-crystallin antibody. Two protein bands of 19-20 kDa were identified as alphaA-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-alphaB-crystallin and antiphospho-alphaB-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating alphaB-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita alpha-crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its alphaA- and alphaB-crystallin content (contain low amount from 5-9% of alphaB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse alpha-crystallins (contain higher amount of alphaB-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the alpha-crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of alphaA- and alphaB-isoforms, as they are at the two extremes in terms of alphaA-and alphaB-crystallin content.

Comparative analysis of crystallins and lipids from the lens of Antarctic toothfish and cow.

Animal model systems of senile cataract and lens crystallin stability are essential to understand the complex nature of lens transparency. Our aim in this study was to assess the long-lived Antarctic toothfish Dissostichus mawsoni (Norman) as a model system to understand long-term lens clarity in terms of solubility changes that occur to crystallins. We compared the toothfish with the mammalian model cow lens, dissecting each species' lens into a cortex and nuclear region. In addition to crystallin distribution, we also assayed fatty acid (FA) composition by negative ion electrospray ionization mass spectrometry (ESI-MS). The majority of toothfish lens crystallins from cortex (90.4%) were soluble, whereas only a third (31.8%) from the nucleus was soluble. Crystallin solubility analysis by SDS-PAGE and immunoblots revealed that relative proportions of crystallins in both soluble and urea-soluble fractions were similar within each species examined and in agreement with previous reports for bovine lens. From our data, we found that both toothfish and cow crystallins follow patterns of insolubility that mirror each animals lens composition with more γ crystallin aggregation seen in the toothfish lens nucleus than in cow. Toothfish lens lipids had a large amount of polyunsaturated fatty acids that were absent in cow resulting in an unsaturation index (I(U)) four-fold higher than that of cow. We identified a novel FA with a molecular mass of 267 mass units in the lens epithelial layer of the toothfish that accounted for well over 50% of the FA abundance. The unidentified lipid in the toothfish lens epithelia corresponds to either an odd-chain (17 carbons) FA or a furanoid. We conclude that long-lived fishes are likely good animal models of lens crystallin solubility and may model post-translational modifications and solubility changes better than short-lived animal models.

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.