The effect of calcium upon the reaggregation of bovine alpha crystallin. Abraham Spector and Carl Rothschild

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1 The effect of calcium upon the reaggregation of bovine alpha crystallin Abraham Spector and Carl Rothschild Calcium is capable of discriminating between low and high molecular weight species of bovine alpha crystallin. If native alpha crystallin of molecular weight 15 x 10 6 or greater is deaggregated in 7M urea and then reaggregated by removal of the dissociating agent only low molecular tceight aggregates are obtained. However, in the presence of calcium, reaggregation of the subunits to species of 50 x 10 6 or greater is observed. With native low molecular weight alpha crystallin, calcium has no effect upon the reaggregation process. Reaggregation of the isolated polypeptide chains of low molecular weight alpha crystallin is also not affected by calcium. Only an atypical A polypeptide fraction from high molecular toeight alpha crystallin has been shown to reaggregate to high molecular weight aggregates in the presence of calcium. A magnesium concentration three times greater than that of calcium is required to produce a similar effect upon the reaggregation process. Equimolar concentrations of ethylenediaminetetraacetate (EDTA), glutathione, oxidized glutathione, or alanine were shown to completely inhibit the calcium effect. Key words: alpha crystallin, calcium, aggregation, deaggregation, subunits, A polypeptide chains, lens. R,lecent studies upon bovine alpha crystallin have shown that it is composed of a number of populations of different-sized From the Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, N. Y. Supported by grants from the National Eye Institute, National Institutes of Health, Department of Health, Education and Welfare, and the John A. Hartford Foundation, Inc. Manuscript submitted for publication Nov. 7, 1972; manuscript accepted for publication Dec. 18, A preliminary report of this work was given at the Association for Research in Vision and Ophthalmology meeting in Sarasota, Fla., April, aggregates which do not appear to be in equilibrium with each other. 1 Utilizing Biogel Agarose columns, macromolecular aggregates of greater than 50 x 10 Daltons have been observed. 2 Investigation of the effect of aging upon the size distribution of the alpha crystallin macromolecules clearly indicates that the high molecular weight aggregates increase in concentration, but only in the nuclear region of the lens. 2 ' 3 It has been suggested that this increase in the size of the alpha crystallin macromolecule may possibly be involved in the development of human geriatric nuclear opacities. 2 It is of considerable importance to determine the mechanism by which the transformation of alpha crystallin from a low to

2 226 Spector and Rothschild Investigative Ophthalmology March 1973 A 280mji A rcoggregoted in PRESENCE Of 0.005M 4A reaggregated in ABSENCE Of M CoCI Fig. 1. Profile of reaggregated protein from high molecular weight alpha crystallin on Bio-gel A-50M in the presence and absence of 0.05 M C'aCl". See text for further information. high molecular weight species occurs. Since even the low molecular weight species of alpha crystallin contains more than 30 polypeptide chains in an average-sized aggregate it was thought that such large macromolecules might be slow in responding to changes in their environment. Therefore, a sensitive model system was developed in which the effect of various agents upon the reaggregation of deaggregated alpha crystallin subunits could be studied. This system allows reaggregation to the same or smaller populations than the initial preparation. 3 ' ' Previously, it was demonstrated that glucose is associated with the high molecular weight species of alpha crystallin and that the presence of glucose during the reaggregation step causes the formation of high molecular weight aggregates while in its absence only low molecular weight macromolecules are observed. 3 Furthermore, it is possible to transform the high molecular weight aggregates to low molecular weight macromolecules by removing dialyzable components from the deaggregated polypeptide chains before reaggregation. While glucose usually causes a transformation from low to high molecular weight, by the procedure described above, with alpha crystallin isolated from the nuclear region of the lens the results obtained with alpha crystallin prepared from the peripheral zone are erratic. Since glucose is present in all parts of the lens, the absence of an increasing proportion of high molecular weight alpha crystallin in the peripheral region cannot be easily explained without assuming that an alteration in the polypeptide chains themselves is required for the glucose effect. An exploration of changes in the polypeptide chains as well as the effect of other factors has therefore been initiated. It has been known for many years that the calcium level in the lens increases markedly during the development of cataract induced by galactose feeding in rats. 5 Jedziniak and co-workers G have investigated the effect of calcium upon bovine lens proteins and found that alpha crystallin is specifically induced to aggregate to larger macromolecules. In the present paper the effect of calcium upon the reaggregation of alpha crystallin is reported. It has been found that calcium will indeed affect the reaggregation of alpha crystallin, but unlike glucose, completely discriminates between the alpha crystallin subunits generated from high and low molecular weight species. Materials and methods These studies were conducted with alpha crystallin prepared from bovine lens weighing 2.2 to 2.8 Cm. The purification of the protein was accomplished by diethylaminoethyl (DEAE) cellulose chromatography and Bio-gel A-15M fractionation as described previously. 1 The elution buffer for the gel filtration was modified to con-

3 Volume 12 Number 3 Reaggregation of bovine alpha crystallin 227 A 280mjj r O Q reaggregated in PRESENCE of 0.005M CoCI 2 <lc reaggregated in ABSENCE of 0.005M CoCI Fig. 2. Profile of reaggregated protein from low molecular weight alpha crystallin on Bio-gel A-50M in presence and absence of M CaCU. See text for further information. 160 tain Tris, 0.01 M, ph 7.6, and KC1, 0.1 M. The deaggregation and reaggregation experiments were performed by modifying a previously described method.* The alpha crystallin obtained from the A or C region of a Bio-gel A-15 column was used at a concentration of 2 to 5 mg. per milliliter. The protein was dissolved in 7 M urea, 10- :t M mercaptoethanol, and 10-3 M Tris HC1, ph 7.4. The urea was passed through amberlite MB-3 shortly before use. Two milliliter portions of the protein solution were dialyzed against 1 L. of the above solution at room temperature for two hours followed by a further 2 hour dialysis at 4 C. with 1 L. of fresh solution. Except where indicated, reaggregation was performed at 4 g C, ph 7.4, by a 2 hour dialysis against 1 L. of 10~ 3 M mercaptoethanol in addition to other components such as CaCl : as described in the text. The preparation was then dialyzed overnight at 4 C. against 0.01 M Tris, ph 7.6, 0.1 M KC1; and then passed through a Bio-gel A-50M column 2.6 by 26 cm. at 4 C. with 0.1 M KC1 and 0.01 M Tris HC1, ph 7.6, as the eluting buffer. Fractions of 3 to 5 ml. were collected. Carboxymethylation was performed as previously described. 7 Results When the first elution peak of alpha crystallin obtained from Bio-gel 15-M columns, a A, is subjected to A-50M gel filtration, a significant proportion of the macromolecules can be eluted with the void volume. This a A protein therefore contains macromolecules greater than 50 x 10 Daltons. If the «A fraction is dissociated with 7 M urea and then reassociated as described in the methods section, there is a complete shift to a population of macromolecules with an elution volume similar to that obtained with low molecular weight alpha-crystallin, a<> However, if the reaggregation solution contains M CaCL then reaggregation to a population of macromolecules resembling the initial distribution occurs. These results are shown in Fig. 1. If a similar experiment is performed with the low molecular weight a G fraction obtained from Bio-gel A-15M no effect upon the size of the reaggregated macromolecules can be detected in the presence of M CaCL (Fig. 2). The populations of polypeptides obtained from a A and «c thus show a striking difference in their response to the presence of CaCL during the reaggregation process. Such experiments suggest that CaCL is acting cooperatively with altered alpha crystallin peptides to produce the high molecular weight species. If this is so, then CaCL should cause a transformation to high molecular weight components from low molecular weight aggregates that were produced from a A by utilizing the deaggregation-reaggregation procedure a second time. Experiments supporting this contention have been successfully performed. If the low molecular weight material generated from aa is again deaggregated and then reaggregated

4 228 Spector and Rothschild Investigative Ophthalmology March 1973 A 280m>u ^A reoggregoted in in presence of CoClg and equimolar EDTA 4A '^aggregated in presence of C0CI2 4A reaggregoted in presence of C0CI2 and equimolar MERCAPTOETHANOL C)A reaggregated in presence of C0CI2 and equimolar GLUTATHIONE Fig. 3. The effect of EDTA, mercaptoethanol, and glutathione all at concentration of M upon the reaggregation of the polypeptides from high molecular weight alpha crystallin in the presence of M CaCl 2. The above compounds were added to the reaggregation system at the same time as the Cad*. See text for further information. but now in the presence of CaCL, a profile similar to that of the initial population is obtained. The effect of the concentration of calcium upon the reaggregation of the a A polypeptide chains is shown in Table I. At concentrations of M CaCl 2 or less little aggregation to high molecular weight species is observed. A significant proportion of the macromolecules are in the high molecular weight population when M calcium chloride is used. Increasing the concentration of CaCL ten times to 0.05 M causes a further twofold increase in high molecular weight species. In all of these experiments the protein concentration is maintained at approximately 3 mg. per milliliter. When similar experiments are performed with MgCl 2 little effect is noted below M and only at unphysiologically high concentrations is an appreciable amount of the high molecular weight material generated. It would appear that calcium ion is approximately 3 times more effective than magnesium in causing the transformation to the high molecular weight components. Studies upon the effect of NaCl clearly indicate that even at concentrations as high as 0.05 M essentially no transformation occurs. Thus it would appear that under these conditions divalent metal ion is required to produce the larger species of alpha crystallin. Table I. Effect of salts on reaggregation of high molecular weight alpha crystallin Molarity CaCL Peak I II MgCh NaCl Peaks I and II refer to the material isolated from a Bio-gel A-50M column after reaggregation of dissociated ara in the presence of the indicated concentration of salt. Peak I represents the high molecular weight region and Peak II the low molecular weight region. It is of interest to obtain some idea of how effectively the alpha crystallin subunits compete for calcium ion in the presence of chelating agents. Experiments performed with M CaCL and equimolar quantities of EDTA result in the formation of no large molecular weight species (Fig. 3). Although the presence of M mercaptoethanol does not effect the action of calcium, increasing the mercaptoethanol concentration to M causes the elimination of any calcium effect (Fig. 3). Such an observation suggests that calcium may be binding to a SH site on the polypeptide chains. To check this possibility the a A SH groups were blocked by reaction with iodoacetic acid to form carboxymethylated a A (CMa A ) and then examined for the effect of calcium on reag-

5 Volume 12 Number 3 Reaggregation of bovine alpha crystallin 229 gregation. No change in the sensitivity of the polypeptide chains to calcium could be detected. The CM«A gave the same high molecular weight profile as had been obtained with the noncarboxymethylated material, It is also pertinent to note that the reaggregation of CMa A or a A in the absence of calcium gives essentially the same profile as low molecular weight alpha crystallin. Thus the blocking of the SH groups does not appear to significantly affect the aggregation process. Such observations suggest that the mercaptoethanol action is directed at binding calcium rather than upon the polypeptide chains. Not only is mercaptoethanol an effective agent for preventing the calcium effect but equi molar concentrations of glutathione also are effective (Fig. 3). Furthermore, studies with oxidized glutathione at concentrations of M have been found to effectively eliminate the calcium action. Such observations suggest that calcium ion can be sufficiently chelated not only by SH groups but by amino acids so that its action is blocked. Indeed, experiments performed in the presence of M alanine confirmed this viewpoint. No calcium effect is observed when alanine is present during the reaggregation of the a A polypeptide. Alpha crystallin has been shown to be composed of 4 polypeptide chains, 2 A chains and 2 B chains. 8 " 10 Recently methods have been developed to isolate these polypeptides in 7 M urea by a combination of affinity chromatography utilizing paraamino phenylmercuric acetate (APMA) and DEAE chromatography. 11 The isolated material was characterized by urea gel and SDS gel electrophoresis. When the urea was removed, the isolated subunits were capable of reaggregation. 4 As might be expected from the lack of effect of calcium on the total population of low molecular weight subunits, calcium had no effect upon the reaggregation of the individual subunits. However, when similar experiments were carried out with the polypeptide chains isolated from high molecular weight alpha crystallin a different response to calcium was observed. The reaggregation of the B chains was not affected by calcium nor were the A chains that bind to the affinity column. However, an atypical population of A chains which appear to have their SH groups masked and thus do not bind to the APMA cellulose were found to reaggregate to a higher molecular weight population in the presence of calcium (Fig. 4). These A chains appear to have normal levels of SH groups (one SH per chain) but the SH groups are apparently not available to the immobilized Hg in the APMA column. Discussion The absence of any calcium effect upon the reaggregation of dissociated low molecular weight alpha crystallin clearly indicates that other factors may be involved in the formation of the high molecular weight species in addition to calcium. These factors appear to be intrinsically related to the polypeptide chains. After exhaustive dialysis the deaggregated polypeptide chains from high molecular weight alpha crystallin can still be reaggregated with calcium to the high molecular weight components. Furthermore, examination of the purified chains of alpha crystallin indicates that none of the polypeptides arising from low molecular weight material will react with calcium to form high molecular weight aggregates. Analyses of the APMA fractions from high molecular weight a crystallin indicate that only the atypical A chains are affected by calcium. The latter observation suggests that it is the atypical A chains which are responsible for the interaction with calcium ion. Further work is now in progress to clarify the difference in the chemistry of normal and atypical A polypeptides. Palmer and Papaconstantinou 12 have presented evidence that the A, chain is not synthesized but arises by chemical alteration of the A L. macromolecule. The altered polypeptide was detected because there was a change in the charge of the polypeptide. It is conceivable that other changes in alpha crystallin previously unobserved, occur in the central region of

6 230 Spector and Rothschild Iuvestigative Ophthalmology March r A 1 POLYPEPTIDE CHAIN reaggregoted in presence of 0.005M CaCl2(~) absence of CaClg (--) Fig. 4. Reaggregation of the A' polypeptide chain isolated from high molecular weight alpha ciystallin with an APMA column. (3 x 22) 100 mg. of «..\ was added to the column in 7M urea, 5 mm. Tris, ph 7.6. The A' material was eluted with the same buffer. Reaggregation was carried out in the presence and absence of M CaCl 2 and then passed through a Bio-gel A-50 column. 160 lens and make the macromolecule susceptible to interaction with calcium. Changes in the environment of the alpha crystallin aggregates in addition to calcium may also be of primary importance. It is well established that the glutathione concentration is lower in the central region of the bovine lens ]:t and decreases sharply in the whole lens in many forms of cataract 14 and that the amino acid concentration also decreases in galactose-induced cataract. ir> The probability of calcium interaction with a ciystallin macromolecules is enhanced as the competitors for calcium ion decrease in concentration and the calcium ion increases to the level observed in cataractous lens. Jedziniak and co-workers" have recently examined the calcium-induced aggregation of nondissociated alpha crystallin. On the basis of their experiments calcium appears to act primarily on alpha crystallin with little effect upon the beta and gamma crystallins. Their data indicate that calcium is capable of causing a shift to high molecular weight species without requiring a preliminary deaggregation to subunits. Their system is considerably different from the one described in this communication. Even low molecular weight alpha crystallin is a large macromolecule consisting of approximately thirty or more interacting subunits. The sites available to calcium in such a macromolecule may differ markedly from those present in a mixture of free subunits. Furthermore, the calcium may very well influence the mechanism of aggregation of the subunits in a manner quite different from that involved in the aggregation of already formed macromolecules. It is, nevertheless, of interest to note some of the differences observed with the two systems. While an equimolar concentration of mercaptoethanol completely inhibits the calcium-induced transformation to high molecular weight when starting with dissociated polypeptide chains, with the nondissociated system, a solution containing approximately 0.01 M mercaptoethanol and calcium continues to show a pronounced Ca ++ effect. The reason for this difference is not immediately apparent. Another interesting difference between the two systems is the observed size distribution of the high molecular weight aggregates. Using Bio-rad A-50M columns capable of separating macromolecules of 50 x 10 (i or less, the calcium-induced high molecular weight aggregates generated from dissociated alpha crystallin are found in the V o region with reasonably good recoveries of the material added to the column. With nondissociated alpha crystallin the aggregates produced by calcium are so large (3 x 10 s to 2 x 10 9 ) that they fail to be eluted from a sepharose 2B column

7 Volume 12 Number 3 Reaggregation of bovine alpha crystallin 231 capable of delineating macromolecules of less than 2.5 x It is unlikely that the physical differences between the two gels can account for this difference. The effectiveness of EDTA in reversing the calcium effect also differs in the two systems. When the high molecular weight calcium-induced aggregates were treated with EDTA a reversal was obtained by Jedziniak and co-workers 6 thus suggesting that the calcium is not tightly bound. In the case of native high molecular weight components or of high molecular weight reaggregated material, a 16 hour dialysis with 0.01 M EDTA had no effect on decreasing the aggregate size. Such results indicate that calcium binding is much stronger in reaggregated material. These results also imply that if calcium is involved in vivo in the formation of such aggregates, the binding is different from that obtained by inducing aggregation of already existing aggregates in vitro. Still another dissimilarity is found in the metal ion specificity inducing higher molecular weight species. With the reaggregation system magnesium, although less effective, is capable of producing the high molecular weight species while only calcium is reported to affect the size of already existing alpha aggregates. The differences cited above suggest that different mechanisms may be involved in inducing the transformation to high molecular weight species in the two systems. We thank Dr. Jedziniak for making the manuscript, "Calcium-induced aggregation of bovine lens alpha crystallin" by Jedziniak and co-workers available before publication. REFERENCES 1. Spector, A., Li, L-K. Augusteyn, R. C, et al.: a-crystallin, the isolation and characterization of distinct macromolecular fractions, Biochem. J. 124: 337, Spector, A.: Aggregation of alpha crystallin and its possible relationship to cataract formation, Proc. Israel Nat. Acad. Sci. 8: 1577, Spector, A., Freund, T., Li, L-K., et al.: Age-dependent changes in the structure of alpha crystallin, INVEST. OPHTHALMOL. 10: 677, Li, L-K., and Spector, A.: The reaggregation of purified subunits of alpha-crystallin, Exp. Eye Res. 15: 239, Salit, P. W., Swan, K. C, and Paul, W. D.: Changes in mineral composition of rat lenses with galactose cataracts, Am. ]. Ophthalmol. 25: 1482, Jedziniak, J. A., Kinoshita, J. H., Yates, E. M., et al.: Calcium-induced aggregation of bovine lens alpha crystallins, Invest. Ophthalmol. 11: 905, Augusteyn, R. C, and Spector, A.: Fractionation of subunits and sequence studies on an isolated polypeptide, Biochem. J. 124: 345, Bjork, I.: Isoelectric focusing of a-crystallin subunits. Acta. Chem. Scand. 22:1355, Schoenmakers, J. C, Matze, J. S. S., Van Poppel, M., et al.: Isolation of nonidentical polypeptide chains of a-crystallin. Int. J. Protein Res. 1: 19, Waley, S. G.: Nomenclature for the polypeptide chains of «-crystallin, Exp. Eye Res. 8: 477, Stauffer, J., Li, L-K., and Spector, A.: Manuscript in preparation. 12. Palmer, W. C, and Papaconstantinou, J.: Aging of a-crystallin during development of the lens. Proc. Nat. Acad. Sci. 64: 404, Kinoshita, J. H., and Merola, L. O.: The distribution of glutathione and protein sulfhydryl groups in calf and cattle lenses. Am. J. Ophthalmol. 46: 36, van Heyningen, R.: In The Eye, Davson, H., editor, New York, 1962, Academic Press, Vol. I, p Kinoshita, J. H.: Cataracts in galactosemia, INVEST. OPHTHALMOL. 4: 786, 1965.