An extrinsic membrane polypeptide associated with high-molecular-weight protein aggregates in human cataract.

A 43,000-dalton polypeptide has been isolated from the high-molecular-weight disulfide-rich fraction of the water-insoluble protein of human cataractous lenses. On the basis of immunochemical reactivity and fluorescent antibody binding, this polypeptide is localized in the membrane region of the lens cell. This observation suggests an interaction between the soluble lens proteins and membrane-associated polypeptides in the formation of large protein aggregates which may cause cataract.

Methylisocyanate and actin polymerization: the in vitro effects of carbamylation.

Uremia has been implicated in cataractogenesis due to protein carbamylation by cyanate derived from urea. The present study was designed to directly identify the effects of carbamylation on actin polymerization and the possible contribution to cataract formation. The susceptibility of actin to carbamylation is expected because of the 19 lysines distributed along its length. The lysines of actin were selectively carbamylated by methylisocyanate (MIC) at pH 8.0 and 4 degrees C and actin polymerization assayed by high-shear viscometry, fluorescence and transmission electron microscopy. Our results provide evidence that non-enzymatic carbamylation of the lysine residues prevents the polymerization of actin. In addition, this carbamylated actin inhibited the polymerization of nascent, unmodified actin. High-shear viscosity measurements demonstrated decreased initial apparent rates and decreased steady-states (final specific viscosities) of polymerization. Fluorescence measurements showed decreased relative intensities of fluorescence versus control and confirmed the inhibitory effects of carbamylation by MIC on the steady state of F-actin. Transmission electron microscopy (TEM) showed the presence of disorganized filaments when carbamylated actin was added to polymerizing unmodified actin. Our results suggest that carbamylation of actin can cause a loss of ordered filament structure and shape of the lens fiber cell, thus predisposing it to cataract development.

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy.

alpha-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of alpha A and alpha B subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37 degrees C: binding sites per 400 kDa aggregate = 10 and Ka = 8.1 x 10(3) M-1; and an essentially identical Ka of 7.84 x 10(3) M-1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, alpha H, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22 degrees C and was absent at 4 degrees C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05-0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum of 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P(i), AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/alpha-crystallin binding.

Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins.

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.

alpha-Crystallin quaternary structure: molecular basis for its chaperone activity.

alpha-Crystallin, the major protein in all vertebrate lenses, functions as a chaperone. In the present analysis, an 'open' micellar structure composed of alpha A subunits is used to simulate chaperoning of partially heat denatured soluble gamma-crystallin. The interaction is both electrostatic and hydrophobic and satisfies experimental evidence for a 1:1 alpha/gamma molar ratio, a doubling of molecular mass and a minimal increase in the dimensions of the complex [J. Biol. Chem. (1994) 269, 13601-13608; Invest. Opthalmol. Vis. Sci. (1995) 36, 311-21]. These data are also in accord with Farahbaksh et al. [Biochemistry (1995) 34, 509-16]; i.e. the bound gamma-crystallin monomers are not in a central cavity, but are separated by alpha A subunits.

Effect of catalytically active recruited crystallins on lens metabolism.

The impact on duck lens metabolism of recruited epsilon-crystallin/LDH-B4 and tau-crystallin/enolase was investigated by NMR spectroscopy. A comparison of the duck lens metabolite profile with that of the calf in which recruited crystallins are absent revealed significant increases in ATP, alpha-glycerophosphate (alpha-GP) and pyridine dinucleotide concentrations. The alterations in the concentrations of ATP and alpha-GP appear to be related to both the high concentration of NAD and the elevated reduced to oxidized NADH/NAD+ ratio.

Localization of B-DNA and Z-DNA in terminally differentiating fiber cells in the adult lens.

We examined histochemically and immunohistochemically the distribution of B- and Z-DNA in the epithelium and terminally differentiating dog lens fiber cells. On the basis of anti-DNA antibody reactivity, qualitative and quantitative data on B- and Z-DNA in cells were determined. Anti-B-DNA immunoreactivity gradually declined throughout nucleated fibers, with a precipitous decrease at approximately 90 microns. Anti-Z-DNA antibody binding decreased with a sudden loss of immunoreactivity at approximately 90 microns. The pattern of anti-B- and Z-DNA staining correlates with the loss of alpha-crystallin immunoreactivity, the major lens crystallin, and decreased eosin staining of proteins. Germinative zone cell nuclei showed the highest DNA probe binding values, followed by the superficial fibers, central zone, middle fibers, and deep fibers. The presence of single-stranded (ss)DNA in deeper fibers was detected by anti-ss-DNA antibodies. This is indicative of DNA degradation. These observations suggest that a dramatic reorganization of lens fiber cells' supramolecular order occurs at approximately 90 microns, the phase transition zone.

Ultrastructural abnormalities in a Marfan's syndrome lens.

The surface ultrastructure of the posterior of the Marfan's syndrome lens was studied by scanning electron microscopy. All aspects of the architecture and construction of the capsule, the zonules and their insertions, the epithelium, and the lens fibers showed a sharp deviation from normal. The capsular fibrils were abnormally large and grossly granular. The zonular fibrils, similar in size and granularity to those of the capsule, contained numerous large aggregates and showed a lack of parallel orientation. The epithelial cells displayed a coarser than normal surface granularity and a lack of regularity of form and orientation. The cortical and nuclear lens fibers displayed a relatively normal surface pattern but showed poorly defined borders and interdigitating processes.

Tomographic measurements of in vivo cataracts by slit-lamp photography.

A practical method for detecting in vivo cataracts has been developed using a commercially available slit-lamp camera and by utilizing a measuring grid photographic technique. The basis of the method is to photograph a series of slit-beam sections through a lens at varying angles to the optic axis. The areas of opacity show up as localized regions of light back-scatter where the opacities intersect the path of the beam. A set of measuring grids compensated for angle distortion have been prepared. These are superimposed on the appropriate photograph. By taking a sufficient number of sections through a cataractous lens, a tomographic representation of the opacities can be constructed using a lens map. An example of the procedure of three-dimensional mapping is presented using actual lens pictures. The photographic parameters used in this method are explained in detail. The method achieves the goals of ease of use, reproducibility, and applicability to research and clinical studies.

An apparent small cluster of choroidal melanoma cases.

Three choroidal melanomas were detected in a 2.5-year period in a small community of 3,592 persons. This small cluster represented an incidence about 20 times that expected (P = .0006). The community has an isolated water supply and very little industry. We determined the incidence of cancer in this and two adjacent communities and found no other unexpectedly high incidence. The three patients had no common exposures. Analyses of air and water from the involved community by mass spectroscopy, chromatography, and Ames (mutagenicity) tests were noncontributory. Nine of 60 mice given community water after weaning developed lens opacities eight to 16 months later; electron microscopy showed an abnormal monolayer of cells on the outer surface of the anterior lens capsule. The genesis of this monolayer was not clear. None of the 30 controls showed such lesions.

Progress toward the establishment of nuclear magnetic resonance measurements as an index of in vivo lens functional integrity.

Cataract prevention, delay, or reversal requires in vivo detection of lens changes that initiate the opacification process. The determination of lenticular biochemistry by a noninvasive methodology would represent a major step in the in vivo assessment of lens normalcy. Nuclear magnetic resonance spectroscopy appears to be a promising noninvasive technique to meet this goal. However, the correlation of lenticular "invasive" morphological and physiological data with NMR spectroscopic data in organ culture is necessary for establishing the validity of NMR spectral measurements as an index of lens functional integrity. The implementation of lenticular NMR spectroscopic studies will provide quantitative information such as intralenticular pH, Na+ and K+ ion distributions, lenticular levels of phosphorus-containing metabolites, glycolytic pathway intermediates and the end product lactate, sorbitol pathway activity, and possibly reduced glutathione levels. Development in surface coil NMR spectroscopy which enables the spatial localization of tissue metabolites and advances in the refinements required for quantitation of these results are discussed. These measurements of in vivo lenticular biochemistry will provide the information necessary for the scheduling of anticataract drug therapy as well as an ongoing method for monitoring the efficacy of such treatment.

Immunofluorescent localization of two intrinsic membrane polypeptides in adult human lenses.

Two major intrinsic membrane polypeptides of molecular weights 22K and 26K have been isolated and antisera for each prepared against highly purified preparations. The lack of reactivity of these antibodies in several other tissues suggests that they are unique to the lens. These antisera were used for localization of these polypeptides in adult human lenses by indirect immunofluorescent staining. Neither of the polypeptides is found in the epithelial cells; however those cells commencing differentiation show some fluorescent staining. The elongating fiber cells of the bow region show cytoplasmic staining for both 22K and 26K. This may reflect their sites of synthesis. In this region the species of 22K studies showed no localization in the membrane; however, 26K is concentrated at the membrane as evidenced by the intense band of fluorescent staining in this region. In the lens inner cortical region, there is a loss of cytoplasmic staining and both polypeptides appear to be membrane bound. This redistribution of the membrane polypeptides occurring in the inner cortex is at the site of considerable lens fiber contour alterations.

L-alpha-glycerophosphate binding to bovine gamma-crystallin: a potential link between metabolism and supramolecular order.

The highly selective nature of protein-ligand interactions provides a sensitive mechanism for the modulation of cellular activity by proteins. In the eye lens the supramolecular order of the lens crystallins, which is expected to be susceptible to protein electrostatic charge, in part defines transparency. The binding of charged ligands to proteins is one way of achieving an alteration in protein electrostatic charge. Evidence is presented that L-alpha-glycerophosphate, a major phosphorus metabolite of eye lens metabolism, binds to the globular protein, gamma-crystallin with moderately high affinity and in a positive cooperative manner. The following binding parameters were obtained from equilibrium measurements: minimum number of binding sites, n = 2; Kassoc = 6.2 +/- 0.5 x 10(3) M-1; cooperativity parameter, alpha H = 1.9 +/- 0.1. Interactive computer graphics display techniques were used to locate putative ligand binding sites, and in turn, to identify the possible molecular interactions responsible for the binding of ligand to protein at one of the sites. One putative binding site was located in the cleft between the two domains of gamma II-crystallin. Arginyl residues 79 and 147 are involved in ligand binding as are the peptide carbonyl oxygens of residues Tyrosyl-50 and Aspartyl-156. Five hydrogen bonds between the ligand and the protein structure are predicted for the binding of L-alpha-glycerophosphate, whereas only 3 occur for the binding of the "unnatural" D-enantiomorph. Modulation of both lens protein supramolecular organization and lens metabolism is predicted to be a consequence of L-alpha-glycerophosphate binding to gamma-crystallin in the lens.

Prion protein expression in mammalian lenses.

PURPOSE: Aging and oxidative stress resulting from over-expression of Alzheimer precursor protein (betaAPP) have been studied as important factors contributing to the major age-related (sporadic), and minor (hereditary) forms of Alzheimer's disease (AD), and muscle inclusion body myositis, (IBM). AD and prion proteins accumulate in plaques linked with AD and scrapie diseases, and in rimmed vacuoles of IBM. Soluble beta-amyloid (Abeta) fibrillar forms are now thought to play a critical role in and outside of cells by producing oxidative stress. In lens, betaAPP and Abeta increase in cultured lenses exposed to oxidative stress, and in areas of lens fiber cell degeneration in thiamine (vitamin B1) deprived mice, a classic model of systemic oxidative stress. The purpose of the present study is to extend our studies of amyloid disease-related protein expression in mammalian lenses. METHODS: Western blot, immunohistochemical detection, and RT-PCR methods were used to identify and quantitate prion protein expression in human, monkey, and guinea pig lenses. RESULTS: We demonstrate for the first time that prion protein gene expression increases with oxidative stress in cultured human lens epithelial cells. In addition, we detected greater prion protein gene expression in fiber cells than epithelial cells in vivo. This is consistent with increases in prion protein expression demonstrated in myoblasts and neuronal cells induced to differentiate. Our initial investigations of prion protein in human lens cataracts identified increased prion protein immunoreactivity in regions of lens fiber cell degeneration. CONCLUSIONS: The present data indicate that prion protein expression increases during lens development, and is substantially increased in cultured human lens epithelial cells exposed to oxidative stress. We also provide evidence that prion protein immunoreactivity can be increased in regions of fiber cell disorganization. These data suggest a potential role for prion protein as a marker for some types of lens pathology, and in the mechanism of oxidative stress-related lens degeneration.

Electrostatic parameters of the theoretical quaternary structure of bovine alpha-crystallin.

By changing the ionic strength, the effects of charge modification on the electrostatic properties of our predicted "open' micellar quaternary structure composed of bovine alpha A subunits were determined. The electrostatic potential values (phi) at 6 arbitrary points surrounding the protein and at all atomic sites of the protein were calculated using the non-linear Poisson-Boltzmann equation. The effective charge (q) of our predicted aggregate ranged from 16 at 0.0022 M to 45 at 0.1472 M ionic strengths. The variation of potential (phi), as well as charge, is a hyperbolic function of ionic strength (R2, 0.901). Plotting the inverse charge (l/q) against inverse ionic strength (1/l) it is possible to calculate maximum charge (q(max)) (approximately 48) at saturation. This value is close to previously reported experimental (50 +/- 5), and our theoretical charge (45), values at physiological ionic strength (0.145 M). These data indicate that maximal repulsion among the alpha-crystallin aggregates occurs at or near physiological ionic strength. Also, half-maximal charge (q(max)/2) at 0.003-0.004 M indicates a transition state at very low ionic strength. The calculated volume available for the mobile solvent in our quaternary structure is approximately 70%. These data are in good agreement with experimental values for bovine alpha-crystallin in solution reported by Xia et al. (Biophys. J., 1994; 66: 861-872). This agreement provides support for our predicted models of alpha-crystallin and a level of confidence in the reliability of the theoretical calculations. Since an ionic gradient exists between the lens cortical and nuclear regions, the modification of charge on alpha-crystallin by varying ionic strength could contribute to the function of alpha-crystallin as a modulator of lens supermolecular order during fiber cell maturation.

Interaction of DNA with bovine lens alpha-crystallin: its functional implications.

Under normal conditions, lens aggregates of alpha-crystallin subunits, alpha A and alpha B, are found in the cytoplasm. However, during stress in nonlenticular tissues, alpha B translocates to the nucleus. A sequence study revealed that both subunits share a consensus sequence with other DNA binding proteins. These observations prompted us to investigate DNA binding with alpha-crystallin by UV-mediated photo-crosslinking. The data show that both single and double stranded DNA crosslink mainly with tetramers of alpha-crystallin subunits. The formation of tetramers appears to modify alpha-crystallin interactive properties and, therefore, its induction may have functional significance. These observations suggest that alpha-crystallin may have a nuclear function which includes DNA binding.

A comparison of structural relationships among alpha-crystallin, human Hsp27, gamma-crystallins and beta B2-crystallin.

The 3D structures of alpha-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that alpha-crystallin is primarily a beta-sheet globular protein similar to alpha-crystallin (Siezen and Argos, Biochim. Biophys. Acta, 1983, 748, 56-67) containing sequence repeats in its two domains (Wistow, FEBS Lett. 1985, 181, 1-6). Positional flexibility of amino acid residues and far UV-circular dichroism spectroscopy were used to investigate structural relationships among these proteins. The utility of flexibility plots for predicting protein structure is demonstrated by the excellent correlation of these plots with the known 3D X-ray structures of beta/gamma-crystallins. Similar analyses of alpha-crystallin subunits, alpha A and alpha B, and human heat shock protein 27 show that the C-terminal domains and connecting segments of these proteins are very similar while the N-terminal domains have significant structural differences. Unlike beta/gamma-crystallins, both Hsp27 and alpha-crystallin subunits are asymmetrical with highly flexible C-terminal domains. Flexibility is considered essential for protein functional activity. Therefore, the C-terminal region may play an active role in alpha-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in alpha-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that alpha-crystallin and members of beta/gamma superfamily are structurally dissimilar.

Refinement of 3D structure of bovine lens alpha A-crystallin.

In absence of 3D structures for alpha-crystallin subunits, alpha A and alpha B, we utilized a number of experimental and molecular modeling techniques to generate working 3D models of these polypeptides (Farnsworth et al., 1994. In Molecular Modeling: From Virtual Tools to Real Problems (Eds. Kumosinski, T.F. and Liebman, M.N.) ACS Symposium Series 576, Ch. 9:123-134, 1994, ACS Books, Washington DC). The refinement of the initial bovine alpha A model was achieved using a more accurate estimation of secondary structure by new/updated methods for analyzing the far UV-CD spectra and by neural network secondary structure predictions in combination with database searches. The spectroscopic study reveals that alpha-crystallin is not an all beta-sheet protein but contains approximately 17% alpha-helices, approximately 33% beta-structures and approximately 50% turns and coils. The refinement of the alpha A structure results in an elongate, asymmetric amphipathic molecule. The hydrophobic N-terminal domain imparts the driving force for subunit aggregation while the more flexible, polar C-terminal domain imparts aggregate solubility. In our quaternary structure of the aggregate, the monomer is the minimal cooperative subunit. In bovine alpha A, the highly negatively charged C-terminal domain has three small positive areas which may participate in dimer or tetramer formation of independently expressed C-terminal domains. The electrostatic potential of positive areas is modulated and become more negative with phosphorylation and ATP binding. The refined bovine alpha A model was used to construct alpha A models for the human, chick and dogfish shark. A high degree of conservation of the three dimensional structure and the electrostatic potential was observed. Our proposed open micellar quaternary structure correlates well with experimental data accumulated over the past several decades. The structure is also predictive of the more recent data.

Effects of temperature and concentration on bovine lens alpha-crystallin secondary structure: a circular dichroism spectroscopic study.

Elucidation of the structure of alpha-crystallin, the major protein in all vertebrate lenses, is important for understanding its role in maintaining transparency and its function in other tissues under both normal and pathological conditions. Progress toward a unified consensus concerning the tertiary and quaternary structures of alpha-crystallin depends, in part, on an accurate estimation of its secondary structure. For the first time, three algorithms, SELCON, K2D and CONTIN were used to analyze far ultra-violet circular dichroism (UV-CD) spectra of bovine lens alpha-crystallin to estimate the secondary structure and to determine the effects of temperature and concentration. Under all experimental conditions tested, the analyses show that alpha-crystallin contains 14% alpha-helix, 35% beta-sheet and the remainder, random coil and turns. The results suggest that alpha-crystallin is best classified as a mixed protein. In addition, increased temperature and concentration of alpha-crystallin result in increased alpha-helices with a compensatory decrease in beta-sheets. Such structural alterations in alpha-crystallin may be functionally important during terminal differentiation of the lens fiber cells that is accompanied by increased protein concentrations and its role as a chaperone-like protein.

Autogenous vein graft thrombosis following exposure to calcium-free solutions (calcium paradox).

The morphological and functional effects of calcium-free and calcium-containing solutions on canine jugular vein intima were examined under conditions which closely resemble those techniques currently employed in peripheral vascular and aortocoronary bypass surgery. Veins that had been exposed only to calcium-containing solutions remained patent for the duration of the experimental period. Vein perfusion with a calcium-free solution, however, resulted in disruption of the jugular vein intima once calcium ions were reintroduced. Autogenous as a femoral arterial graft became thrombosed within 60 minutes. It is therefore suggested that vein grafts of autogenous origin be irrigated with calcium-containing solutions to prevent intimal damage and thrombosis.