Proc. Nati. Acad. Sci. USAVol. 81, pp. 2878-2881, May 1984Medical Sciences

Nondisulfide polymerization of v- and f3-crystallinsin the human lens

(43,000-dalton polypeptide/photochemistry/cataract)

DEBDUTTA Roy, JAMES DILLON, EIKO WADA, WILLIAM CHANEY, AND ABRAHAM SPECTOR*

Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University,New York, NY 10032

Communicated by Zacharias Dische, January 13, 1984

ABSTRACT The water-soluble 43,000-dalton fraction(WS43) of the human lens has been shown to be heteroge-neous. It appears to contain, in addition to actin, componentsrelated to the crystallins. Immunoblot reactions indicate thatthis polypeptide fraction is composed of dimers containing (i-and y-crystallin components. It has been estimated that 10-30% of this fraction arises by dimerization of y-crystallin. Apossible route for the formation of the 43,000-dalton fraction issuggested by the observation that photolysis of y-crystallinwith light >295 nm leads to polymer formation, including the43,000-dalton fraction. The polymerization products reactwith anti-WS43. The results suggest that photochemical reac-tions may lead to the accumulation of polymers of some of thecrystallins with aging of the human lens. Similar covalentlylinked polypeptides have previously been shown to be presentin the high molecular weight aggregates associated with cata-ract formation.

It is now well established that oxidative insult is fundamen-tally involved in the cataractous process (1-4). This has beendemonstrated by the extensive oxidation of methionine andcysteine that is found during the development of cataract (4).There is also the formation of high molecular weight (HMW)disulfide-linked aggregates that are believed to contribute tothe loss of transparency (3). It has been suggested that theaggregation process is initiated at or near the plasma mem-brane and that a 43,000-dalton extrinsic membrane polypep-tide fraction is one of the polypeptides involved in the forma-tion of these aggregates (5).

Previous work has shown that the major component con-taining an atypical protein fluorescence (excitation, 340; 360nm: emission, 410; 440 nm) was the 43,000-dalton fraction(6). This fluorescence was found to increase with aging andcataract formation and to be present in both soluble and in-soluble protein fractions in older lenses (6). Examination ofthe abundance and distribution of the 43,000-dalton fractionindicated that in very young human lenses, it was only pres-ent in the water-insoluble fraction, appearing in the water-soluble fraction at approximately the age of 5 yr and increas-ing in abundance up to approximately the age of 30 yr whereit stabilizes at -8%, a concentration similar to that found inthe water-insoluble fraction (7). Studies on the water-insolu-ble 43,000-dalton fraction indicate that this protein fractionhas a similar amino acid composition to the water-soluble43,000-dalton fraction (WS43) and that these two proteinfractions are immunochemically similar (8).What is the origin of this fraction? Lens culture experi-

ments with labeled amino acids demonstrated incorporationinto the 43,000-dalton fraction, suggesting polypeptide syn-thesis (7). The only known polypeptide of this size present inthe lens is actin (9, 10). However, the amino acid composi-

tion of the 43,000-dalton protein fraction is markedly differ-ent from that reported for actin (8). Furthermore, with bothLaemmli (11) and Weber-Osborn (12) PAGE systems, broadCoomassie blue-stained bands are observed with the 43,000-dalton fraction from both water-soluble and -insoluble mate-rial, whereas actin isolated from chicken gizzard produces asingle sharp band. Such observations suggest that this mate-rial is a complex mixture containing directly synthesizedpolypeptides, such as actin, as well as polypeptides arisingfrom post-translational modification (6).

In this report, it is shown that the WS43 fraction containsf3- and y-crystallin components and that polymerization ofcrystallin monomers can be produced by photochemical re-actions.

MATERIALS AND METHODSThe WS43 polypeptide fraction was prepared from the wa-ter-soluble protein isolated from batches of 50-60 nonclassi-fied human cataractous lenses as described (8). a-, ,3-, and y-crystallins were isolated from bovine outer cortex by Sepha-dex G-200 chromatography (13). Chicken gizzard actin wasprepared by the method of Lazarides (14). Antibody to theWS43 fraction was raised in rabbits by subcutaneous injec-tion in Freund's complete adjuvant (8). a- and y-crystallinantibodies were produced in rabbits as described (8). Thetwo-dimensional gel electrophoresis was done according toHaley et al. (15). For immunological analysis of the polypep-tides, the blot technique as described by Reiser and Wardale(16) was used. NaDodSO4/PAGE was run with 3% acrylam-ide/0.2% Bis in 0.054 M Tris HCI/0.025 M H2SO4, pH 6.12,buffer in the stacking gel and 12% acrylamide/0.19% Bis in0.42 M Tris HCl (pH 8.8) in the separating gel. The upperbuffer reservoir contained 0.04 M TrisHCl/0.04 M boricacid/0.1% NaDodSO4, pH 8.5, and the lower buffer reser-voir contained 0.21 M Tris HCl (pH 8.8). For quantitive anal-ysis of antigen in the WS43 preparation, two RIA methodswere used. In the first method, known amounts of calf y-crystallin and WS43 were run on NaDodSO4/PAGE gels andthe amount of reaction was determined by measurements ofthe exposure of the x-ray film with a densitometer after im-munoblot. In the second RIA method (17), calf 'y-crystallin(50 ,ul at 1 mg/ml) in 0.01 M phosphate/0.9% NaCl, pH 7.2(Pi/NaCl), was allowed to bind to wells in polyvinyl chloridemicrotiter plates (Dynatech, Alexandria, VA) for 2 hr at25°C. The plates were then washed 3 times with 0.5% bovineserum albumin in P1/NaCl (albumin/PjiNaCl) to prevent fur-ther nonspecific binding. A 1:200 dilution of a rabbit anti-y-crystallin antiserum in albumin/PINaCl was added, contain-ing various concentrations of the protein being assayed. Af-ter a 2-hr incubation at 25°C, the plates were washed withalbumin/P1/NaCl, and were then further incubated with 1251_

Abbreviations: WS43, water-soluble 43,000-dalton fraction; HMW,high molecular weight; Pi/NaCI, phosphate-buffered saline.*To whom requests for reprints should be addressed.

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Proc. Natl. Acad Sci. USA 81 (1984) 2879

labeled protein A (25,000 cpm per well) for 1 hr. Unbound125I-labeled protein A was removed with four albumin/PfNa-Cl washes, and the wells were cut apart for determination ofradioactivity. Estimation of the amount of y-crystallin in theWS43 polypeptide fraction was accomplished by comparingthe inhibition of the binding of the anti-y-crystallin to the y-crystallin immobilized on the microtiter plate wells by dilu-tions of the WS43 fraction and of y-crystallin (17). Photolysisof calf y-crystallin was carried out for 6 hr under conditionspublished previously (18). The isolated crystallin fractions(2.0 mg/ml) were dissolved in 0.1 M phosphate (pH 7.4) andphotolyzed with a 450 W medium pressure mercury lamp;the light was filtered through a 5% solution of copper sulfate(1% transmission at 297 nm). All protein determinationswere done by amino acid analysis.

RESULTS AND DISCUSSIONIt is apparent from previous work that the 43,000-dalton frac-tion is heterogeneous (6, 8). To more clearly demonstratethis heterogeneity, the 43,000-dalton fraction isolated fromwater-soluble lens protein was subjected to fractionation us-ing isoelectric focusing and NaDodSO4/PAGE. The resultsare shown in Fig. 1. To identify actin in this electrophoresispattern, actin isolated from chicken gizzard was run in a par-allel gel as shown in Fig. 1B. Superimposition of such gels aswell as coelectrophoresis of actin with WS43 indicates thatthe region delineated by the arrow in Fig. LA corresponds toactin. Two-dimensional gel electrophoresis of labeled poly-peptides isolated from the water-soluble and water-insolublefractions of human lenses after organ culture in the presenceof [355]methionine, demonstrated that the major 43,000-dal-ton polypeptide synthesized in these lenses is actin (unpub-lished observations). Radioactivity was not detected in any

kDa92.5

66.2

43

31

21.5A

r-IEF

wr

:q

92.566.2

43

31

21.5

B

FIG. 1. Two-dimensional gel electrophoresis of WS43 (A) andchicken gizzard actin (B). The arrow indicates the position of actinin the WS43 preparation.

other component in the 43,000-dalton region. Thus, it can beconcluded that nonactin polypeptides are present in theWS43 fraction (Fig. 1A) and are not synthesized de novo.The linking of lower molecular weight polypeptides via di-sulfide bond can be ruled out because all the gels were rununder reducing conditions, and reduction and alkylation ofthe 43,000-dalton fraction does not change its molecularweight (6, 8). Also, no other lens polypeptides are known tohave molecular weights and isoelectric points similar to thatfound for the nonactin polypeptides in the WS43 fraction. Itwas, therefore, of interest to examine the possibility thatsome of these components may arise from nondisulfide co-valent bonding of the lower weight crystallin polypeptides.

Fig. 2 shows the reaction of the WS43 antibody with bo-vine a-, f,-, and y-crystallins, chicken gizzard actin, and hu-man WS43 fraction. It is apparent from this experiment that43,000-dalton antibody reacts with bovine ,8- and y-crystallinbut not with the a-crystallin or actin. Weak reaction is alsoobserved with 30,000-dalton polypeptides present in the bo-vine a-crystallin fraction and with 43,000-dalton polypep-tides present in the ,B-crystallin fraction. Since the WS43fraction isolated from human lenses does not contain any de-tectable polypeptide in the 20,000-dalton region (Fig. 1), it isprobable that this fraction contains antigens that are derivedfrom ,3- and y-crystallin components that have been incorpo-rated into the WS43 fraction. The WS43 antibody containsno detectable antibody directed toward actin. This is consist-ent with previous observations that non-NaDodSO4 dena-tured actin is a poor antigen (14). The WS43 fraction used toraise anti-WS43 was not denatured with NaDodSO4.To further substantiate the presence of crystallin antigens

in the WS43 fraction, immunoblot reactions with anti-a- andanti-y-crystallins were performed. The results are shown inFigs. 3 and 4, respectively. Anti-a-crystallin reacts weaklywith NaDodSO4-denatured a-crystallin polypeptides, as ex-pected, since only 16% of the anti-a-crystallin antibody isdirected toward the primary structure (19). No reaction withthe WS43 fraction or with (3- or y-crystallin are observed,indicating the probable absence of a-crystallin antigens inthe WS43 preparations. It cannot be stated unequivocallythat a-crystallin antigens are not present in the WS43 prepa-ration, because it is possible that dimerized a-crystallin poly-peptides are nonimmunogenic and are not cxpressed in therabbit. In contrast to the anti-a-crystallin, anti-y-crystallingives a definitive positive reaction with the WS43 fraction(Fig. 4). These observations support the conclusion that they-crystallin antigens reacting in the WS43 fraction may have

kDa

43 -

25-420-_

Ac tin WS43

FIG. 2. NaDodSO4/PAGE and immunoblot profiles of calf a-,f3-, and y-crystallin, chicken gizzard actin, and WS43. On the leftside of each pair is shown the NaDodSO4/PAGE profile, and on theright side is the immunoblot reaction. The antibody used for the im-munoreaction was anti-WS43.

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100-

c0

._-00

Ws43

i0 40 60 80 100Protein added to anti-rcrystallin, Ag/ml

FIG. 3. NaDodSO4/PAGE (left side) and immunoblot (rightside) of calf a-, A-, and y-crystallin, and WS43. Anti-calf a-crystallinwas used for the immunoblot.

been formed by dimerization of y-crystallin monomers or bylinkage with p-crystallin monomers. This is further substan-tiated by the observation that the bovine y-crystallin thatwas used for antibody production does not contain polypep-tides in the 43,000-dalton molecular size range.For quantitation of the 'y-crystallin antigen in the 43,000-

dalton preparation, two methods have been used. In the firstmethod, known amounts of 'y-crystallin and WS43 were run

on polyacrylamide gel and blotted with a diazotized paper;they were then reacted with calf anti-y-crystallin and 1251_labeled protein A. The level of y-crystallin antigen in theWS43 preparation was determined by densitometric scan-ning of the autoradiographs. By comparison with reactionwith known levels of y-crystallin, it was estimated that-10% ± 2% of the WS43 polypeptides react with anti-y crys-tallin. In the second method, an estimate of the amount of y-crystallin in the WS43 fraction was obtained through fluid-phase cross-blocking of the binding of rabbit anti-y-crystallinto bound 'y-crystallin using 125I-labeled protein A to detectbound immunoglobulins. The inhibition by serial dilutions of

kDa

43 -*

25

20 -4

. l

FIG. 5. Percent inhibition of anti-y-crystallin binding to immobi-lized y-crystallin. The anti-y-crystallin was treated with increasingamounts of y-crystallin (o) or WS43 (e) before the binding study.

y-crystallin or the WS43 fraction of the binding of the anti-^y-crystallin antibody was measured. As shown in Fig. 5, the y-

crystallin inhibition to the binding of anti-y-crystallin is lin-ear from 10% to 50%, while the WS43 inhibition is linear upto -90%. Since the slopes of the two inhibition curves differ,only a range of y-crystallin content in the 43,000-dalton prep-

aration can be determined. Analyses of the data yield a range

in the experimentally reliable region from 10% to 30%, with a

midpoint value of =20%. These results may be a low esti-mate because immunological reactivity could be decreaseddue to (i) species variation, (ii) modification of the antigenduring dimerization, and (iii) the aging process in which oth-er post-translational changes may occur.

It has been shown previously that old and cataractous hu-man lenses contain non-disulfide-linked HMW proteins (3).Some of these proteins do not enter NaDodSO4/polyacryl-amide gels and many of them can only be resolved as

streaks. It was considered possible that the polypeptidescontributing to the formation ofWS43 may also be present insuch HMW proteins. The 43,000-dalton fraction may then bean intermediate in a polymerization process leading to theformation ofHMW components. Therefore, the total water-soluble fraction of 63-yr-old human lenses was separated by

A

kDa

43 --

25-4

20-)

Ohr 6hrWS43

FIG. 4. NaDodSO4/PAGE profile and immunoblot reaction ofcalf a-, f3-, and y-crystallin and WS43. On the left side of each pair isthe NaDodSO4/PAGE profile, and on the right side is the immuno-blot reaction. The immunoblot reaction was performed with anti-calfy-crystallin.

B

i

Ws

FIG. 6. (A) Immunoblot reaction of calf y-crystallin photolyzedfor 0 and 6 hr. (B) The immunoblot reaction of total water-solubleproteins (WS) from 63-yr-old human lenses. Anti-WS43 was used forboth reactions.

kDa

'662a

43

318

18 -4.~-:r

-I-) Ws1, ~ vv)

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Proc. Natl. Acad. Sci. USA 81 (1984) 2881

NaDodSO4/PAGE and subjected to immunoblot analysiswith anti-WS43. As shown in Fig. 6B, a streak of reactiveprotein is observed extending from the top of the gel. Suchdata suggest that polymerization of polypeptides to a com-plex mixture ofHMW components has occurred in older hu-man lenses.At present, the mechanism of formation of the 43,000-dal-

ton fraction and HMW components in the human lens is ob-scure. Polyamines in the presence of transglutaminase mayform dimers of polypeptides (20). The presence of poly-amines in both bovine and human lenses has been demon-strated (21), and lens transglutaminase has also been report-ed by Lorand et al. (22). Another mechanism by which pro-tein polymers may be produced involves photolysis (18).Since the lens is subjected to light throughout life, this routeof polymerization was investigated. Bovine y-crystallin wasphotolyzed and analyzed by the immunoblot technique withanti-WS43 antibody. The results are shown in Fig. 6A. It isapparent that photopolymerization of y-crystallin leads toformation of polypeptides in the 43,000-dalton range as wellas a spectrum of components of higher and lower molecularweight and these products are immunoreactive with the anti-WS43. It is, thus, probable that under appropriate condi-tions, the pattern obtained in Fig. 6B showing the anti-WS43immunoblot of water-soluble protein could be duplicated.

This communication is the first demonstration of in vivonondisulfide polymerization of the crystallin proteins in thehuman lens. Only with aged collagen and elastin has it beenshown that polymerization of polypeptides occur (23, 24).The importance of the 43,000-dalton polypeptide in the hu-man lens has previously been demonstrated (5). This poly-peptide has been shown to be present in disulfide-linkedHMW protein found only in cataractous lenses (3). The poly-merization of the crystallins with aging may be a factor incataractogenesis, because this process could lead to a par-tially unfolded protein, thereby increasing its susceptibilityto oxidation.

This work was supported by grants from the National Eye Insti-tute, National Institutes of Health.

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24.7. Garner, W. H. & Spector, A. (1979) Doc. Ophthalmol. Proc.

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(1981) Proc. Natl. Acad. Sci. USA 78, 1356-1360.24. Bornstein, P. (1974) Annu. Rev. Biochem. 43, 567-603.25. Gallop, P. M. & Paz, M. A. (1975) Physiol. Rev. 55, 418-487.

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