Exp. Eye Res. (1976) 22,273--279

High Molecular Weight Protein from Human Lenses DEBDUTTA

ROY

AND

ABRAHAM

SPECTOR

Department of Ophthalmology, College of Physicians and Surgeons, Columbia Uwiversity, New York, N. Y. 10032, U.S.A. (Recelived 3 November 1975, Neu~ York) :\ new rapid method based upon differential centrifugation is described for the isolation of high molecular weight (HMW) protein from human lenses. Similar material is obtained with this method or by gel filtration. It was found that the HMW protein is almost identical in composition to the insoluble protein and is probably an intermediate in the formation of the insoluble fraction. There appears to be a spectrum of increasingly large components ranging from the relatively small HMW species to the largest particles in the insoluble fraction. Analyses of the polypeptides composing these proteins indicate the presence of 11 000. 20 000, 22 000, 27 000 and 45 000 dalton components. It is concluded that the human HMW and insoluble proteins are composed of a mixture of lens proteins as well as a major fract,ion consisting of smaller degraded polypeptides.

1. Introduction With aging of the bovine lens, there is a transformation of alpha-crystallin to high molecular weight (HMW) species greater than 50 x lo6 daltons (Spector, 1972). This aggregation occurs primarily in the central region of the lens (Spector, Freund, Li and Augusteyn, 1971) and has been of particular interest since the process may be related to the formation of senile cataract in the human lens characterized by central sclerosis and opacity. Bovine alpha-crystallin is composed of two different kinds of polypeptides designated A and B chains. The newly synthesized macromolecule contains only the A, and B, species held together by noncovalent forces to form a macromolecular aggregate with a molecular weight of approximately 7 x lo5 (Spector, Wandel and Li, 1968; Stauffer, Rothschild, Wandel and Spector, 1974). With aging post-translational changes occur and a number of altered A and B chains are observed (Stauffet. Rothschild, Wandel and Spector, 1974; Van Kleef, 1975). There is also the development of physical heterogeneity and a shift to higher molecular weight species (Spector, Freund, Li and Augusteyn, 1971). Investigation of the different sized alpha-crystalljn macromolecules indicates that subtle chemical changes are involved in the transformation to HMW (Spector, Freund, Li and Augusteyn, 1971; Jedziniak, Kinoshita, Yates. Hacker and Benedek, 1972; Li and Spector, 1973; Spector and Rothschild, 1973). LIeaggregation-reaggregation experiments utilizing urea or guanidine indicate that low molecular weight components may act cooperatively with certain altered A chains to produce the HMW species (Spector and Rothschild, 1973). The situation in the human lens is not clearly understood. With aging HMW protein species are formed (J-edziniak, Yates, Hacker and Benedek, 1973; Spector, Stauffer and Sigelman, 1973; Spector, Li and Sigelman, 1974; Jedziniak, Kinoshjta, Yates and Benedek, 1975) primarily in the nuclear region of the lens where they represent a significant proportion of the total soluble protein fraction (Spector, .Li and

251

I).

ROY

ANJ,

.A. SI’k;c’T(,J!

Sigehnan, 19i4; Jedziniak, Kinoshit,a. Yates anal Keu:?tlck. 19i5). However. ~~Y:MISI~ of the difficulty in characterizing the polypcptidc comporit ion of thrsfa aggregatesan11 1)ecuusethe structural proteins of the human lens have until recently been poori! defined. it has not been possible to relahtethis fractioii to otJhrr proteins. The illsoluble protein fraction of the lens also iucrc,aticswith age (Pirio. 196~: i ‘iark. Zigman and Lerman, 1969; Spector. Roy a.ntl Star&r. 19’7.3).It IM I~et~n~~ropo~r~~l that in t)hc bovine lens the HMW protein. which i.bc:sclusivt~l~;tlI)11~1-(:r~sfi~.llin. i- ;I precursor to the insoluble protein (Spector. 1952: Jw3ziiiiak. Kinoshitil. ;Ul(l Benedek, 1975). Examination of the composition a.n(l ii~llnlllloChellli(‘;L1charac.tc>tisticah of t,he H&IN’ and insoluble fractiorls super-t t,hat ~tlI)tla-o~!stallil~ is t,l~c~~~~jor VOIII~ poncn~ ia the iilSOhlhle fraction (Clark. Zigman an(1 llerinai~. 1969). Iro\vevc~r. ot \I,~I, Imt,eins in atltlit~ion to ,zlpha-c~~st,;lllirl II~T’C I~(~(~~1 ol~sw~\~c~l (~lmski ;rl~tl J1;ll.i iilvz. 1971). In the huirian lens little was previously 1~iiow11 al~or~tttrck ctwlpositioii of 111~ insoluble fraction. In the present communication, a new method for isolat,in$thtL HM\Y prot,c’in S~JCY’IIY frown human lensesis described basedupon tliffercnGa1 urnt~rifugatiorl. It is showrl tIllat the HMW speciesare essentially itlentical to the insolul)l~~prottairl. IntlrccI thcb(I I,‘-tinction lwtween the HMW prot,ein uud the so-called irlsolul IIPfraction ih I~aseclripoff the size of the particlcs suspendedin the preparatiou. Tllct sntallcr 1)ilL&cleS(HM\\ fraction) remain in suspmsion under conditions \vlwrc t tie laqyr (iiisolil l)ltn fraction) arc rcnioved. A major corliponent of tlww prott,iu J’rilc+ioil:: ff I 1~. COII~~OS~VI oi’ tleg~~cletlpolypeptides of i~pp~OXil~l~~t(‘~~ I I 000 tI;~ll olix, Yiltf'h

]litl'ti(~lPS

il)l~?>itl'b

2. Material and Methods The nuclear region (approximately the inner 4(1 4h”,,) wah isc)lirt~e(l froiu cibtitrit(.tou~ huuian lemes65 bo 70 years of age by utilizing ~1. ,j it1niVlJTli hrrr itricl removing t,hr t>i~tla of t,heexcisedcylinder. Fhe nuclearregiuuwashonlclgenizeclat,() C with a Ten Broeck tissuls homogenizer;it a,concentrxtiou of 0.7- 1.0 g (Ivet weight) per 10 1111 of (1.01.\l-Tris. (1.1\I KCl, pH 7.6. The homogenate\\-aricentrifuged at 12 000 rev;tliiti (I:! K), :iver;Ige wwt~rJl~~l,filI,-:! ult~;~c.rntrifil#~,ilsing it tylw fugal forcr. ,.:9900g for 15 niin in ;I r2drlr~~Ll~ 50 titauiunl rot,or at 4°C. The supernatant WM rewtrtrifupe~l ;I,t 20 000 rev/nliil (20 I\:) a\rerage cei1trifugal force ~*‘I%4OOgfor 15 tnin ill111ttw resulting super’natilnt was w ceutrifuged at 30 000 rev/min (30 Ii) for 15 min, ;averxgecwltiifu,g:al forcr ;.:59 4OUg. The 12, 20 and 30 Ii pellets were gently7n-ashedt’hreetinw with the Trix KC1hut&r ant1\v+artn then resuspendedin this buffer ivy gentle holrlogerriz;rtioll. The frwt,ions were the11 chrornato,qnphed 011a Rio-Gel &5Om co1u11m (3 ‘. &! c*nr). A Hio-CM A-15m IY~~IIII IIrv weights \Yerecletertnine(l (2.5 :‘50 ~ cm) was sometimesusedfor the 12 after dialysis against H,O for 24 hr. IDS-polq-acr!-larnide ,gel eleetroplioresis,reductio1l and alkylation and amino acid analyses of the protein were performed :L.Stlescrilwl previously (Specbr, Roy and Stauffer, 1975). K

SUpe~~lilt~~flt~.

3. Results Figure 1 illustrates the Bio-Gel A-15m filtration of the 12 K supernatant obt,ainecl from a homogenate of the nuclear region of the lens. The first peak which is eluted at the void volume representsapproximately 359:, of the material basedupon ahsorbmcc~ When it was rerun on Bio-Gel A-50111a single sharp peak was obtained at at A 280nm. the void volume indicating that this fraction cant-ains protein having a molecular weight greater than 50 x 106. The samehigh molecular weight protein can be prepared by differential ccntrit’uga-

HI(:H

MOLECULAR

WEIGHT

PROTEIK

FROM

HVMAN

LENSES

375

tion with the same buffer utilized in the gel filtration experiments. After removing the insoluble protein fraction by centrifugation at 12 K, the supernatant was recentrifuged at 20 K. A translucent pellet was obtained. The 20 K supernatant on recentrifugation at 30 K yields a transparent pellet. The 30 K supernatant was chromatographed on Rio-Gel A-50m (Fig. 2) and was found to be almost completely free of any HMW protein. Resuspended 20 K and 30 K pellets on chromatography on Bio-Gel A-50m (Fig. 2) were found to contain only HMW protein. The protein was eked in the void volume indicating macromoleculesgreater than 50 x lo6 daltons. The recovery from Bio-Gel A-50111 columns with the 30 K supernatant was usually greater than 95% but with the rehomogenized 20 K and 30 K pellets considerably lower recoveries were found. 3.6 32I 282.4 -

IZO-8 -

i

120

60

180

240

Volume

300

340

300

(mll

I; .. 30

60

90

120

150

..,” 180 Volume

210 imll

.. 240

270

300

330

360

276

I). ROY AND

A. SI’EC’J’OI:

Examination of the fractionation obtained by this procedure indicates that 55,:.5” r, of the dry weight of the nuclear region from cataractous lensesis represented I)y thv Id K insoluble fraction. The 20 K HMW fraction is approximately 2.5 times greater than the 30 K HMW fraction. The two HMW fractions obtained by different,inl centrifugation contribute approximately 30:/, of the material basedupon absorbence at A 280nm and approximately IS:/, of the dry weight of t,he soluble fraction.

45555

1

2

SDS polyacrylamide gel electrophorcsis (Pig. 3) of thcb 12 E;. 20 K antI SOIi ~~c,llt~l after reduction and alkylation gave the sameprofiles itlc.lica,tillg the prt:st~ncc’ol fi\‘t~ different sized polypeptides. The relat,ivct proport,iuns of’ tfrtl pol~peptidr.~ ;tlalt\ino a&l conlpobit io11 of alpha-crystallin is apparent.

HIGH

MOLECULAR

WEIGHT

PROTEIN

FROM

HUMAN

LENSES

277

TABIJC 1

Awho

acid analysis

of insoldde

and HMW

protein

Fraction 21) K

4. Discussion Either gel filtration or differential centrifugation can be utilized for the isolation of HMR protein. The centrifugat’ion method is preferred since it requires lesstime and is suitable for large-scalepreparations. The amino acid compositions, SDS gel elect’rophoresisa,ntl gel filtration patterns are the samefor HMW protein prepared by either procedure. It is apparent from these experiments that the definition of human lens HMW protein is an operational one. At high centrifugal forces it can be completely removed from solmion while at low centrifugal forces incomplete pelleting of the insoluble fraction occurs. Thus the conditions for ohta.inin~ the HMW fraction are somewhat, arbitrary. However, there are two observations that have guided these experiments. Under conditions previously described utilizing pel filtrat,ion, HMW protein is found t,o be insignificant in young lensesand increasesmarkedly with aging (Spector, Li and Sigelman. 1974). This processoccurs primarily in the nuclear region and only in very old lensesare significant amounts of HJIW protjein observed in the periphery. The conditions utilized in this communication give results similar to those obtained previously. On the basis of the observations reported in this communication. t,he insoluble fraction is defined as the precipitate obtained by centrifugation of a solution comaining 0.7-1.0 g (wet weight) of lens per 10 ml of buffer at an average centrifugal force of x 9900 g for 15 min. The HMW fraction is isolated by centrifuging the supernatant, obtained above at an average centrifupal force oi x 59 400 g for 15 min. It has been found in t’his investigation that the HMW protein constitutes a sienifica,nt fraction of the total protein of the nuclear region. Similar observations of

278

I).

ROY

ANI)

A. SPE’:(:‘TOK

appreciable quantities of HMW protein in bun-I~IL lrnscs have previously tJecn rtiported (Spector, Li and Sigelman. 1974; Jedziniak. Kinoshit,a. Ya,tes and Kenedek. 1975). It should also be noted that! the presence of 50 mm dithioerythitol during the isolation does not appear to have an appreciable effect upon the yield of HMW protein. R*ecently it has been reported that only very small amounts of HMW protein ibrc present. in the human lens and that, therefore aggregat,ion of human lerls prot,cin cannot be involved in cataract formation (Dillq. 1975). III that report. the insolublr fraction was removed at x 26 000 g. Most of the HMW fraction as defined in t,his communication would be removed with the insoluble material hy such a proced urf,. Furthermore, if the HMW fraction is an intermediate in the formation of still larger components which constitute the insoluble fraction. the quantity of HNW protritl map be independent of the amounts of insoluble protein present in the tissurl. N’ course the formation of HMW and insoluble L rotein may possibly he an artif’act produced by the isolation techniyuo. However. sinc(l young lenses have very small amounts of insoluble protein and only small quantities of HMW proteiu are found in the lens periphery, formation of these large macromolecules must bc dependent 71porb a fundamental change in the chemistry of the lens prot,eins. The similarity in the chemical properties of the HMW and the insoluble fract,ions supports the concept of a precursor relationship. There mav be a gradual incre;tse in the size of the HMW macromolecules which finallg result*s in insolubilization. How ever. it appears from gel filtration experimentIs t,liat lft~~roIllOleG~~litr aggregates between 15 and 50 x lo6 daltons are not present’. Thus HJ’IW protein is formed by the interaction of low molecular weight components without significant formatiorl of intermediates (15 to 50 x lo6 daltons). It has previously been found that, human ;tlyA-crystallin isolatetl from t,ht: I)(briphery contains aggregates larger than 5 x 10” daltons (Roy ant1 Spector. 1975). Such preparations have essentially none of the 27 000 arid I I 000 tlaltou componont~s found in the HMW and insoluble fractions of t,he nuclear region. The relationship of these alpha-crystallin aggregates to t*hc formation of’ H&lW and insoluble protjein i> not understood at the present time. The composition and the size distribution oj’ t,hc polypeptitle chains of the HMV and insoluble fractions clearly indicate that ttlc>y ar(’ not composetl exclusively of alpha-crystallin as is the case in bovine lenses (it should LJC noted thst very old t)ovinr lenses have not as yet been examined). Based on the known sizes of the polypq:.t,ide+ oi the lens proteins it is probable that alpha-crystallin contributes to t’ho 20 000 am I 22 000 dalton polypeptides and t,he 27 000 dalton polypeptitle is a IJeta-orystallin component. The origin of the 11 000 dalton fmction remains to be elucitlat~trd. It5 abundance in the nuclear region whcrc protein synthesis is insi&icant, sug#csts t3hnt these polypeptides are degradation products. perhaps. formed t)y proteolysis. Proteolytjic enzymes have recently I)c~t:n reportecl in human lens (Tray hum anct van Heyningcn. 1975). Polypepticles in the 45 000 dalton range have p~iorisl~ IJCW found to t)e ussociat,rtl with a fluorescent component which may link smallt>r IJolypept,itlos t)y ~mtlrfirwtl reactions (Speotol. Roy and Stauffer. 1975). Recent work sug.q~ s that; the fluorcsccnt materials are probably /I-carbolines (Dillon. Spector mcl Nakanishi. 1975). Thc~ abundance of such components in the insolu hlc fmctiou suggests that it 111ay I,tx involvccl in the insolublization process (Spector. Roy and Stauffer. 1975). lt, is aspparent from previous work with the hovine lens th;lt the fact,ors invo1vc.d ilr the formation of HMm’ protein and the insoluble fraction are complex depenclin~

H LGH

MClLEC’ULAR

\\‘EIGHT

PROTEIS

FROM

H 1lMAN

LEXSES

"79

upon a number of factors. The presence of fluorescent components, degraded polypeptitlcs and presumptive /3-crystallin polypeptides in the human HMW and insoluble fractions a.tid additional complexity tG the problem.

The skilled technical assistanceof Kathv Huc2ngand Gloria Jenkins is grstjefullv a~ck!~o\vledgetl. This \vork was supported by grants from the Xational Eve Tnstitute, t,he K>ltiod llrstitutes of Health, Department of Health, Education and Iqelfare.

REFERENCES Ib., Zigman. S. and Lerman. S. (1969). Stndieeon the strnctld proteinsof the hum:rn lens. k.rp. Eye Res. 8, 172%5’7. TXllep. K. (1975). The proportion of protein from the normal and cataractous human lens whicil exists as high molecular weight aggregates in vitro. Exp. Eye ti’es. 20, 738. Dillon. J.. Spector, A. and Sakanishi, ;2. (1975). Identification of /?-carholines isolated frcin, fluorescent human lens proteins. Nnfule. (In press.) Jetlziniak. J. .+., Kinoshita, J. H.. Yates, E. M.. Rocker, L. 0. and Benedek. G. B. (1972). C’alciuminduced aggregat.ion of bovine lens alpha crystallins. Inrest. Ophth~nlmol. 11, 905-15. .Jedziniak, J. A., Kinoshita. J. H.. Yates, E. BI., Hacker, I,. 0. and Benedek, G. B. (1973). On the presence and mechanism of formation of heavy molecular weight aggrega,tes in human normal and cataractous lenses. Exp. Eye Res. 15, 185-92. Jedziniak, J.. Kinoshita, J. H.. Yates. E. M. and Benedek, G. B. (1975). The roncent,ration and localizat~ion of heavy molecular weight aggregates in aging normal and cat,aractous humw~ lenses. Exp. Eye Rc.9. 20, 367-Y. Li. Lu-Ku and Spector, A. (1973). The reaggregation of pnrifietl subunits of alpha-crystalhi,. Exp. I;‘yt Res. 15, 179-8::. Manski. \I’. and Martinez. C’. (1971). hnml~noc~hemical studies on albuminoid, II. (hanges associated with age. Exp. Eye Res. 12, X&11. I’iric, A. (I 96X). Color and solubilitg of the proteins of human cataracts. Incest. Ophthtrlwol. 7, K14-51 I. Boy. D. and Spector, A. (1975). Human alpha crystallin, Il. Characterization of the protein isolated from the periphery of cataractous lenses. Biochemistry. (In press.) Spector, A., \\‘andel, T. and Li, Lu-Ku (1968). The purification and characterization of the highly labeled protein fract,ion from calf lens. Inwsf. O~~~~~Z~o~. 7, 179-90. Spector. A.. Freund. T.. Li. Lu-Ku and Augusteyn, R,. f. (1971). a4ge dependent. changes in the st,ruct.rtre of alpha crystallin. In,wf. Ophthnltnol. 10, 677-W. Spector. X. (1972). Aggregation of alpha crystallin and its possible relationship to cataract formation. Is. J. Med. Sci. 8, 1577-S Spector. A., Stauffer. J. and Sigelman. J. (1973). Preliminary observations upon the proteins ot human lens. Cibn E’oundntion Synlposium 19 (Sew Series), 187-206. ASP, Amsterdam. Spector. A. and R,othschild, C.‘. (1973). The effect of calcium upon the reaggregation of borinc alpha crystallin. Incest. OphthnZrr,oZ. 12, 225-31. Spector. A., Li. S. and Sigelman. J. (1974). Age-dependent changes in the molecular size of human lens protein and their relationship to light scatter. Iwest. Ophthnln~ol. 13, 795%. Spector, -1.. Roy. D. and Stauffer. ,J. (1975). Isolation and characterization of an age dependent polypeptide from human lens with non-tryptophan fluorescence. Exp. Eye Rex. 21, 9-S. Stouffer. J.. Rothschild, C.. Handel. T. and Spect)or. A. (1974). Transformation of alpha-crystalliu polyprptide chains with aging. Incest. Ophfhnlmol. 13, 135.-46. Trayhum. P. and van Heyningen, 13. (1976). Seutral proteinase activity in the human Icns. Exp. L:ye Res. 22. van Kleef, F. S. 31. (1975). Post-synfhefic modijicuficms o~boGw dphn-rrystnllin. Thesis. I~nircrait~y of Sijmegen. The Netherlands.

Clark.