MALDI imaging mass spectrometry of ß- and γ-crystallins in the ocular lens.

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of ß- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas ß-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.

Statistical-thermodynamic model for light scattering from eye lens protein mixtures.

We model light-scattering cross sections of concentrated aqueous mixtures of the bovine eye lens proteins γB- and α-crystallin by adapting a statistical-thermodynamic model of mixtures of spheres with short-range attractions. The model reproduces measured static light scattering cross sections, or Rayleigh ratios, of γB-α mixtures from dilute concentrations where light scattering intensity depends on molecular weights and virial coefficients, to realistically high concentration protein mixtures like those of the lens. The model relates γB-γB and γB-α attraction strengths and the γB-α size ratio to the free energy curvatures that set light scattering efficiency in tandem with protein refractive index increments. The model includes (i) hard-sphere α-α interactions, which create short-range order and transparency at high protein concentrations, (ii) short-range attractive plus hard-core γ-γ interactions, which produce intense light scattering and liquid-liquid phase separation in aqueous γ-crystallin solutions, and (iii) short-range attractive plus hard-core γ-α interactions, which strongly influence highly non-additive light scattering and phase separation in concentrated γ-α mixtures. The model reveals a new lens transparency mechanism, that prominent equilibrium composition fluctuations can be perpendicular to the refractive index gradient. The model reproduces the concave-up dependence of the Rayleigh ratio on α/γ composition at high concentrations, its concave-down nature at intermediate concentrations, non-monotonic dependence of light scattering on γ-α attraction strength, and more intricate, temperature-dependent features. We analytically compute the mixed virial series for light scattering efficiency through third order for the sticky-sphere mixture, and find that the full model represents the available light scattering data at concentrations several times those where the second and third mixed virial contributions fail. The model indicates that increased γ-γ attraction can raise γ-α mixture light scattering far more than it does for solutions of γ-crystallin alone, and can produce marked turbidity tens of degrees celsius above liquid-liquid separation.

Crystallin distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei lenses.

The eye lens remains transparent because of soluble lens proteins known as crystallins. For years γ-crystallins have been known as the main lens proteins in lower vertebrates such as fish and amphibians. The unique growth features of the lens render it an ideal structure to study ageing; few studies have examined such changes in anuran lenses. This study aimed to investigate protein distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei species. Lenses were fractionated into concentric layers by controlled dissolution. Water-soluble proteins were separated into high (HMW), middle (MMW) and low molecular weight (LMW) fractions by size-exclusion HPLC and constituents of each protein class revealed by 1DE and 2DE. Spots were selected from 2DE gels on the basis of known ranges of subunit molecular weights and pH ranges and were identified by MALDI-TOF/TOF MS following trypsin digestion. Comparable lens distribution patterns were found for each species studied. Common crystallins were detected in both species; the most prominent of these was γ-crystallin. Towards the lens centre, there was a decrease in α- and ß-crystallin proportions and an increase in γ-crystallins. Subunits representing taxon-specific crystallins demonstrating strong sequence homology with ζ-crystallin/quinone oxidoreductase were found in both L. infrafrenata and P. sauvagei lenses. Further work is needed to determine which amphibians have taxon-specific crystallins, their evolutionary origins, and their function.

Racemization of two proteins over our lifespan: deamidation of asparagine 76 in γS crystallin is greater in cataract than in normal lenses across the age range.

PURPOSE: Long-lived proteins are widespread in man, yet little is known about the processes that affect their function over time, or their role in age-related diseases. METHODS: Racemization of two proteins from normal and cataract human lenses were compared with age using tryptic digestion and LC/mass spectrometry. Asp 151 in αA crystallin and Asn 76 in γS crystallin were studied. RESULTS: Age-dependent profiles for the two proteins from normal lenses were different. In neither protein did the modifications increase linearly with age. For αA crystallin, racemization occurred most rapidly during the first 15 years of life, with approximately half of L-Asp 151 converted to D-isoAsp, L-isoAsp, and D-Asp in a ratio of 3:1:0.5. Values then changed little. By contrast, racemization of Asn 76 in γS crystallin was slow until age 15, with isoAsp accounting for only 5%. Values remained relatively constant until age 40 when a linear increase (1%/year) took place. When cataract lenses were compared with age-matched normal lenses, there were marked differences in the time courses of the two crystallins. For αA crystallin, there was no significant difference in Asp 151 racemization between cataract and normal lenses. By contrast, in γS crystallin the degree of conversion of Asn 76 to isoAsp in cataract lenses was approximately double that of normals at every age. CONCLUSIONS: Modification of Asn and Asp over time may contribute to denaturation of proteins in the human lens. An accelerated rate of deamidation/racemization at selected sites in proteins, such as γS crystallin, may contribute to cataract formation.

A novel mutation in γD-crystallin associated with autosomal dominant congenital cataract in a Chinese family.

PURPOSE: To identify the pathogenic gene mutation in a Chinese family with autosomal dominant congenital nuclear cataract. METHODS: After obtaining informed consent, detailed ophthalmic examinations were performed and genomic DNAs were obtained from eleven family members in a three-generation Chinese family with five affected. All exons of candidate genes associated with congenital nuclear cataract were amplified by polymerase chain reaction (PCR) and the PCR products were sequenced in both directions. The hydrophobic property of the mutant protein was analyzed with bioinformatics program ProtScale. The structure homology modeling of the mutant protein was based on Swiss-Model Serve, and its structure was displayed and compared with native γD-crystallin (CRYGD) using the RasMol software. RESULTS: By sequencing the encoding regions of the candidate genes, a novel mutation (c.110G>C) was detected in exon 2 of CRYGD, which resulted in the substitution of a highly conserved arginine by proline at codon 36 (p.R36P). The mutation co-segregated with all patients and was absent in 100 normal Chinese controls. Bioinformatics analysis showed an obvious increase of the local hydrophilicity of the R36P mutant γD-crystallin. The homology modeling showed that the structure of the mutant protein was similar with that of native human γD-crystallin. CONCLUSIONS: The study identified a novel mutation (c. 110G>C) in CRYGD associated with autosomal dominant congenital cataract in a Chinese family. It expands the mutation spectrum of CRYGD in association with congenital cataract.

Crystallin distribution patterns in concentric layers from toad eye lenses.

Protein distribution patterns across eye lenses from the Asiatic toad Bufo gargarizans were investigated and individual crystallin classes characterised. Special fractionation that follows the growth mode of the lens was used to yield nine fractions corresponding to layers laid down at different chronological (developmental) stages. Proportions of soluble and insoluble crystallins within each fraction were measured by Bradford assay. Water-soluble proteins in all fractions were separated by size-exclusion HPLC and constituents of each class further characterised by electrophoresis, RP-HPLC and MS analysis. In outer lens layers, alpha-crystallin is the most abundant soluble protein but is not found in soluble proteins in the lens centre. Water-soluble beta-crystallins also decrease from their highest level in the outer lens to negligible mounts in the central lens. The proportion of soluble gamma-crystallin increases significantly towards the lens centre where this is the only soluble protein present. Insoluble protein levels increase significantly towards the lens centre. In B. gargarizans lenses, as with other anurans, the predominant water-soluble protein class is gamma-crystallin. No taxon-specific crystallins were found. The relationship between the protein distribution patterns and the functional properties of the lens this species is discussed.

Differential protein expression in lens epithelial whole-mounts and lens epithelial cell cultures.

PURPOSE: Lens fibergenesis is a problem in several types of cataract and in the posterior capsular opacification following cataract surgery. To correct improper fiber differentiation or to prevent unwanted growth on the posterior capsule following cataract surgery requires a thorough understanding of normal and abnormal fiber formation. To this end, studies were initiated to characterize fiber differentiation in the bovine lens and in lens epithelial cell cultures. METHODS: Indirect immunofluorescence and immunoblot analysis were employed to study the expression of vimentin, beta-crystallin, gamma-crystallin, filensin, aquaporin 0 and the Na, K-ATPase catalytic subunit isoforms (alpha1, alpha2, alpha3) in bovine lens epithelium whole-mounts as well as lens epithelial cell cultures propagated in medium containing 10% bovine serum or in medium supplemented with bovine serum concentrations < or =4%. RESULTS: Three distinct cell types were observed in the bovine lens epithelium. The cells of the central zone were identified by a polarized distribution of two distinct Na, K-ATPase catalytic subunit isoforms, alpha1 to the apical (fiber side) and alpha3 to the basal (aqueous humor side) membranes. Lateral to the polarized central zone, was the germinative zone of cells, best characterized by perinuclear vimentin basket-like structures and the loss of polarized Na, K-ATPase catalytic subunit isoforms. Lateral to the germinative zone were the cells of the transition zone (meridinal rows) where expression of the lens specific proteins beta-crystallin, gamma-crystallin, filensin and aquaporin 0 as well as the lens fiber-, adipocyte- and brain glia-specific Na, K-ATPase catalytic subunit, alpha2 are expressed. The cultured cells propagated in medium supplemented with 10% serum bore no resemblance to any of the cells of the bovine lens epithelium whole-mounts. The cells propagated in the medium supplemented with the lower bovine serum levels resembled the differentiating fibers of the transition zone of the bovine lens epithelium whole-mounts as well as superficial cortical fibers. CONCLUSIONS: Since the low-serum lens epithelial cell cultures bear a remarkable resemblance to early differentiating fibers, they are reasonable models for the study of early fiber differentiation or prevention of differentiation. The culture conditions employed do not yield the polarized cells of the central zone. Nor has the function of these polarized cells in lens fluid, nutrient and ion homeostasis been determined.

Ontogeny and localization of the alpha, beta, and gamma crystallins in newt eye lens development.

The alpha-, beta-, and gamma-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the alpha-, beta-, and gamma-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. beta-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. gamma-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and alpha-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for beta-crystallins until late in lens morphogenesis, and alpha- and gamma-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.