Physicochemical characterization of high-molecular-weight alpha-crystallin subpopulations from the calf lens nucleus

Calf lens nuclear alpha-crystallin was separated into five molecular weight subpopulations by exclusion chromatography on Bio-Gel A-5m. These subpopulations were compared by amino acid analysis, ultraviolet absorption analysis, fluorescence, far- and near-ultraviolet circular dichroism, isoelectric focusing, SDS-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Although only minor differences were detectable in most physicochemical properties, progressive changes were found in the near-ultraviolet circular dichroism spectra and in pellet hardness after centrifugation. Minute amounts of beta-crystallin polypeptides and a 43 kDa component were present in all five subpopulations. In addition, the highest molecular weight aggregates contain some gamma-crystallin polypeptides. A slow re-equilibration of separated subpopulations towards the initial distribution was observed by rechromatography.     

Trehalose effects on alpha-crystallin aggregates

alpha-Crystallin in its native state is a large, heterogeneous, low-molecular weight (LMW) aggregate that under certain conditions may progressively became part of insoluble high-molecular weight (HMW) systems. These systems are supposed to play a relevant role in eye lens opacification and vision impairment. In this paper, we report the effects of trehalose on alpha-crystallin aggregates. The role of trehalose in alpha-crystallin stress tolerance, chaperone activity and thermal stability is studied. The results show that trehalose stabilizes the alpha-crystallin native structure, inhibits alpha-crystallin aggregation, and disaggregates preformed LMW systems not affecting its chaperone activity.     

Self-similarity properties of alpha-crystallin supramolecular aggregates.

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin.     

The dephosphorylation of lens alpha-crystallin A chain

The present communication reports the presence of a phosphoprotein phosphatase activity in bovine lens preparations which dephosphorylates alpha Ap, the phosphorylated form of alpha A, one of the alpha-crystallin polypeptides, in a Ca2+/calmodulin dependent manner. The activity was found in soluble preparations from epithelial cells but it could not be detected in similar preparations from fiber cells. A 60,000 Mr calmodulin binding polypeptide and a 15,000 Mr polypeptide found in the epithelial cell preparations comigrated in SDS-PAGE with the A and B subunits of bovine brain calcineurin (phosphoprotein phosphatase 2B) respectively. The 15,000 Mr was specifically recognized by an anti-bovine brain calcineurin antiserum. Bovine brain calcineurin was as effective in dephosphorylating alpha Ap as the lens preparations. Thus, it is likely that the activity present in the lens is related to this enzyme. The results indicate that the lens specific polypeptide alpha A may be subject to metabolic control through phosphorylation and dephosphorylation pathways regulated by cAMP and calcium respectively. Changes in the activities of these pathways appear to occur during differentiation of the lens epithelial cell and may be related to gene regulation during the differentiation process.     

Age-related changes of alpha-crystallin aggregate in human lens

Summary. Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.     

Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.     

Calf lens alpha-crystallin quaternary structure. A three-layer tetrahedral model

Calf lens alpha-crystallins are polydisperse globular particles made of a large number of two types of subunits, A and B, both of molecular weight congruent to 20,000. alpha-Crystallin populations consisting on average of 40 subunits or more were subjected to various changes in pH, ionic strength, temperature and urea concentration. Modifications in quaternary structure induced by variation of these physicochemical parameters were followed by means of X-ray and quasi-elastic light-scattering and quantified in terms of weight average molecular weight (M), radius of gyration (Rg) and hydrodynamic radius (Rh). High-pressure liquid chromatography was used as a control of polydispersity. Increasing the pH, decreasing the ionic strength and incubating at temperatures from 20 degrees C to 45 degrees C all resulted in the formation of particles of decreasing M, Rg and Rh values. These effects are cumulative. All monomodal alpha-crystallin populations encountered in this study, which covers a wide range of sizes and molecular weights, may be accounted for by a three-layer model with partial filling up of the layers. Applying basic principles of symmetry and postulating specific contacts between protein subunits to construct this three-layer model leads to tetrahedral symmetry, with 12, 24 and 24 sites in the first, second and third layers, respectively. Variations in probabilities of site occupancy account for both the observed quaternary structure modifications and the intrinsic polydispersity of alpha-crystallins     

Biophysical characterization of alpha-crystallin aggregates: validation of the micelle hypothesis.

The size of alpha-crystallin aggregates, as well as the structural organization of each particle's subunits, is currently unknown, although a number of different laboratories have suggested both structures and average molecular weights (Thomson, J.A. and Augusteyn, R.C. (1984) Proc. Int. Soc. Eye Res. 3, 152). One hypothesis, compatible with literature reports and consistent with what is known of subunit primary structure and physiological function, is that alpha-crystallin exists in vivo as a naturally occurring protein micelle (Sen, A.C. and Chakrabarti, B. (1991) Biophysical J. 59, 108a.) To test this hypothesis, experiments were performed on this protein to determine its behavior under increased hydrostatic pressure and the effect of its concentration on aqueous surface tension. With increasing hydrostatic pressure, the turbidity of an alpha-crystallin solution increases exponentially to a plateau at about 6000-8000 psi; upon release of pressure, the samples slowly return to their original turbidity level. Other naturally aggregating proteins, such as skeletal muscle myosin, demonstrate a decrease in turbidity under the same conditions. The surface tension of alpha-crystallin in aqueous solution decreases to a plateau with increasing protein subunit concentration, with an inflection point over the range 0.18-0.25 mM; cholate and other amphiphiles exhibit similar behavior. In contrast, plots of surface tension over the equivalent concentration range for other protein aggregates in the same buffer more closely approximate the types of curve obtained with short chain aliphatic acids. These results indicate that alpha-crystallin behaves like the protein version of a micelle.     

Complete protection by alpha-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

Sorbitol dehydrogenase (l-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1. 14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens alpha-crystallin. An alpha-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 degrees C. Moreover, an alpha-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degrees C for at least 40 min. The protection by alpha-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the alpha-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of alpha-crystallin against the inactivation of SDH induced at 55 degrees C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of alpha-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with alpha-crystallin. Two different adducts between alpha-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of alpha-crystallin. Even though it had a reduced efficiency with respect to alpha-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of alpha-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of alpha-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.     

Interaction of alpha-crystallin with lens plasma membranes. Affinity for MP26

The binding of the major water-soluble lens protein alpha-crystallin to the lens plasma membrane has been investigated by reassociating purified alpha-crystallin with alpha-crystallin-depleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. alpha-Crystallin reassociates at high affinity (Kd = 13 X 10(-8)M) with alkali-washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles or erythrocyte membranes. Binding to lens plasma membranes is dependent on salt, temperature and pH and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of alpha-crystallin binding suggests the involvement of this transmembrane protein. Binding ist not influenced by pretreatment of membranes with proteases, suggesting that the 4-kDa cytoplasmic fragment of MP26 is not necessary for alpha-crystallin binding. Labeling experiments using (trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine as a probe for intrinsic membrane proteins further showed that alpha-crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized alpha-crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of alpha-crystallin aggregates does not take place in a membrane-bound mode.     

Association of partially folded lens betaB2-crystallins with the alpha-crystallin molecular chaperone

Age-related cataract is a result of crystallins, the predominant lens proteins, forming light-scattering aggregates. In the low protein turnover environment of the eye lens, the crystallins are susceptible to modifications that can reduce stability, increasing the probability of unfolding and aggregation events occurring. It is hypothesized that the alpha-crystallin molecular chaperone system recognizes and binds these proteins before they can form the light-scattering centres that result in cataract, thus maintaining the long-term transparency of the lens. In the present study, we investigated the unfolding and aggregation of (wild-type) human and calf betaB2-crystallins and the formation of a complex between alpha-crystallin and betaB2-crystallins under destabilizing conditions. Human and calf betaB2-crystallin unfold through a structurally similar pathway, but the increased stability of the C-terminal domain of human betaB2-crystallin relative to calf betaB2-crystallin results in the increased population of a partially folded intermediate during unfolding. This intermediate is aggregation-prone and prevents constructive refolding of human betaB2-crystallin, while calf betaB2-crystallin can refold with high efficiency. alpha-Crystallin can effectively chaperone both human and calf betaB2-crystallins from thermal aggregation, although chaperone-bound betaB2-crystallins are unable to refold once returned to native conditions. Ordered secondary structure is seen to increase in alpha-crystallin with elevated temperatures up to 60 degrees C; structure is rapidly lost at temperatures of 70 degrees C and above. Our experimental results combined with previously reported observations of alpha-crystallin quaternary structure have led us to propose a structural model of how activated alpha-crystallin chaperones unfolded betaB2-crystallin.     

Conformational changes induced in bovine lens alpha-crystallin by carbamylation. Relevance to cataract.

Carbamylation of lens proteins may contribute to cataractogenesis in certain medical conditions where blood urea is elevated for prolonged periods. This paper reports on the effects of carbamylation on the physicochemical properties of one of the major lens structural proteins, alpha-crystallin. In particular it is shown that carbamylation alters the tertiary and secondary structure of the protein, leading to an increased reactivity of protein thiols, resulting in interchain disulphide bonding.