Exp. Eye Res. (19’i9) 29, 625-635
Immunochemical Studies on Lens Protein-Protein Complexes I. The Heterogeneity and Structure of Complexed a-Crystallin W.
MANSKI,
K.
NALIITOWSKI
AND
G.
BOXITSIS
Departments of Microbiology and Ophthalmology, Collegeof Physicians Columbia University, New York, N.Y. 10032, U.X.A.
and Surgeons,
(Received8 March 1979, &SewYork) A buffered extract of young cattle lenses filtered through Diaflo XM-300 membranes, retaining molecules above 300 000 daltons, was found to contain in the non-filterable fraction constituting 50% of soluble lens proteins cc-and /%crystallins. The non-filterable fraction of soluble proteins from mature cattle lenses, constituting 87% of all crystallins, contained not only cc- and P-crystallins, but y-crystallins as well. Only cc-crystallin, with a molecular weight above 300 000 daltons, was expected to remain in the non-filterable fraction. All of the ,kcrystallins, which have a molecular weight below 200 000 daltons, and y-crvstallins, with a molecular weight of about 20 000 daltons, if free in solution and not complexed, were expected to be in the filtrate. The lens aroteins with a molecular weight above 300 000 daltons from young lens tissue were separaied by gel chrcmatography (l%o-Rad A 15 m) into free cc-erys~allinufollowed by an oc-/?-crystallin complex. By the same procedure, the prot,eins above 300 000 daltons from mature lenses were separated into two complexes of quantitatively different z-fl~crystallin composition. A dependence of lens protein complex formation on the ionic composition of the solvent was observed. The differences bet.ween calf cortical and bovine nuclear complexes, found in buffer extracts, were much less pronounced in water extracts in which both contained y-crystallins. Specific dissociation of the lens protein complexes was obtained by use of an anti-acrystallin immunoadsorbent, as shown by the passage of the previously retained /3- and ycrystallms &rough a Diaflo XM-300 membrane. An anti-/3-crystallin immunoadsorbent reacting with complexes from mature lens tissue yielded a non-filterable a-crystallin and a filterable y-crystal&n in the supernatant, indicating an absence of direct a+ complexes. These results point to a potentially central role of /z-crystallins in complex formation among lens proteins. Key lords: calf lens; bovine lens; lens crystallins; lens protein-protein complexes; heterogeneity of complexed a-crystallin; structure of complexed lens crystallins; protein interaction/salt concentration dependence.
1. Introduction An immunochen~ical approach is being used in a series of investigations on the occurrence of interactions between different protein molecules synthesized within the lens epithelial cell. Such investigations were designed to determine whether such interactions lead to the formation of protein-protein complexes of different composition and stability and whether or not such interactions are dependent on the age of the cell in which they occur. The lens provides a uniquely appropriate model for the study of these particular problems. This tissue is formed from one kind of ectodermal cell, functioning like a monoclonal cell culture in vivo and in the aging process of the lens, older cells are not shed but persist compressed into the center of the lens (Pirie and van Heyningen, 1956). OO14-4835,09/1206%+11
0
SOLOO/ 625
1979 Academic
Press Inc. (London)
Limited
626
W.
3IAXSK1,
K. &fALISOWGKI
A?u’D
G. RONITSIS
Both lens protein fractions, obtained on extraction, the soluble crpstallins, the insoluble albuminoid (Kuck, 1970) have antigenically the same crystallin composition, as shown by the complete cross-absorption of anti-crystallin antiserum by the albuminoid fraction, and vice versa (Manski, Behrens and Martinez, 1968 ; Manski and Martinez, 1971). However, the relative amount of the various proteins was shown in these reports to be different in the crystallin and albuminoid fractions. Immunoelectrophoretic analysis of the crystallins demonstrates the presence of approximately 12 different proteins (Manski, Halbert and Auerbach, 1961). The main structural protein of the lens, a-crystallin, constitutes about 30% of all lens proteins and is characterized by its high molecular weight exceeding 300 000, averaging 800 OOO1000 000 daltons. The molecular weight of the different /3-crystallins falls within a range of 20 000 to 200 000 daltons. The molecular weight of y-crystallins is 20 000 daltons. A review of these different structural proteins of the mammalian lens was published by Harding and Dilley (1976). Underlying research on lens proteins, from the early work of Morner (1894) to recent studies, has been the implicit assumption that the different pre-a-, /3- and y-crystallins are present in the lens in a free state. It is one of the aims of this report to demonstrate that formation of complexes involving or-crystallin does occur in the lens and that this formation progresses with age. Another aim of this report is to gain insight into the heterogeneity and structure of such complexes. 2. Materials
and Methods
Dissecticw of calf lens cortex and bovine lens wucbezu
Immediately after slaughter, lenseswere removed from 336 month old calves and from cattle about 24 months old, Carewastaken to free the lensesfrom capsular,vitreous and aqueous material. The clean lenses were immediately frozen. The equatorial section of the frozen lenses was removed wit’h a stainless steel cork borer of 12 mm id. in the case of the calf lenses and 14 mm i.d. in the case of the bovine lenses. The inner nuclear part of the remaining lens (about 30%) was separated from the outer cortical part (about 38%) wit,h a 7 mm i.d. stainless steel cork borer for calf lenses and a 9 mm id. borer for the bovine lenses. Isolation
of the cx-crystallin
j%action
The cortical portion of lenses from 3-6 month old calves was homogenized and extracted at 4°C with PBS. The insoluble albuminoid and cryoprotein fractions were removed by centrifugation at 18 000 x g for 30 min at 4°C in a Sorvall centrifuge with an SS-34 rotor (Ivan Sorvall, Inc., Norwalk, Conn.). Alpha crystallin was isolated from the soluble fractions by repeated isoelectric precipitation at pH 5.0 (Francois, Rabaey and Wieme, 1955), followed by ultrafiltration through an XM-300 Diaflo membrane (cut-off point 300 000 daltons) (Amicon Corp., Lexington, Mass.), as described in a previous report (Malinowski and Manski, 1977). Isolation
of the P-csystallin fractiolz
The procedure used is a modification of the procedures reported by Herbrink, van Westreenen and Bloemendal(l975) and by Spector (1964). After the removal of cc-crystalhn by isoelectric precipitation (pH 5.0), the supernatant was equilibrated with a solution of O-1 &I-Tris, 1 M-NaCl and O-005 r+EDTA, pH 7.5, then applied to a Bio-gel A 0.5 m column (5-O x 44.0 cm) equilibrated with the same buffer. The third fraction eluted from the column was concentrated and equilibrated with a buffer 0.005 M-Tris-Hcl and 0,005 M-
ALPHA
CRYBTALLIIZ-
COVPLEXES
62i
KCI, pH 7.3, then applied to a Sephadex G-50 column (50 x 44.0 cm) equilibrated with the same buffer. The second of the four chromatographic fractions contained ,&crystallins free from Z- and y-crystallins. This was determined by immunoelectrophoretic analysis in which the isolated P-crystallin fraction was reacted with rabbit anti-total cattle lens protein antiserum. bsolatio~
of has u-crystal&n
conzplexes
by ultrajltration
dmicon ultrafiltration cells with X31-300 Diaflo membranes were used for the separation of lens proteins based on their different molecular weights. According to the manufacturers, the retention capacity of these ultrafilters is approximately 98%. Twenty g of calf lens cortex or bovine lens nucleus were homogenized at 4°C in a N.Y.) with 100 ml of an 0.01 nr-‘I&-WC1 buffer, homogenizer (Virtis Inc., Gardiner, pH 7.2, containing 0.09 ~-Kc1 and 0.05 x-NaCl. These salts approximate the intracellular ionic environment of lens proteins in vivo. The insoluble albuminoid fraction was removed by centrifugation at 18 OOOxg for 30 min at 4°C in a Sorvall centrifuge. The soluble fraction was filtered at 4°C in an Amicon cell with a Diaflo XM-300 membrane under 20 psi of nitrogen pressure, while a constant volume was maintained by the addition of the same Tris-buffered salts. Filtration was continued until the absorba,nce of the filtrate at 280 nm was below 0.02 o.d., approximately after 4 days. The non-filterable fraction was concentrated to 20 mg/ml on the same X31-300 Diaflo membrane. The filterable fract.ion, in turn, was concentrated in an Amicon cell using a PM-10 Diafio membrane (cut-off point 10 000 daltons). In another procedure, 20 g of calf lens cortex or bovine lens nucleus were homogenized wirlth 100 ml of distilled water. The tissues were treated thereafter in the same manner as the buffered preparation, except that constant volume was maintained during filtration by the addition of distilled water instead of buffer. Iunwa7cn~e sera
Adult chinchilla rabbits weighing 5-6 lb each were immunized by the subcutaneous injection of 5 mg of a cattle lens homogenate in complete Freund’s adjuvant (&I. butyricum, Difco Labs., Detroit, JIich.) at five separate body locations. The first three immmizations were administered at a-week intervals. Subsequent immunizations were administered at monthly intervals. The immune sera were collected under sterile conditions after about ten immunizations. Isolation
of anti-a-
or anti-/!-crptalh
antibodies
Anti-K-crystallin antibodies were isolated from rabbit anti-total calf lens protein antiserum by mixing 500 ml of rabbit antiserum with 30 g of a calf crystallin immunoadsorbent containing 510 mg of bound calf cc-crystallin. Mixing was carried out overnight, at room temperature. Similarly, the anti-p-crystallin antibodies were isolated by reacting 500 ml of rabbit anti-total calf lens protein antiserum (after remova of anti-u-crystal&n antibodies) with 30 g of a P-crystallin immunoadsorbent conta,ining 390 mg of bound ,/3-orystallin. The immunoadsorbent suspensions were filtered and washed on a Buchner funnel with PBS until the absorbance at 280 nm was lower than 0.02 o.d. For the desorption of bound antibodies, the washed immunoadsorbents were mixed for 10 min with 50 ml of 0.5 wCH,COOH and then filtered. The desorption procedure was repeated three times. The collected filtrates were immediately brought to pH 7.2 by the addition of 0.1 M-XaOH. The amount of isolated anti-calf oc-crystallin antibodies was 98.7 mg; the amount of isolated anti-@rystallin antibodies was 69.5 mg. The purity of ihe isolated antibodies was tested by immunoelectrophoresis, using total cattle lens proteins.
W. MANSKI,
628
Prepwatiorz
PC. MALIIYOWSKI
AYD
G. BONITSIS
of irnnaunoadsorbents
The a- and P-crystallin immunoadsorbents were prepared according to the method of Cuatracasas and Anfinsen (1971), as described in a previous report (Manski and Malinowski, 1979). Approximately 17 mg of cc-crystallin and 13 mg of fi-crystallin were bound per gram of Sepharose 4B gel (Pharmacia Fine Chemicals, Uppsala, Sweden). For preparation of the antibody immunoadsorbents, the desorbed antibodies were equilibrated with 0.1 iv-NaHCO,, pH 8.1, in an Amicon filtration cell with a P%lO Diaflo membrane (cut-off point 10 000 daltons). The isolated anti-cc- or anti-P-crystallin antibodies were coupled separately to activated Sepharose by Cuatracasas and Anfinsen’s procedure, The yield of coupling was 8.2 mg of anti-calf cc-crystallin antibodies and 67 mg of ant,i+crystallin antibodies per gram of Sepharose 4B gel. Gel chromatography
of lens protein
complexes
For the procedure, 0.5 g of the non-filterable lens proteins in 10 ml of a 0.01 3r-TrisHCl buffer, pH 7.2, solution containing 0.09 ~-Kc1 and 0.05 M-NaCl, was applied to a Bio-gel A 15 m agarose column (Bio-Rad Labs., Richmond, Calif.) equilibrated with the same buffer. The flow rate of the elution buffer was 1 ml per min. Separation of proteins into fractions was monitored on a multi-channel spectrophotometer (Gilford Instrument Labs., Inc., Oberlin, Ohio). Xpec$c
dissociation
of fpvotei+~rotein
complexes
Sixty mg of the non-filterable bovine nuclear lens protein in 60 ml of salt-containing Tris buffer with 0.02% NTahT,, were added to 36 g of an immunoadsorbent with 295 mg of bound anti-calf a-crystallin antibodies or with 241 mg of bound anti-calf j-crystallin antibodies. The complexes and immunoadsorbents were mixed in a roller culture apMillville, N.J.) for 72 hr at room temperature. The paratus (1Vheaton Instruments, immunoadsorbents were then separated by filtration through sinter glass funnels. The proteins not bound to the immunoadsorbents were washed out with the salt-containing Tris buffer. The filtrates were combined and concentrated, using a UM-2 Diaflo membrane (cut-off point 2000 daltons). The proteins bound to the immunoadsorbents were desorbed in five 25ml portions of 0.5 M-CH,COOH. neutralized with Th e acid was immediately 0.1 r;-NaOH. Both the filtrates and the desorbed solutions were dialyzed against the above Tris buffer, using UM-2 Diaflo membranes; then concentrated to 20 mg of protein per ml.
The method used is based on the micro-technique of Scheidegger (1955). Agar-coated slides were covered with a 1.5% Bacto gel agar (Difco Labs., Detroit, Mich.) in 0.015 MTris-Na, EDTA-boric acid buffer, pH 8.4. The distance between the antigen hole and the antiserum well was 3 mm. The volume of the antiserum was 0.25 ml. The volume of the antigen was 0.02 ml and its concentration 20 mg/ml. A potential of 5 V/cm across the slide was applied for 90 min. The immunoelectrophoretic patterns were developed at 4°C and observed for 7 days. Prot&
determination
Absorption at wavelengths of 260 and 280 nm was measured in a DU-2 spectrophotoCalif.). The protein concentration was meter (Beckman Instruments, Inc., Fullerton, calculated according to the formula: protein in mg/ml = 1.45 E,,,-0.74 E,,, (Kalckar, 1947).
ALPHA
CRYSTALLIN
COXPLEXES
629
3. Results Occuwence of lens protein-protein
complexes
When the soluble calf cortical lens proteins in a salt-containing Tris buffer were fiitered through an XM-300 membrane: these proteins were found to contain 500/ of a non-filterable fraction. The non-filterable fraction of bovine nuclear lens proteins in the same buffer was 87%. When the lens extracts were prepared in water, the nonfilterable fraction of the calf cortical lens proteins increased significantly, to il)i/,. The corresponding fraction of bovine nuclear lens proteins in water was SS%, practically unchanged from the amount obtained in buffer. All the fractions were analyzed by immunoelectrophoresis. The non-filterable fraction of the calf cortical lens proteins (Fig. 1, column a) in buffer (molecular weight above 300 000 daltons) contained not only an alpha but also /Scrystallin lines. By comparison, the non-filterable fraction of bovine nuclear iens proteins in the same buffer (Fig. 1, column b) contained at least two /I-crystallin lines and, in addition, two y-crystallin lines. The immunoelectrophoretic pattern of a non-filterable calf cortical fraction obtained by water extraction (Fig. 1, column c) was more complex than the one obtained by buffer extraction, showing a greater Gmilarity to the pattern of the corresponding water-extracted non-filterable bovine nuclear fraction (Fig. 1: column d). The non-filterable fractions of the bovine nuclear lens proteins extracted by buffer and by water showed no pronounced differences in composition. The presence of proteins other tha.n a-crystallin in the non-filterable fractions was confirmed by comparison of the multiple lines formed by anti-total lens crystalhn antiserum with the single lines developed in immunoelectrophoresis with anti-acrystallin antiserum (Fig. 1, second row)] hens protein
fraction
Calf cortex
(20
mg/ml)
Bovine
with
o molecular
weight
above
300
000
doltons
Calf cortex
nucleus
Bovme
nucleus
In water
In buffer (b)
(0)
(d)
(cl
AM-total
lens protein
a
ant&serum *
I& I Anti-a-crystolhn
Lens protein
fraction
-
-lu
_o.
0
(20
mg/ml)
wih
o molecular
lens protein
a nmlecular
weight
above
and
below
300
000
daltons
antiserum
IO Ant!-a-crystallin
FIG. 1. Irnnrunoelectrophoretic
weight
c
Anti-total
with
antiserum
analysis below
0
antiserum
of the calf cortical 300 000 daltons.
and
bovine
nuclear
lem
erystallin
fraction
630
W. MAXSKI>
K. MALISOWSKI
AND
G. BONITSIS
Immunoelectrophoretic analysis of the filterable fraction of calf cortex in buffer (molecular weight below 300 000 daltons) showed the presence of both ,& and ycrystallins (Pig. 1, column a, third row). In addition, some .z-crystallin was also present in this filterable fraction, possibly indicating that the cut-off point of the membranes is somewhat higher than indicated, or that there are some cc-crystallin molecules in young lens tissue which have a molecular weight lower than 300 000 daltons. Certainly, this result excludes the possibility that the pore size of the filters is smaller than indicated which would have alIowed retention of some ,& or even some y-crystallins. In the buffer extracted bovine nuclear lens proteins, both /3- and y-crystallins were found in the filtrate (Fig. 1, column d, third row). By comparison9 the filtrate of bovine nucIear Iens crystallins extracted in water showed the presence of beta lines (Fig. 1, column b, third row). It can thus be concluded that in a water extract all the bovine nuclear y-crystallins must have been complexed. Heterogeneity of lens protein-protein
complexes
Pigure 2 shows that the non-filterable fraction obtained by buffer extraction of CrystalEns from a calf cortical lens preparation separated into two peaks by gel chromatography. Peak 1 contained 2l+3o/oof the total protein collected and peak 2 contained 78.2%. By comparison, the corresponding buffer-extracted bovine nuclear fraction (Fig. 3) separated into three peaks, with peak 1 containing 226% of the total protein collected; peak 2 containing 6.2%; and peak 3 containing 15.4%. The gel
400
800
1200
1600
2000
ml FIG. 2. Gel chromatographic analysis on a Bio-gel A 15 m column lens protein fraction non-filterable through an X&I-300 membrane.
(50 x 44.0 cm) of the calf cortical
ALPHA
CRYSTALLIN
400
COxPLEXE8
800
1200
631
I600
2000
ml E'IG. 3. Chromatographic analgsis on a Bio-gel A 15 m column Iens protein fraction non-filterable through an XM-300 membrane.
(50 x44.0
nuclear
Concentrotlon of antigens hng/ml)
Peak 2
Peak I
cm) of the bovine
10-o
_o,,
0 .__._. - -____-0 FIG. 4. Immunoelectrophoretic ilterable calf cortical lens protein
2.5
-
0 titration fraction).
of the chromatographic
0.6
0.3
fractions
shown
in Fig.
2 (non-
W. bUNSKI,
632
K. iUALIP\‘OWSKI
AND
G. BOSITSIS:
chromatographic analyses shown in Figs 2 and 3 were done under the same experimental conditions, The fractions marked by the shaded areas were collected, concentrated and analyzed by immunoelectrophoresis. The photographs of the patterns obtained for each peak are shown in Figs 2 and 3. Their dia.grammatic presentation is given in the titration experiments, presented in Figs 4 and 5. As can be seen in Fig. 4, peak 1 obtained by gel chromatography of the nonfilterable calf cortical lens protein fraction showed the presence of cr-crystallin only. By comparison, peak 2, consisting of molecules of lower weight, contained both c(- and p-crystallins. Th’IS was confirmed by the immunoelectrophoretic reaction of peak 2 with anti-or-crystallin antibodies, which then showed a single line of cr-crystallin.
Peak
Peak
I
Peak 3
2
b
_o,
b
AL-
o
0,
0
_o I_0
0,
FIG.
5. Immunoelectrophoretic titration of the bovine nuclear lens protein fraction).
chromatographic
5.0
--
2.5
-
1.2
-
0
0
.0..... .___“. filterable
-
0
--------
Concentration of ontlgens hg/m
0.6
-.__.. fractions
shown
in Fig.
o-3
3 (non-
There was no separation of free from complexed or-crystallin in the first two gel chromatographic peaks obtained from the non-filterable bovine nuclear lens proteins in buffer (Fig. 5). Immunoelectrophoretic analysis demonstrated in both the presence of cc- and P-crystallins as well as y-crystallins. Peak 3, however, contained only p- and y-crystallins. Th’1s f raction was filterable through an X$l-300 membrane, indicating that these crystallins must have been only weakly bound to the more stable ~-p-y complexes in the initially non-filterable fraction. The
structure
of lens
protein-protein
complexes
Analysis of the structure of the lens crystallin complexes was based on their reaction with specific antibodies to 01- or P-crystallin bound to matrices. Specific dissociation of the complexes by the antibodies was tested in the supernatants after removal of the immunoadsorbent. Immunoelectrophoresis was done both before and after ultrafiltration of the supernatants through XM-300 Diaflo filters. These analyses are illustrated by the reactions of the lens protein complex in the gel chromatographie peak 2 of the non-filterable bovine nuclear lens proteins (Fig. 5).
SLPHA
CR.YSTALLIN
633
COMPLEXES
As can be seen in Fig. 6, all p- and y-crystallins were observed in the supernatant after exposure to an anti-or-crystallin immunoadsorbent (Fig. 6-2), and all of them were filterable through an X&I-300 membrane (Fig. 6-3). The complete dissociation of the initial lens protein complex was proven by the fact that the protein desorbecl from the immunosdsorbent (Fig. 6-4) was a-crystallin and not an a-crystailin complex.
,-
3-
FIG. 6. Specific dissociation of lens protein complexes by anti-cc-crystallin antibodies. 1: Bovine nuclear lens protein oomplex before reaction with an anti-calf cc-crystallin immunoadsorbent 2: Bovine nuclear lens protein complex after reaction with an anti-calf cr-crystallin immunoadsorbent. 3: Lens proteins in the reaction mixture filterable through an XlW300 membrane. 4: Lens proteins desorbed from the anti-cc-crystallin immunoadsorbent.
,-so
5-
FIG. 7. Specific dissociation of lens protein complexes by anti-F-crystallin antibodies. 1: Bovine nuclear lens protein complex with a molecular weight above 300 000 daltons before reaction with an anti+crystallin immunoadsorbent. 2: Bovine nnclear lens protein complex with a molecular weight shove 300 000 daltons after reaction with an anti-P-crystallin immunoadsorbent. 3 : Lens proteins in the reaction mixture filterable through an XM-300 membrane. 4: Lens proteins in the reaction mixture nonfilterable through an XM-300 membrane. 5: Lens proteins desorbed from the anti-/%crystallin immunoadsorbent.
The same bovine nuclear lens protein complex (Fig. ‘7-l) was also exposed to an excess of an anti-/Scrystallin immunoadsorbent. After centrifugation immunoelectrophoresis of the supernatant of the reaction mixture (Fig. 7-2) demonstrated the presence of 01- and y-crystallins. The y-crystallins were completely filterable through an XM-300 filter (Fig. 7-3). Alph a- cr y,st a11’m was retained on the membrane (Fig. ‘i-4). Only P-crystallins were desorbed from the immunoadsorbent (Pig. 7-5).
4. Discussion There are only a few references in the literature which point to potential proteinprotein complex formation in the lens. Woods and Burky (1933) studied the role of cha.nged ratios of or-crystallin to /3-crystallin in cataract formation. These authors observed that addition of a-crystallin prevented the spontaneous precipitation of pcrystallin in solution. They also noted that a-crystallin in solution precipitated on the addition of CaCl, at a concentration of approximately 9 mg percent. In the presence of ,E-crystallin, a higher concentration of CaCl,, about half of saturation, was required to cause precipitation of cx-crystallin.
634
Vi’. MAn-SKI,
Ii. MALINOWSKI
AKD
G. BONITSIS
The ability of oc-crystallin to react with some non-lens proteins was studied by Crzalesi and Miglior (1957). Using electrophoretic methods, cc-crystallin was found to form stable complexes with strongly electropositive molecules such as protannnes. Proof of complex formation between cc-crystallin and other lens protein rests, in OLW experiments, on the following observations : (1) that the calf cortical lens proteins which did not filter through a membrane having a cut-off point of 300 000 daltons contained not only a-crystallin but also ,&crystallin, which has a molecular weight of no more than 200 000 daltons, and (2) that the corresponding bovine nuclear fraction contained not only ,8-crystallin, but also y-crystallin, which has a molecular weight of only about 20 000 daltons. The non-filterable bovine nuclear lens protein complex of a-, p- and y-crystallins, after reaction with an anti-a-crystallin immunoadsorbent, yielded filterable p- and y-crystallins. The anti-or-crystallin immunoadsorbent could not have had any direct influence on thep- or y-crystallins. Thus, these results strongly indicate that these crystallins initially did not filter through an XM-300 membrane because of their interaction with oc-crystallin rather than because of self aggregation, which might have led to the formation of ,L3-and y-macromolecules of high molecular weight. When an anti-p-crystallin immunoaclsorbent was used, a filterable y-crystallin was liberated from the complexes, indicating that the y-crystallin in these complexes is bound directly to P-crystallin but not to cx-crystallin. This P-crystallin may play a potentially crucial role in lens protein complex formation. The specific dissociation of the protein complexes by antibody immunoadsorbents may be caused by the antibody acting as a competitive ligand for the complexed c/.or P-crystallin, the antibody apparently having an affinity for the specific crystallin which is stronger than the affinity which binds the crystallins into complexes. The specific dissociation may also be caused by conformational changes of the antigen when it is bound to antibodies (Kabat, 1976). These conformational changes may, in turn, cause a dissociation of the bonds involved in the complex formed by the antigen. Age differences were explored by comparing protein-protein complexes from the cortical part of lenses from young animals with the nuclear part of the lenses of mature animals (Pirie and van Heyningen, 1956). When gel chromatography of the non-filterable calf cortical fraction was done, free M-crystallin was eluted first, and was thus shown to have a higher molecular weight than the eomplexed oc+crystallin. An a-crystallin is formed by the association of two SH-containing subunits and one XH-free subunit, forming a molecule of ca. 60 000 daltons. This primary oc-crystallin evidently undergoes self aggregation, forming large macromolecules from hundreds of thousands to millions of daltons. The heavy weight population of cc-crystallins increases with age. For some cx-crystallin molecules~ self aggregation which can be looked upon as homologous complexing apparently becomes interrupted early by heterologous complexing, leading to the formation of macromolecules with lower weights than those produced by homologous complexing. Comparison of lens protein complexes involving cr-crystallins in young and mature animals indicates that complex formation is a continuous process which starts early between part of the v.- and /3-crystallins. In mature lenses, all of the cc-crystallin was found to be complexed, forming at least two types of E-P-~ macromolecules, having different amounts of /3- and y-crystallins. The complex, which has a higher p- but a lower y-crystallin content and comes out in the first gel chromatographic fraction, may thus represent a transitional form between the early K-p-crystallin
BLPHA
CRYSTALLIN
635
COMPLEXES
complex found in the young calf cortical lens tissue and the complex isolated in the second gel chromatographic fraction of the mature bovine nuclear lens preparation. In lenses from young animals, the presence of y-crystallins in the protein complexes extracted w&h water but not in those extracted with buffer is a strong indication of dependence between the formation and stability of the complexes and their ionic environment. In Ienses from mature animals, the composition of the protein complexes was found to depend very little on the ionic composition of the solvent used. The occurrence of separate crystallin lines in the immunoelectrophoresis of a lens protein complex is also a reflection of the protein complexes instability in an electrical field. The relative stability of the various lens protein complexes is the subject of a separate investigation (Malinomiski and Manski, in press). SCKKOWLEDGRLENTS
This inI fest.igation was supported by U.S.P.H.S. Research Grant EY 00189. RsEE’ERENCES Cuatracasas, P. and Anfinsen, C. 13. (1971). Affinity chromatogmphy. In Methods of Enzyrno2ogy. Vol. XXII, pp. 345-78. Academic Press, London, New York. Francois, J., R,abaey, M. and Wieme, R. J. (1955). New method for fractionation of lens proteins. Arch. Ophthalmol. 53, 481-6. Harding, J. J. and Dilley, K. J. (1976). St ructural proteins of the mammalian lens: A review with emphasis on changes in development, aging and cataract. Exp. Eye Res. 22, l-73. Herbrink, P., van Westreenen, H. and Bloemendal, H. (1975). Further studies on the polypeptide chains of P-crystallin. Exp. Eye Res. 20, 541-8. &bat, E. A. (1976). Structural Concepts in Innmzutzology and Ina~uunochenLstry. Pp. 151-2. Holt, Reinehart and Winston, New York. Kalckar, H. M. (1947). Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem. 167, 461. Kuck, J. F. R. (1970). Chemical constituents of the lens. In Biochemistry of the Eye. (Ed. Graymore, C. N.). Pp. 183-261. Academic Press, London, New York. Malinowski, K. a,nd Manski, W. (1977). Antibody response and subunit structure of calf lens c(crystallin. lmmu7~ochemistry 14, 603-9. Malinowski, K. and Manski, W. (1980). On the stability of the protein-protein complexes in the lens. Exp. Eye Res. (In press). Manski, W., Behrens, H. and Martinez, C. (1968). I mmunochemical studies on albuminoid. 1. Exp. Eye Res. 7,164-71. Manski, W., Halbert, S. P. and Auerbach, T. P. (1961). Immunochemical analysis of lens protein separations. Arch. Biochenz. Biophys. 92, 512-24. Manski. W. and Malinowski, K. (1978). The evolutionary sequence and quantitative content of different antigenic determinants of calf lens z-crystallin. Immunochemistry 15, 1781-6. Manski, W. and Martinez, C. (1971). Immunochemical studies on aibuminoid. II. Changes associated with age. Ezp. Eye Res. 12,206-11. Morner, C. T. (1894). Untersuchung der Proteinsubstanzen in den Leichtbrechenden Medien des Auges. 2. Physiol. Chem. 18, 61-106. Orzalesi, F. and Miglior, M. (1957). Sur les interactions entre Proteines Lenticulaires et autres Proteines. Premier mote sur la Possibilite des Precipitations fraction&es des Proteines Lenticulaires au moyen des Proteines Electropositives. Arch. Ophthalmol. 17, 752. Pirie, 9. and van Heyningen, R. (1956). Biochemistry of the Eye. C. C. Thomas, Springfield. Scheidegger, J. J. (1955). Une micro-method de l‘immunoelectrophorese. Int. Arch. Allergy AppZ. Immunol. 7, 103. Spector, A. (1964). Methods of isolation of a-, p- and y-crystallins and their subgroups. Invest. Ophtfdmol.
3, ISZ-93.
Woods, 4. C. and Burky, production of cataract.
E. L. (1933). The Am. J. Ophthalmol.
possible influence 16,951-61.
of immunological
factors
in the
Immunochemical Studies on Lens Protein-Protein Complexes I. The Heterogeneity and Structure of Complexed a-Crystallin W.
MANSKI,
K.
NALIITOWSKI
AND
G.
BOXITSIS
Departments of Microbiology and Ophthalmology, Collegeof Physicians Columbia University, New York, N.Y. 10032, U.X.A.
and Surgeons,
(Received8 March 1979, &SewYork) A buffered extract of young cattle lenses filtered through Diaflo XM-300 membranes, retaining molecules above 300 000 daltons, was found to contain in the non-filterable fraction constituting 50% of soluble lens proteins cc-and /%crystallins. The non-filterable fraction of soluble proteins from mature cattle lenses, constituting 87% of all crystallins, contained not only cc- and P-crystallins, but y-crystallins as well. Only cc-crystallin, with a molecular weight above 300 000 daltons, was expected to remain in the non-filterable fraction. All of the ,kcrystallins, which have a molecular weight below 200 000 daltons, and y-crvstallins, with a molecular weight of about 20 000 daltons, if free in solution and not complexed, were expected to be in the filtrate. The lens aroteins with a molecular weight above 300 000 daltons from young lens tissue were separaied by gel chrcmatography (l%o-Rad A 15 m) into free cc-erys~allinufollowed by an oc-/?-crystallin complex. By the same procedure, the prot,eins above 300 000 daltons from mature lenses were separated into two complexes of quantitatively different z-fl~crystallin composition. A dependence of lens protein complex formation on the ionic composition of the solvent was observed. The differences bet.ween calf cortical and bovine nuclear complexes, found in buffer extracts, were much less pronounced in water extracts in which both contained y-crystallins. Specific dissociation of the lens protein complexes was obtained by use of an anti-acrystallin immunoadsorbent, as shown by the passage of the previously retained /3- and ycrystallms &rough a Diaflo XM-300 membrane. An anti-/3-crystallin immunoadsorbent reacting with complexes from mature lens tissue yielded a non-filterable a-crystallin and a filterable y-crystal&n in the supernatant, indicating an absence of direct a+ complexes. These results point to a potentially central role of /z-crystallins in complex formation among lens proteins. Key lords: calf lens; bovine lens; lens crystallins; lens protein-protein complexes; heterogeneity of complexed a-crystallin; structure of complexed lens crystallins; protein interaction/salt concentration dependence.
1. Introduction An immunochen~ical approach is being used in a series of investigations on the occurrence of interactions between different protein molecules synthesized within the lens epithelial cell. Such investigations were designed to determine whether such interactions lead to the formation of protein-protein complexes of different composition and stability and whether or not such interactions are dependent on the age of the cell in which they occur. The lens provides a uniquely appropriate model for the study of these particular problems. This tissue is formed from one kind of ectodermal cell, functioning like a monoclonal cell culture in vivo and in the aging process of the lens, older cells are not shed but persist compressed into the center of the lens (Pirie and van Heyningen, 1956). OO14-4835,09/1206%+11
0
SOLOO/ 625
1979 Academic
Press Inc. (London)
Limited
626
W.
3IAXSK1,
K. &fALISOWGKI
A?u’D
G. RONITSIS
Both lens protein fractions, obtained on extraction, the soluble crpstallins, the insoluble albuminoid (Kuck, 1970) have antigenically the same crystallin composition, as shown by the complete cross-absorption of anti-crystallin antiserum by the albuminoid fraction, and vice versa (Manski, Behrens and Martinez, 1968 ; Manski and Martinez, 1971). However, the relative amount of the various proteins was shown in these reports to be different in the crystallin and albuminoid fractions. Immunoelectrophoretic analysis of the crystallins demonstrates the presence of approximately 12 different proteins (Manski, Halbert and Auerbach, 1961). The main structural protein of the lens, a-crystallin, constitutes about 30% of all lens proteins and is characterized by its high molecular weight exceeding 300 000, averaging 800 OOO1000 000 daltons. The molecular weight of the different /3-crystallins falls within a range of 20 000 to 200 000 daltons. The molecular weight of y-crystallins is 20 000 daltons. A review of these different structural proteins of the mammalian lens was published by Harding and Dilley (1976). Underlying research on lens proteins, from the early work of Morner (1894) to recent studies, has been the implicit assumption that the different pre-a-, /3- and y-crystallins are present in the lens in a free state. It is one of the aims of this report to demonstrate that formation of complexes involving or-crystallin does occur in the lens and that this formation progresses with age. Another aim of this report is to gain insight into the heterogeneity and structure of such complexes. 2. Materials
and Methods
Dissecticw of calf lens cortex and bovine lens wucbezu
Immediately after slaughter, lenseswere removed from 336 month old calves and from cattle about 24 months old, Carewastaken to free the lensesfrom capsular,vitreous and aqueous material. The clean lenses were immediately frozen. The equatorial section of the frozen lenses was removed wit’h a stainless steel cork borer of 12 mm id. in the case of the calf lenses and 14 mm i.d. in the case of the bovine lenses. The inner nuclear part of the remaining lens (about 30%) was separated from the outer cortical part (about 38%) wit,h a 7 mm i.d. stainless steel cork borer for calf lenses and a 9 mm id. borer for the bovine lenses. Isolation
of the cx-crystallin
j%action
The cortical portion of lenses from 3-6 month old calves was homogenized and extracted at 4°C with PBS. The insoluble albuminoid and cryoprotein fractions were removed by centrifugation at 18 000 x g for 30 min at 4°C in a Sorvall centrifuge with an SS-34 rotor (Ivan Sorvall, Inc., Norwalk, Conn.). Alpha crystallin was isolated from the soluble fractions by repeated isoelectric precipitation at pH 5.0 (Francois, Rabaey and Wieme, 1955), followed by ultrafiltration through an XM-300 Diaflo membrane (cut-off point 300 000 daltons) (Amicon Corp., Lexington, Mass.), as described in a previous report (Malinowski and Manski, 1977). Isolation
of the P-csystallin fractiolz
The procedure used is a modification of the procedures reported by Herbrink, van Westreenen and Bloemendal(l975) and by Spector (1964). After the removal of cc-crystalhn by isoelectric precipitation (pH 5.0), the supernatant was equilibrated with a solution of O-1 &I-Tris, 1 M-NaCl and O-005 r+EDTA, pH 7.5, then applied to a Bio-gel A 0.5 m column (5-O x 44.0 cm) equilibrated with the same buffer. The third fraction eluted from the column was concentrated and equilibrated with a buffer 0.005 M-Tris-Hcl and 0,005 M-
ALPHA
CRYBTALLIIZ-
COVPLEXES
62i
KCI, pH 7.3, then applied to a Sephadex G-50 column (50 x 44.0 cm) equilibrated with the same buffer. The second of the four chromatographic fractions contained ,&crystallins free from Z- and y-crystallins. This was determined by immunoelectrophoretic analysis in which the isolated P-crystallin fraction was reacted with rabbit anti-total cattle lens protein antiserum. bsolatio~
of has u-crystal&n
conzplexes
by ultrajltration
dmicon ultrafiltration cells with X31-300 Diaflo membranes were used for the separation of lens proteins based on their different molecular weights. According to the manufacturers, the retention capacity of these ultrafilters is approximately 98%. Twenty g of calf lens cortex or bovine lens nucleus were homogenized at 4°C in a N.Y.) with 100 ml of an 0.01 nr-‘I&-WC1 buffer, homogenizer (Virtis Inc., Gardiner, pH 7.2, containing 0.09 ~-Kc1 and 0.05 x-NaCl. These salts approximate the intracellular ionic environment of lens proteins in vivo. The insoluble albuminoid fraction was removed by centrifugation at 18 OOOxg for 30 min at 4°C in a Sorvall centrifuge. The soluble fraction was filtered at 4°C in an Amicon cell with a Diaflo XM-300 membrane under 20 psi of nitrogen pressure, while a constant volume was maintained by the addition of the same Tris-buffered salts. Filtration was continued until the absorba,nce of the filtrate at 280 nm was below 0.02 o.d., approximately after 4 days. The non-filterable fraction was concentrated to 20 mg/ml on the same X31-300 Diaflo membrane. The filterable fract.ion, in turn, was concentrated in an Amicon cell using a PM-10 Diafio membrane (cut-off point 10 000 daltons). In another procedure, 20 g of calf lens cortex or bovine lens nucleus were homogenized wirlth 100 ml of distilled water. The tissues were treated thereafter in the same manner as the buffered preparation, except that constant volume was maintained during filtration by the addition of distilled water instead of buffer. Iunwa7cn~e sera
Adult chinchilla rabbits weighing 5-6 lb each were immunized by the subcutaneous injection of 5 mg of a cattle lens homogenate in complete Freund’s adjuvant (&I. butyricum, Difco Labs., Detroit, JIich.) at five separate body locations. The first three immmizations were administered at a-week intervals. Subsequent immunizations were administered at monthly intervals. The immune sera were collected under sterile conditions after about ten immunizations. Isolation
of anti-a-
or anti-/!-crptalh
antibodies
Anti-K-crystallin antibodies were isolated from rabbit anti-total calf lens protein antiserum by mixing 500 ml of rabbit antiserum with 30 g of a calf crystallin immunoadsorbent containing 510 mg of bound calf cc-crystallin. Mixing was carried out overnight, at room temperature. Similarly, the anti-p-crystallin antibodies were isolated by reacting 500 ml of rabbit anti-total calf lens protein antiserum (after remova of anti-u-crystal&n antibodies) with 30 g of a P-crystallin immunoadsorbent conta,ining 390 mg of bound ,/3-orystallin. The immunoadsorbent suspensions were filtered and washed on a Buchner funnel with PBS until the absorbance at 280 nm was lower than 0.02 o.d. For the desorption of bound antibodies, the washed immunoadsorbents were mixed for 10 min with 50 ml of 0.5 wCH,COOH and then filtered. The desorption procedure was repeated three times. The collected filtrates were immediately brought to pH 7.2 by the addition of 0.1 M-XaOH. The amount of isolated anti-calf oc-crystallin antibodies was 98.7 mg; the amount of isolated anti-@rystallin antibodies was 69.5 mg. The purity of ihe isolated antibodies was tested by immunoelectrophoresis, using total cattle lens proteins.
W. MANSKI,
628
Prepwatiorz
PC. MALIIYOWSKI
AYD
G. BONITSIS
of irnnaunoadsorbents
The a- and P-crystallin immunoadsorbents were prepared according to the method of Cuatracasas and Anfinsen (1971), as described in a previous report (Manski and Malinowski, 1979). Approximately 17 mg of cc-crystallin and 13 mg of fi-crystallin were bound per gram of Sepharose 4B gel (Pharmacia Fine Chemicals, Uppsala, Sweden). For preparation of the antibody immunoadsorbents, the desorbed antibodies were equilibrated with 0.1 iv-NaHCO,, pH 8.1, in an Amicon filtration cell with a P%lO Diaflo membrane (cut-off point 10 000 daltons). The isolated anti-cc- or anti-P-crystallin antibodies were coupled separately to activated Sepharose by Cuatracasas and Anfinsen’s procedure, The yield of coupling was 8.2 mg of anti-calf cc-crystallin antibodies and 67 mg of ant,i+crystallin antibodies per gram of Sepharose 4B gel. Gel chromatography
of lens protein
complexes
For the procedure, 0.5 g of the non-filterable lens proteins in 10 ml of a 0.01 3r-TrisHCl buffer, pH 7.2, solution containing 0.09 ~-Kc1 and 0.05 M-NaCl, was applied to a Bio-gel A 15 m agarose column (Bio-Rad Labs., Richmond, Calif.) equilibrated with the same buffer. The flow rate of the elution buffer was 1 ml per min. Separation of proteins into fractions was monitored on a multi-channel spectrophotometer (Gilford Instrument Labs., Inc., Oberlin, Ohio). Xpec$c
dissociation
of fpvotei+~rotein
complexes
Sixty mg of the non-filterable bovine nuclear lens protein in 60 ml of salt-containing Tris buffer with 0.02% NTahT,, were added to 36 g of an immunoadsorbent with 295 mg of bound anti-calf a-crystallin antibodies or with 241 mg of bound anti-calf j-crystallin antibodies. The complexes and immunoadsorbents were mixed in a roller culture apMillville, N.J.) for 72 hr at room temperature. The paratus (1Vheaton Instruments, immunoadsorbents were then separated by filtration through sinter glass funnels. The proteins not bound to the immunoadsorbents were washed out with the salt-containing Tris buffer. The filtrates were combined and concentrated, using a UM-2 Diaflo membrane (cut-off point 2000 daltons). The proteins bound to the immunoadsorbents were desorbed in five 25ml portions of 0.5 M-CH,COOH. neutralized with Th e acid was immediately 0.1 r;-NaOH. Both the filtrates and the desorbed solutions were dialyzed against the above Tris buffer, using UM-2 Diaflo membranes; then concentrated to 20 mg of protein per ml.
The method used is based on the micro-technique of Scheidegger (1955). Agar-coated slides were covered with a 1.5% Bacto gel agar (Difco Labs., Detroit, Mich.) in 0.015 MTris-Na, EDTA-boric acid buffer, pH 8.4. The distance between the antigen hole and the antiserum well was 3 mm. The volume of the antiserum was 0.25 ml. The volume of the antigen was 0.02 ml and its concentration 20 mg/ml. A potential of 5 V/cm across the slide was applied for 90 min. The immunoelectrophoretic patterns were developed at 4°C and observed for 7 days. Prot&
determination
Absorption at wavelengths of 260 and 280 nm was measured in a DU-2 spectrophotoCalif.). The protein concentration was meter (Beckman Instruments, Inc., Fullerton, calculated according to the formula: protein in mg/ml = 1.45 E,,,-0.74 E,,, (Kalckar, 1947).
ALPHA
CRYSTALLIN
COXPLEXES
629
3. Results Occuwence of lens protein-protein
complexes
When the soluble calf cortical lens proteins in a salt-containing Tris buffer were fiitered through an XM-300 membrane: these proteins were found to contain 500/ of a non-filterable fraction. The non-filterable fraction of bovine nuclear lens proteins in the same buffer was 87%. When the lens extracts were prepared in water, the nonfilterable fraction of the calf cortical lens proteins increased significantly, to il)i/,. The corresponding fraction of bovine nuclear lens proteins in water was SS%, practically unchanged from the amount obtained in buffer. All the fractions were analyzed by immunoelectrophoresis. The non-filterable fraction of the calf cortical lens proteins (Fig. 1, column a) in buffer (molecular weight above 300 000 daltons) contained not only an alpha but also /Scrystallin lines. By comparison, the non-filterable fraction of bovine nuclear iens proteins in the same buffer (Fig. 1, column b) contained at least two /I-crystallin lines and, in addition, two y-crystallin lines. The immunoelectrophoretic pattern of a non-filterable calf cortical fraction obtained by water extraction (Fig. 1, column c) was more complex than the one obtained by buffer extraction, showing a greater Gmilarity to the pattern of the corresponding water-extracted non-filterable bovine nuclear fraction (Fig. 1: column d). The non-filterable fractions of the bovine nuclear lens proteins extracted by buffer and by water showed no pronounced differences in composition. The presence of proteins other tha.n a-crystallin in the non-filterable fractions was confirmed by comparison of the multiple lines formed by anti-total lens crystalhn antiserum with the single lines developed in immunoelectrophoresis with anti-acrystallin antiserum (Fig. 1, second row)] hens protein
fraction
Calf cortex
(20
mg/ml)
Bovine
with
o molecular
weight
above
300
000
doltons
Calf cortex
nucleus
Bovme
nucleus
In water
In buffer (b)
(0)
(d)
(cl
AM-total
lens protein
a
ant&serum *
I& I Anti-a-crystolhn
Lens protein
fraction
-
-lu
_o.
0
(20
mg/ml)
wih
o molecular
lens protein
a nmlecular
weight
above
and
below
300
000
daltons
antiserum
IO Ant!-a-crystallin
FIG. 1. Irnnrunoelectrophoretic
weight
c
Anti-total
with
antiserum
analysis below
0
antiserum
of the calf cortical 300 000 daltons.
and
bovine
nuclear
lem
erystallin
fraction
630
W. MAXSKI>
K. MALISOWSKI
AND
G. BONITSIS
Immunoelectrophoretic analysis of the filterable fraction of calf cortex in buffer (molecular weight below 300 000 daltons) showed the presence of both ,& and ycrystallins (Pig. 1, column a, third row). In addition, some .z-crystallin was also present in this filterable fraction, possibly indicating that the cut-off point of the membranes is somewhat higher than indicated, or that there are some cc-crystallin molecules in young lens tissue which have a molecular weight lower than 300 000 daltons. Certainly, this result excludes the possibility that the pore size of the filters is smaller than indicated which would have alIowed retention of some ,& or even some y-crystallins. In the buffer extracted bovine nuclear lens proteins, both /3- and y-crystallins were found in the filtrate (Fig. 1, column d, third row). By comparison9 the filtrate of bovine nucIear Iens crystallins extracted in water showed the presence of beta lines (Fig. 1, column b, third row). It can thus be concluded that in a water extract all the bovine nuclear y-crystallins must have been complexed. Heterogeneity of lens protein-protein
complexes
Pigure 2 shows that the non-filterable fraction obtained by buffer extraction of CrystalEns from a calf cortical lens preparation separated into two peaks by gel chromatography. Peak 1 contained 2l+3o/oof the total protein collected and peak 2 contained 78.2%. By comparison, the corresponding buffer-extracted bovine nuclear fraction (Fig. 3) separated into three peaks, with peak 1 containing 226% of the total protein collected; peak 2 containing 6.2%; and peak 3 containing 15.4%. The gel
400
800
1200
1600
2000
ml FIG. 2. Gel chromatographic analysis on a Bio-gel A 15 m column lens protein fraction non-filterable through an X&I-300 membrane.
(50 x 44.0 cm) of the calf cortical
ALPHA
CRYSTALLIN
400
COxPLEXE8
800
1200
631
I600
2000
ml E'IG. 3. Chromatographic analgsis on a Bio-gel A 15 m column Iens protein fraction non-filterable through an XM-300 membrane.
(50 x44.0
nuclear
Concentrotlon of antigens hng/ml)
Peak 2
Peak I
cm) of the bovine
10-o
_o,,
0 .__._. - -____-0 FIG. 4. Immunoelectrophoretic ilterable calf cortical lens protein
2.5
-
0 titration fraction).
of the chromatographic
0.6
0.3
fractions
shown
in Fig.
2 (non-
W. bUNSKI,
632
K. iUALIP\‘OWSKI
AND
G. BOSITSIS:
chromatographic analyses shown in Figs 2 and 3 were done under the same experimental conditions, The fractions marked by the shaded areas were collected, concentrated and analyzed by immunoelectrophoresis. The photographs of the patterns obtained for each peak are shown in Figs 2 and 3. Their dia.grammatic presentation is given in the titration experiments, presented in Figs 4 and 5. As can be seen in Fig. 4, peak 1 obtained by gel chromatography of the nonfilterable calf cortical lens protein fraction showed the presence of cr-crystallin only. By comparison, peak 2, consisting of molecules of lower weight, contained both c(- and p-crystallins. Th’IS was confirmed by the immunoelectrophoretic reaction of peak 2 with anti-or-crystallin antibodies, which then showed a single line of cr-crystallin.
Peak
Peak
I
Peak 3
2
b
_o,
b
AL-
o
0,
0
_o I_0
0,
FIG.
5. Immunoelectrophoretic titration of the bovine nuclear lens protein fraction).
chromatographic
5.0
--
2.5
-
1.2
-
0
0
.0..... .___“. filterable
-
0
--------
Concentration of ontlgens hg/m
0.6
-.__.. fractions
shown
in Fig.
o-3
3 (non-
There was no separation of free from complexed or-crystallin in the first two gel chromatographic peaks obtained from the non-filterable bovine nuclear lens proteins in buffer (Fig. 5). Immunoelectrophoretic analysis demonstrated in both the presence of cc- and P-crystallins as well as y-crystallins. Peak 3, however, contained only p- and y-crystallins. Th’1s f raction was filterable through an X$l-300 membrane, indicating that these crystallins must have been only weakly bound to the more stable ~-p-y complexes in the initially non-filterable fraction. The
structure
of lens
protein-protein
complexes
Analysis of the structure of the lens crystallin complexes was based on their reaction with specific antibodies to 01- or P-crystallin bound to matrices. Specific dissociation of the complexes by the antibodies was tested in the supernatants after removal of the immunoadsorbent. Immunoelectrophoresis was done both before and after ultrafiltration of the supernatants through XM-300 Diaflo filters. These analyses are illustrated by the reactions of the lens protein complex in the gel chromatographie peak 2 of the non-filterable bovine nuclear lens proteins (Fig. 5).
SLPHA
CR.YSTALLIN
633
COMPLEXES
As can be seen in Fig. 6, all p- and y-crystallins were observed in the supernatant after exposure to an anti-or-crystallin immunoadsorbent (Fig. 6-2), and all of them were filterable through an X&I-300 membrane (Fig. 6-3). The complete dissociation of the initial lens protein complex was proven by the fact that the protein desorbecl from the immunosdsorbent (Fig. 6-4) was a-crystallin and not an a-crystailin complex.
,-
3-
FIG. 6. Specific dissociation of lens protein complexes by anti-cc-crystallin antibodies. 1: Bovine nuclear lens protein oomplex before reaction with an anti-calf cc-crystallin immunoadsorbent 2: Bovine nuclear lens protein complex after reaction with an anti-calf cr-crystallin immunoadsorbent. 3: Lens proteins in the reaction mixture filterable through an XlW300 membrane. 4: Lens proteins desorbed from the anti-cc-crystallin immunoadsorbent.
,-so
5-
FIG. 7. Specific dissociation of lens protein complexes by anti-F-crystallin antibodies. 1: Bovine nuclear lens protein complex with a molecular weight above 300 000 daltons before reaction with an anti+crystallin immunoadsorbent. 2: Bovine nnclear lens protein complex with a molecular weight shove 300 000 daltons after reaction with an anti-P-crystallin immunoadsorbent. 3 : Lens proteins in the reaction mixture filterable through an XM-300 membrane. 4: Lens proteins in the reaction mixture nonfilterable through an XM-300 membrane. 5: Lens proteins desorbed from the anti-/%crystallin immunoadsorbent.
The same bovine nuclear lens protein complex (Fig. ‘7-l) was also exposed to an excess of an anti-/Scrystallin immunoadsorbent. After centrifugation immunoelectrophoresis of the supernatant of the reaction mixture (Fig. 7-2) demonstrated the presence of 01- and y-crystallins. The y-crystallins were completely filterable through an XM-300 filter (Fig. 7-3). Alph a- cr y,st a11’m was retained on the membrane (Fig. ‘i-4). Only P-crystallins were desorbed from the immunoadsorbent (Pig. 7-5).
4. Discussion There are only a few references in the literature which point to potential proteinprotein complex formation in the lens. Woods and Burky (1933) studied the role of cha.nged ratios of or-crystallin to /3-crystallin in cataract formation. These authors observed that addition of a-crystallin prevented the spontaneous precipitation of pcrystallin in solution. They also noted that a-crystallin in solution precipitated on the addition of CaCl, at a concentration of approximately 9 mg percent. In the presence of ,E-crystallin, a higher concentration of CaCl,, about half of saturation, was required to cause precipitation of cx-crystallin.
634
Vi’. MAn-SKI,
Ii. MALINOWSKI
AKD
G. BONITSIS
The ability of oc-crystallin to react with some non-lens proteins was studied by Crzalesi and Miglior (1957). Using electrophoretic methods, cc-crystallin was found to form stable complexes with strongly electropositive molecules such as protannnes. Proof of complex formation between cc-crystallin and other lens protein rests, in OLW experiments, on the following observations : (1) that the calf cortical lens proteins which did not filter through a membrane having a cut-off point of 300 000 daltons contained not only a-crystallin but also ,&crystallin, which has a molecular weight of no more than 200 000 daltons, and (2) that the corresponding bovine nuclear fraction contained not only ,8-crystallin, but also y-crystallin, which has a molecular weight of only about 20 000 daltons. The non-filterable bovine nuclear lens protein complex of a-, p- and y-crystallins, after reaction with an anti-a-crystallin immunoadsorbent, yielded filterable p- and y-crystallins. The anti-or-crystallin immunoadsorbent could not have had any direct influence on thep- or y-crystallins. Thus, these results strongly indicate that these crystallins initially did not filter through an XM-300 membrane because of their interaction with oc-crystallin rather than because of self aggregation, which might have led to the formation of ,L3-and y-macromolecules of high molecular weight. When an anti-p-crystallin immunoaclsorbent was used, a filterable y-crystallin was liberated from the complexes, indicating that the y-crystallin in these complexes is bound directly to P-crystallin but not to cx-crystallin. This P-crystallin may play a potentially crucial role in lens protein complex formation. The specific dissociation of the protein complexes by antibody immunoadsorbents may be caused by the antibody acting as a competitive ligand for the complexed c/.or P-crystallin, the antibody apparently having an affinity for the specific crystallin which is stronger than the affinity which binds the crystallins into complexes. The specific dissociation may also be caused by conformational changes of the antigen when it is bound to antibodies (Kabat, 1976). These conformational changes may, in turn, cause a dissociation of the bonds involved in the complex formed by the antigen. Age differences were explored by comparing protein-protein complexes from the cortical part of lenses from young animals with the nuclear part of the lenses of mature animals (Pirie and van Heyningen, 1956). When gel chromatography of the non-filterable calf cortical fraction was done, free M-crystallin was eluted first, and was thus shown to have a higher molecular weight than the eomplexed oc+crystallin. An a-crystallin is formed by the association of two SH-containing subunits and one XH-free subunit, forming a molecule of ca. 60 000 daltons. This primary oc-crystallin evidently undergoes self aggregation, forming large macromolecules from hundreds of thousands to millions of daltons. The heavy weight population of cc-crystallins increases with age. For some cx-crystallin molecules~ self aggregation which can be looked upon as homologous complexing apparently becomes interrupted early by heterologous complexing, leading to the formation of macromolecules with lower weights than those produced by homologous complexing. Comparison of lens protein complexes involving cr-crystallins in young and mature animals indicates that complex formation is a continuous process which starts early between part of the v.- and /3-crystallins. In mature lenses, all of the cc-crystallin was found to be complexed, forming at least two types of E-P-~ macromolecules, having different amounts of /3- and y-crystallins. The complex, which has a higher p- but a lower y-crystallin content and comes out in the first gel chromatographic fraction, may thus represent a transitional form between the early K-p-crystallin
BLPHA
CRYSTALLIN
635
COMPLEXES
complex found in the young calf cortical lens tissue and the complex isolated in the second gel chromatographic fraction of the mature bovine nuclear lens preparation. In lenses from young animals, the presence of y-crystallins in the protein complexes extracted w&h water but not in those extracted with buffer is a strong indication of dependence between the formation and stability of the complexes and their ionic environment. In Ienses from mature animals, the composition of the protein complexes was found to depend very little on the ionic composition of the solvent used. The occurrence of separate crystallin lines in the immunoelectrophoresis of a lens protein complex is also a reflection of the protein complexes instability in an electrical field. The relative stability of the various lens protein complexes is the subject of a separate investigation (Malinomiski and Manski, in press). SCKKOWLEDGRLENTS
This inI fest.igation was supported by U.S.P.H.S. Research Grant EY 00189. RsEE’ERENCES Cuatracasas, P. and Anfinsen, C. 13. (1971). Affinity chromatogmphy. In Methods of Enzyrno2ogy. Vol. XXII, pp. 345-78. Academic Press, London, New York. Francois, J., R,abaey, M. and Wieme, R. J. (1955). New method for fractionation of lens proteins. Arch. Ophthalmol. 53, 481-6. Harding, J. J. and Dilley, K. J. (1976). St ructural proteins of the mammalian lens: A review with emphasis on changes in development, aging and cataract. Exp. Eye Res. 22, l-73. Herbrink, P., van Westreenen, H. and Bloemendal, H. (1975). Further studies on the polypeptide chains of P-crystallin. Exp. Eye Res. 20, 541-8. &bat, E. A. (1976). Structural Concepts in Innmzutzology and Ina~uunochenLstry. Pp. 151-2. Holt, Reinehart and Winston, New York. Kalckar, H. M. (1947). Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem. 167, 461. Kuck, J. F. R. (1970). Chemical constituents of the lens. In Biochemistry of the Eye. (Ed. Graymore, C. N.). Pp. 183-261. Academic Press, London, New York. Malinowski, K. a,nd Manski, W. (1977). Antibody response and subunit structure of calf lens c(crystallin. lmmu7~ochemistry 14, 603-9. Malinowski, K. and Manski, W. (1980). On the stability of the protein-protein complexes in the lens. Exp. Eye Res. (In press). Manski, W., Behrens, H. and Martinez, C. (1968). I mmunochemical studies on albuminoid. 1. Exp. Eye Res. 7,164-71. Manski, W., Halbert, S. P. and Auerbach, T. P. (1961). Immunochemical analysis of lens protein separations. Arch. Biochenz. Biophys. 92, 512-24. Manski. W. and Malinowski, K. (1978). The evolutionary sequence and quantitative content of different antigenic determinants of calf lens z-crystallin. Immunochemistry 15, 1781-6. Manski, W. and Martinez, C. (1971). Immunochemical studies on aibuminoid. II. Changes associated with age. Ezp. Eye Res. 12,206-11. Morner, C. T. (1894). Untersuchung der Proteinsubstanzen in den Leichtbrechenden Medien des Auges. 2. Physiol. Chem. 18, 61-106. Orzalesi, F. and Miglior, M. (1957). Sur les interactions entre Proteines Lenticulaires et autres Proteines. Premier mote sur la Possibilite des Precipitations fraction&es des Proteines Lenticulaires au moyen des Proteines Electropositives. Arch. Ophthalmol. 17, 752. Pirie, 9. and van Heyningen, R. (1956). Biochemistry of the Eye. C. C. Thomas, Springfield. Scheidegger, J. J. (1955). Une micro-method de l‘immunoelectrophorese. Int. Arch. Allergy AppZ. Immunol. 7, 103. Spector, A. (1964). Methods of isolation of a-, p- and y-crystallins and their subgroups. Invest. Ophtfdmol.
3, ISZ-93.
Woods, 4. C. and Burky, production of cataract.
E. L. (1933). The Am. J. Ophthalmol.
possible influence 16,951-61.
of immunological
factors
in the
