Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis.

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Theoretical and experimental basis for the inhibition of cataract.

Aggregation of the lens proteins to form high molecular weight clusters is a major contributing factor in age-onset nuclear cataract [Benedek, G. B. (1971) Theory of transparency of the eye. Appl. Optics, 10, 459-473]. This aggregation occurs continually throughout life and contributes to an exponential increase, as a function of age, in the intensity of the light backscattered out of the lens. The time constant deltaT for this exponential increase in human populations is a valuable index, helpful for conducting clinical trials. In-vitro studies have identified reagents capable of inhibiting high molecular weight aggregate formation, as well as the non-covalent interprotein interactions responsible for phase separation. These reagents are also found experimentally to be effective cataract inhibitors in animal model systems in vivo. We believe that the stage is now set for human clinical trials of putative cataract inhibitors. We present rough quantitative estimates of the trial parameters needed to assure an unambiguous determination of efficacy in a trial population. Such a trial simply requires a measurement of the time constant deltaT in the treated population relative to the untreated population. A successful outcome of the trial is indicated if deltaT increases by 20% over that found for the untreated population. Our estimates suggest efficacy could be determined in a two year trial involving about 300 subjects in the treated group.

Monitoring protein assembly using quasielastic light scattering spectroscopy.

This article discussed the principles and practice of QLS with respect to protein assembly reactions. Particles undergoing Brownian motion in solution produce fluctuations in scattered light intensity. We have described how the temporal correlation function of these fluctuations can be measured and how mathematical analysis of the correlation function provides information about the distribution of diffusion coefficients of the particles. We have explained that deconvolution of the correlation function is an "ill-posed" problem and therefore that careful attention must be paid to the assumptions incorporated into data analysis procedures. We have shown how the Stokes-Einstein relationship can be used to convert distributions of diffusion coefficients into distributions of particle size. In the case of fibrillar polymers, this process allows direct determination of fibril length, enabling nucleation and elongation rates to be calculated. Finally, we have used examples from studies of A beta fibrillogenesis to illustrate the power these quantitative capabilities provide for understanding the molecular mechanisms of the fibrillogenesis reaction and for guiding the development of fibrillogenesis inhibitors.

The effects of glycols, aldehydes, and acrylamide on phase separation and opacification in the calf lens.

Glycols, aldehydes, and acrylamide inhibit the in vitro formation of cold cataracts in calf lenses. The inhibition is reversed when the glycols or the acrylamide are diffused out of the lenses. The inhibition by aldehydes or polacrylamide is irreversible, suggesting that the crosslinking reagents may permanently modify lens structure to prevent the development of lens opacities due to a phase transition.

Aggregation in aqueous solutions of bovine lens gamma-crystallins: special role of gamma(s).

PURPOSE: To study the aggregation of bovine gamma-crystallins in aqueous solutions and the effect of gamma(s)-crystallin on the aggregation of other gamma-crystallins. METHODS: Aggregation in aqueous solutions of gamma(s)-, gammaII-, and gammaIVa-crystallin was monitored by quasielastic light scattering. Aggregation in mixtures of gamma(s)- and gammaII-crystallin, and gamma(s)- and gammaIVa-crystallin, was also monitored by light scattering. RESULTS: All of the gamma-crystallins studied formed large aggregates (or "megamers") in aqueous solutions. However, each protein differed in the relative rates of formation of megamers. Gamma(s)-crystallin formed megamers much more slowly than gammaII- and gammaIVa-crystallin. In solutions containing mixtures of gammaII and gamma(s), and gammaIVa and gamma(s), gamma(s)-crystallin significantly suppressed the aggregation of gammaII- and gammaIVa-crystallin. Megamerization seemed to be associated with thiol oxidation in these proteins. CONCLUSIONS: Gamma-crystallins undergo aggregation in which a small fraction of the proteins form a few large aggregates, whereas the larger proportion of the proteins remain monomeric. This suggests that the megamerization is preceded by an initial modification of the protein. The modification is associated with the thiol groups, and only such modified protein species participate in megamerization. The presence of gamma(s) significantly slows the megamerization of gammaII- and gammaIVa-crystallin. This fact, together with the previous finding that gamma(s) strongly reduces the phase separation temperatures of the gamma-crystallins, suggests that gamma(s)-crystallin plays an important role in suppressing the formation of light-scattering elements and helps maintain lens transparency.

Quantitative verification of the existence of high molecular weight protein aggregates in the intact normal human lens by light-scattering spectroscopy.

The method of quasi-elastic light-scattering spectroscopy was used to establish quantitatively the concentration of high mmolecular weight (HMW) aggregates present in the normal human intact lens as a function of age. The concentration of HMW proteins increases monotonically with age. HMW proteins are absent in the infant lens, but represent 3% of the total soluble lens protein at age 60 years. The percent concentration of HMW proteins measured in intact lenses of various ages by quasi-elastic light scattering is in striking agreement with values determined biochemically.

Phase separation of X-irradiated lenses of rabbit.

The phase separation temperature (Tcat) was studied as a function of time (age) after the administration of a single dose of radiation (2000 rad), which induces cataract in the rabbit lens. In the normal unirradiated lens, Tcat decreases linearly with age at a rate (DTcat/dt) approximately 2.2 degrees/week. In the irradiated lens, Tcat initially decreases with age much less than the normal lens, then rises sharply with age at the time of the appearance of opacity in the living rabbit eye. We suggest that the phase separation temperature may serve as a sensitive and early indicator of cataractogenic processes in the lens.

Oligomerization and phase separation in globular protein solutions.

We have chemically crosslinked a globular protein, gamma IIIb-crystallin, to produce a system of well-defined oligomers: monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid-liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for gamma II-crystallin, for which oxidation-induced oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization.

The effect of naturally occurring cellular constituents on phase separation and opacification in calf lens nuclear homogenates.

We have measured the change in the phase separation temperature, Tc, in calf nuclear homogenate produced by a variety of naturally occurring cellular constituents. For all of the compounds studied, increasing concentrations of test compound were found to lower Tc. Phosphorylated nucleotides had the greatest effect, lowering Tc by 165-305 degrees/mole of test compound. Reduced and oxidized glutathione reduced Tc by 69 and 100 degrees C/mole, respectively. Smaller effects were observed for amino and ascorbic acids and sugars. The pH of the homogenate was also found to affect Tc. In the normal lens, the concentration of each of these constituents is small. Therefore, for each individual component, the cataract associated decrease in concentration is insufficient to produce the large increase in Tc that has been observed during cataractogenesis in some model systems. However, the superposition of the effects of changes in many cellular constituents (including pH and hydration) could possible produce a change in Tc more consistent with the experimentally observed one. In support of this, we have found that combinations of test compound have an additive effect on the Tc of the nuclear homogenate.

Controlled modulation of the phase separation and opacification temperature of purified bovine gamma IV-crystallin.

In the bovine lens the gamma IV-crystallin fraction is a principal determinant of the phase separation and opacification temperature, Tc (Siezen et al, Proc. Natl. Acad. Sci. USA 82, 1985, 1701). We have now measured the effect on Tc of purified gamma IV-crystallin solutions produced by a variety of reagents which affect protein-protein, protein-water and water-water interactions. Ionic strengths less than physiological increase Tc dramatically, while higher ionic strength has very little effect. Calcium ion concentrations up to 8 mM produce no change in Tc. Glycerol and acrylamide both depress Tc linearly with reagent concentrations; Tc depression of gamma IV-crystallin by these compounds is quantitatively the same as for whole lens. Sulfhydryl reducing agents such as glutathione and dithiothreitol lower Tc, while hydrogen peroxide increases Tc. Changes in opacification temperature of gamma IV-crystallin produced by oxidizing and reducing agents are time-dependent and highly non-linear with reagent concentration. Our results clearly show that bovine gamma IV-crystallin is an important target protein for various reagents which are known perturbants of the opacification temperature of whole lens. The relevance of these findings to human diabetic and senile cataract formation is discussed.

In vivo measurement of the aging rabbit lens using quasielastic light scattering.

We have been able to analyze the autocorrelation function of the light scattered from the rabbit lens in vivo in terms of a two component, exponential fit. We have measured the intensity and the decay rate associated with each of these two components as a function of age and position in the lens. As far as is possible we have interpreted these results in terms of molecular changes in the state of association of the protein constituents in the aging rabbit lens. These studies suggest that the method of quasielastic light scattering spectroscopy can provide a useful probe of the protein modifications that occur in both normal and cataractous human lenses.

Pantethine inhibits the formation of high-Tc protein aggregates in gamma B crystallin solutions.

PURPOSE: Solutions of the bovine lens protein gamma B (or gamma II) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species, gamma IIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (approximately equal to 32% of total protein by weight) and loosely associated dimers (approximately equal to 66%). gamma IIH has a phase separation temperature (Tph), at least 40 degrees C higher than that of native gamma II crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation of gamma IIH. METHODS: gamma II crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The Tph of the solutions was monitored as a function of incubation time. Corresponding to each Tph measurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount of gamma IIH formed. RESULTS: Incubation of gamma II crystallin with increasing amounts of pantethine lowers Tph and suppresses the formation of gamma IIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the Tph of gamma II crystallin is lowered from 8 degrees C in the native protein, to 2 degrees C, -3 degrees C respectively, at a protein concentration of approximately equal to 200 mg/ml. The amount of gamma IIH accumulated decreases from approximately 25% in the native protein to 10%, 1% and 0% respectively in these pantethine-treated protein solutions. For complete suppression of the rise in Tph and inhibition of gamma IIH formation, a 2:1 mole ratio of pantethine to protein is required. CONCLUSIONS: We suggest that pantethine reacts with two cysteine residues of gamma IIH crystallin by forming a mixed disulfide, and effectively suppress protein aggregation and lowers Tph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.

Quantitative detection of the molecular changes associated with early cataractogenesis in the living human lens using quasielastic light scattering.

We have used quasielastic light scattering to detect and quantitatively characterize the molecular changes associated with the early stages of cataractogenesis in the living human lens. The autocorrelation function of the fluctuations in the light scattered by the lens shows the presence of two major species responsible for the scattering. The first, fast diffusing species (f), has a diffusivity of approximately 3 x 10(-7) cm2/sec and corresponds to the alpha crystallin proteins. The second, slow diffusing species (s), has a diffusivity of approximately 10(-9) cm2/sec and corresponds to the diffusivity of a large aggregate. The intensity of light If and Is scattered into the collection optics by each of these species was also measured. We studied a group of 49 individuals ranging in age from 21 years to 82 years. In this group 40 presented with preoperative cataract development. In this patient population we found that regardless of age, or position in the lens that a plot of Itot = If+Is versus Is could be well fitted by a straight line with a slope less than unity and a positive intercept Ifo. It has been possible to explain this finding using a two state model for the molecular changes associated with early cataractogenesis. In this model the proteins in the slow diffusing species are aggregates each containing a definite number of rapidly diffusing proteins. The early development of cataract is represented by the redistribution of protein between the unaggregated form (f) and the aggregated form (s). The prediction for the relationship between Itot and Is based on this two state model is in very good agreement with our experimental data. Indeed the measured position of the point (Itot, Is) along this line provides a sensitive, and quantitative measure of the degree of cataract development at any selected location in the lens.

Quasielastic light scattering study of the living human lens as a function of age.

PURPOSE: To determine contributions of molecular scattering elements to the increase with age in the light scattered from the human ocular lens in vivo. METHODS: We used quasielastic light scattering to measure autocorrelation functions of the intensity of light scattered in vivo from three locations (anterior, nuclear and posterior) along the optic axis in ocular lenses of 225 subjects, ranging from 17 to 63 years of age. We deduced probability distributions of key parameters (Is, If, Ii, IT), which describe contributions of slowly diffusing (Is), rapidly diffusing (If) and relatively immobile (Ii) scattering elements to the total light intensity (IT) scattered into the collection optics. We deduced characteristic time tau s and tau f that describe the Brownian motion of scattering elements. RESULTS: Probability distributions for each age decile show clearly defined shifts in key parameters with age. IT at the nucleus increases by a factor of three from age 20 to 60 years. This increase is produced principally by an approximate five-fold increase is Is. IT and Is and can be detected with an accuracy of approximately +/- 10%. We estimate threshold values for IT, which mark the boundary beyond which clinical cataract becomes manifest. This boundary represents 6 to 8 times the light scattering efficiency expected from the newborn lens. CONCLUSIONS: This methodology permits a sensitive, quantitative, clinically useful representation of the pre-cataractous molecular changes associated with aging in the living human lens.