Human Alpha-Crystallin-III Isolation and Characterization of Protein from Normal Infant Lenses and Old Lens Peripheries

Human alpha-crystallin-III isolation and characterization of protein from normal infant lenses and old lens peripheries

Debdutta Roy and Abraham Spector

Alpha-crystallin isolated from the peripheries of old normal or cataractous lenses appears to be identical, consisting of eleven polypeptides, five B, and six A chains. In contrast, alpha- crystallin isolated from normal six-week-old human lenses has only three major polypeptides, corresponding to B,, A,, and At of the old human lens protein as well as small amounts of some of the other components. Comparisons with bovine alpha-crystallin are, also reported. Based on gel filtration experiments with Bio-Gel A-1.5m, two distinct populations of alpha- crystallin were found in old lens periphery, one containing species greater than 1.5 x 10" daltons and another of approximately 9 x 10s daltons. In the cataract preparations, the higher molecular weight fraction is predominant. This fraction is not present in young lenses:

Key words: lens, cataract, alpha-crystallin, aging aggregation, polypeptide chains.

. lpha-crystallin is one of the major cule of approximately 7 x 105 daltons.0'7 It structural proteins of the lens. In the bo- contains two different 2 x 104 dalton poly- vine tissue, it has been demonstrated that peptide chains designated A2 and B2, held with aging, this protein aggregates to high together by noncovalent forces. Very molecular weight (HMW) species1 greater shortly after formation, post-translational G 2 3 than 50 x 10 daltons ' and is also the reactions produce a modified A2 polypep- 10 major component of the so-called insoluble tide designated AJ and a modified B2 fraction.4- r> This transformation occurs pri- polypeptide designated B,.7 Transforma- marily in the nuclear region of the lens.1 tion to a heterogeneous macromolecular Newly synthesized (NS) alpha-crystallin population with an increase in the weight is a physically homogeneous macromole- average molecular weight (Mw) is con- comitantly observed.7 In older lenses, ad- ditional changes in the A and B polypep- From the Department of Ophthalmology, College 7 of Physicians and Surgeons, Columbia Univer- tides have been reported ' " as well as a sity, New York, N. Y. shift to HMW aggregates1 and the forma- Supported by grants from the National Eye In- tion of increasing amounts of the insoluble stitute, The National Institutes of Health, De- protein.12 Because of the possible relation- partment of Health, Education, and Welfare. ship between the development of HMW Submitted for publication Dec. 3, 1975. and insoluble protein and cataract forma- Reprint requests: Dr. Abraham Spector, Depart- tion there is considerable interest in the ment of Ophthalmology, College of Physicians and Surgeons, Columbia University, 630 W. structure of alpha-crystallin and its rela- 168th St., New York, N. Y. 10032. tionship to these macromolecular species. 394

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6 WEEKS OLD Table I. Amino acid composition of 2.0 alpha-crystallins * WHOLE LENS 1 .6 Old normal Old cataract periphery periphery Infant 1.2 CMCys 11 10 13 Asp 89 92 95 0.8 Thr 48 45 51 Ser 87 92 94 0.4 Glu 117 116 105 Pro 64 62 61 Gly 67 69 55 £ 4.0 65-70 YEARS OLD Ala 49 51 36 NORMAL LENS Val 63 61 61 PERIPHERY Met 13 15 12 3.0 280 r

ILeu 49 47 47 :Y Leu 95 92 93 Tyr 33 34 29 2.0 Phe 71 72 81 Lys 49 44 46 ORBANC His 37 40 41 Arg 75 72 77 "Results are expressed as residues per 1,000 residues. Values are not corrected for destruction during hydrolysis. 65-70 YEARS OLD CATARACT PERIPHERY Recently, human alpha-crystallin has been characterized.13-14 Based upon sedi- mentation equilibrium experiments the NS protein isolated from very young normal human lenses appears to be physically homogeneous and has a molecular weight of approximately 7.5 x 105.13 It contains three polypeptides, two presumptive A chains, and one presumptive B chain. It is not known at present whether both A 30 60 90 120 150 180 240 chains are synthesized or if a rapid post- VOLUME (ml) translational transformation causes the ap- Fig. 1. Bio-Gel A-1.5m chromatography of soluble pearance of the second A chain. proteins from six-week-old whole human lens, 65- to 70-year-old normal human lens periphery, Alpha-crystallin has also been isolated and 65- to 70-year-old cataract lens periphery. from old cataractous lens periphery.14 This The eluting buffer is 0.01 M Tris, pH 7.6, 0.1 M preparation is physically heterogeneous KC1. with species ranging from less than 1 x 10G to greater than 5 x 10° daltons. A complex Materials and methods group of eleven polypeptides was ob- Normal human eyes were obtained from the served. Although the cataractous lenses New York Eye Bank within approximately 24 used for that study contained opacities hours of death. Cataractous lenses were obtained with little pathology in the peripheral re- as described previously.14 The lenses were im- gion, there is a question of whether alpha- mediately removed, inspected for clarity and then stored at -84° C. The periphery, defined as crystallin obtained from old normal lens the outer 50 to 60 per cent of the lens, was periphery has a similar chemistry. This separated from the nucleus of the lens with a communication deals with the isolation No. 3 cork borer. Peripheries or whole lenses and characterization of alpha-crystallin were homogenized in 0.01 M Tris, pH 7.6, 0.1 M from old lenses. This protein was found KC1, and centrifuged at 27,000 g for 15 minutes. The supernatants were fractionated on a 1.5 x 150 to be identical to that obtained from cata- cm. Bio-Gel A-1.5m column utilizing 0.01 M Tris, ractous lenses. pH 7.6, 0.1M KC1. Pooled fractions were dialyzed

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Results In order to evaluate some of the age- dependent changes in alpha-crystallin in normal human lenses, the protein was isolated from six-week-old lenses and 65- to 70-year-old lens periphery. The protein was also isolated from 65- to 70-year-old cataractous lens periphery for comparative purposes. After homogenizing the lenses, the HMW and the insoluble fractions were removed and the supernatants from the three different preparations were then passed through a Bio-Gel A-1.5m column. The profiles obtained with the three supernatants are shown in Fig. 1. Based on previous work, alpha-crystallin would be expected to appear in the first peak.14 It will be demonstrated that this presump- tion is valid in these experiments. The first peak was eluted somewhat later with the six-week-old lens supernatant than in the preparations of the older lenses appearing in the region anticipated for a macromolecular population of approximately 9 x 105 daltons. In the old lens preparations, a corresponding peak was observed as well as a component which appeared in the void volume (40 ml.) indicating protein with a molecular weight greater than 1.5 x 106. This fraction appeared in considera- bly greater concentration in the preparation from old cataractous lenses than from normal tissue. Earlier work has shown that this fraction contains predominantly alpha- crystallin aggregates greater than 5 x 10° daltons.14 Investigation of the gel electrophoresis profiles in 6M urea and SDS as well as I 2 3 amino acid analyses indicates that the first Fig. 2. SDS polyacrylamide gel electrophoresis of two peaks from both the old normal and alpha-acrystallin isolated from (1) six-week-old cataractous lenses have identical composi- human lens, (2) 65- to 70-year-old normal human tion. Thus the presumptive alpha-crystallin lens periphery, and (3) 65- to 70-year-old cataract is present in different sized populations, lens periphery. the higher molecular weight species being absent in young lens. Because of the limited supply of normal human lenses, the against water and then lyophilized. Sodium two peaks isolated from old normal lens dodecyl sulfate (SDS) and urea gel electrophoresis, DEAE chromatography, amino acid periphery were pooled for further charac- analysis, and fingerprinting were performed as terization and fractionation of polypeptide described previously.14 chains.

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The amino acid composition of these preparations is shown in Table I. The old normal and cataractous proteins are essen- IT tially identical and quite similar to the six- B2 week-old material where significant differences are found in only a few amino acids B3 such as phenylalanine, alanine, glycine, and glutamic acid. B5 SDS gel electrophoresis of the material obtained from the three preparations is shown in Fig. 2. The material from the six-week-old lenses (gel No. 1) contains 4 only two bands corresponding to molecu- A5 lar weights of 22,000 and 20,000. The older preparations (normal gel No. 2, cataract gel No. 3) also contain significant amounts of a component of approximately 17,000 daltons as well as minor species of higher and lower molecular weight. The pattern obtained from the young lenses is similar BOVINE to that of calf alpha-crystallin while some of the other bands found in the older prep- Fig. 3. Alkaline urea gel electrophoresis of alpha- arations have been observed in HMW crystallin isolated from calf lens from (1) six- bovine alpha-crystallins.7 week-old human lens, from (2) 65- to 70-year-old normal human lens periphery, and from (3) 65- The alkaline urea gel patterns of the to 70-year-old cataract lens periphery. three preparations and bovine alpha-crystallin are shown in Fig. 3. The nomencla- ture used for defining the human alpha- To further establish that the normal and crystallin is based upon recent work,14 with cataractous preparations are the same, the protein isolated from old cataractous some of the polypeptides were isolated by lens periphery where eleven polypeptide DEAE chromatography in the presence of chains were observed (Fig. 3, No. 3)'. An 6 M urea. Profiles similar to those previ- identical pattern was observed with the ously reported for alpha-crystallin isolated material isolated from old normal lens pe- from cataractous lenses were obtained.14 riphery (Fig. 3, No. 2). The material iso- Because of the limited amount of normal lated from young human lenses contains human material available, only sufficient three major components corresponding to quantities of B1? B2, B3, and Ai and A2 Bu Al5 and Aa and minor bands corre- could be obtained from lenses of older sponding to B2, B3j Bj, and A3 (Fig. 3, No. individuals for further investigation. Their 1). No evidence of other polypeptides was amino acid compositions correspond to observed. their counterparts isolated from cataract- 14 Electrophoresis in urea of bovine alpha- ous lenses. Two dimensional fractionation crystallin reveals a pattern that is signifi- of the tryptic digests of these polypeptides cantly different from that of the human, indicate no differences between normal with bovine B2 corresponding to human and cataractous material. Fig. 4 illustrates B13 bovine Bt to human B3, and bovine A2 the patterns obtained with human Bi and to human A,. Bovine Ai does not appear B3 and Ax and A2. Bt and B3 each contain to have a mobility corresponding to any one peptide not found in the other. B2 was human A chain migrating between human found to contain two peptides not found A2 and A3. in either Bx or B3. At and A, are also al-

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HUMAN Bj an Unfortunately, considerable material is re- quired for such investigation and only a limited number of normal lenses was avail- O able for the present investigation. Never- 0 0 theless, five polypeptides Bi, B2, B3, Al5 and A2 were isolated in sufficient quantity from normal lens periphery for characterization and comparative studies with the 0 Q * B1 protein from cataractous lenses (Insuffi- 0/~B3 f 0 cient material was available from six-week- o °0 old lenses for such study). Besides the al- most identical amino acid compositions and SDS and urea gel patterns of all the alpha-crystallins, the polypeptides isolated -A/ HUMAN A f and A2 from normal tissue were identical to their 0 cataractous lens counterpart. Thus it can be concluded that alpha-crystallin isolated o from old normal and cataractous lens pe- 0 ripheries have identical polypeptide compositions. 0 Correlation of the observations upon hu- >° 0 \ man NS, young and old alpha-crystallin 0 suggests post-translational reactions simi- 0 0 lar to those found in the bovine lens. NS o o °0° protein contains only three polypeptides 0 while in six-week-old lenses, seven poly- 4- peptides are observed and in older lenses, eleven polypeptides. Observations upon ELECTROPHORESIS both cataractous and normal lens alpha- # Yellow after Ninhydrin Staining crystallin indicate that the A and B chains • Origin are each a group of closely related poly- Fig. 4. Fingerprinting of tryptic digests of human peptides with only minor changes in their Bi, B3, Ai, and A2. The designation -Bi indicates that particular peptide is absent in Bi. chemistries. It appears that alpha-crystallin in human lenses undergoes a similar age-dependent most identical with A, containing two pep- size transformation to that found in the tides not found in A2 and A2, one peptide bovine lens increasing from approximately not found in A,. 7.5 x 105 daltons to more than 5 x 10G daltons.11 The HMW species (greater than Discussion 50 x 10° daltons) found primarily in the Previous work with alpha-crystallin iso- nuclear region also appears to contain lated from the peripheries of cataractous alpha-crystallin.15 However, in contrast to lenses has unequivocally demonstrated that the bovine lens, the human HMW fraction of the eleven polypeptides found in this is composed of other proteins as well as macromolecule, four are B chains and six degraded polypeptides with a molecular are A chains.14 One polypeptide designated weight of approximately 11,000.15 The B4 requires further investigation to confirm so-called insoluble fraction appears to be its assignment. The identification of these very similar to the HMW material suggest- polypeptides was based upon the isola- ing that the HMW species may be pre- tion and characterization of each chain. cursors to the insoluble protein.15 Observa-

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tions on the proteins of the nuclear region peptide chains with aging, INVEST. OPHTHAL- will be reported in later communications. MOL. 13: 135, 1974. 8. Schoenmakers, J. G. G., and Bloemendal, H.: Subunits of alpha-crystallin from adults The competent technical assistance of Ms. and embryonic cattle lens, Nature 220: 790, Gloria Jenkins and Ms. Kathy Huang are grate- 1968. fully acknowledged. 9. Palmer, W. G., and Papaconstantinou, J.: REFERENCES Aging of alpha-crystallin during development of the lens, Proc. Nat. Acad. Sci. 64: 404, 1. Spector, A., Freund, T., Li, L-K., et al.: Age- 1969. dependent changes in the structure of alpha- 10. Delcour, J., and Papaconstantinou, J.: Bio- crystallin, INVEST. OPHTHALMOL. 10: 677, chemistry of bovine lens proteins, IV. Synthe- 1971. sis and aggregation of alpha-crystallin sub- 2. Spector, A.: Aggregation of alpha-crystallin units in differentiating lens cells, J. Biol. and its possible relationship to cataract for- Chem. 247: 3289, 1972. mation, Isr. J. Med. Sci. 8: 1577, 1972. 11. van Kleef, F. S. M., Nijzink-Maas, M. J. 3. Jedziniak, J. A., Kinoshita, J. H., Yates, E. C. M., and Hoenders, H. J.: Intercellular M., et al.: Calcium-induced aggregation of degradation of alpha-crystallin, Eur. J. Bio- bovine lens alpha-crystallin, INVEST. OPH- chem. 48: 563, 1974. THALMOL. 11: 905, 1972. 12. Krause, A. C: The Biochemistry of the 4. Clark, R., Zigman, S., and Lerman, S.: Eye. Baltimore, 1934, John Hopkins Press. Studies on the structural proteins of the 13. Spector, A., Stauffer, J., Roy, D., et al.: human lens, Exp. Eye Res. 8: 72, 1969. Human alpha crystallin. I. The isolation and 5. Manski, W., Behrens, M., and Martinez, C: characterization of newly synthesized alpha- Immunochemical studies on albuminoid, crystallin, INVEST. OPHTHALMOL. In press. INVEST. OPHTHALMOL. 7: 164, 1968. 14. Roy, D., and Spector, A.: Human alpha 6. Spector, A., Wandel, T., and Li, L-K.: The crystallin. II. Characterization of the protein purification and characterization of the highly isolated from the periphery of cataractous labeled protein fraction from the calf lens, lenses, Biochemistry 15: 1180, 1976. INVEST. OPHTHALMOL. 7: 179, 1968. 15. Roy, D., and Spector, A.: High molecular 7. Stauffer, J., Rothschild, C, Wandel, T., et weight protein from human lenses, Exp. Eye al.: Transformation of alpha-crystallin poly- Res., In press.

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