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Purification and crystallization of mammalian lens γ-crystallins
Ezp.
Eye
Res.
(1983)
37,
urification
517-530
and Crystallization y-Crystallins C. SLINCSBY
Department
of Crystallography,
(Received 23 March
AND
Birkbeck London,
of Mammalian
Lens
L. R. MILLER
College, University WClE 7HX
of London,
Malet
Street,
1983 and accepted 25 May 1983, London)
Monomeric crystallins, ps and several members of the y-crystallin family, occw in different relative proportions in a way which is related to the age of the lens cells. Methods of purification of large amounts of these different low molecular weight protein components from young and old bovine lenses are described along with details of crystallization of several y-fractions. Purification procedures have been developed for rabbit y-crystallins. The chromatographic methods achieve separation of several bovine and rabbit y-crystallins which have very similar electrophoretic mobilities. However, on storage, many electrophoretic variants are generated from some of these fractions. Key words: y-crystallin; /Is-wystallin; crystals; ageing of y-crystallin.
1. Introduction Eye lenses contain high concentrations of proteins in order to achieve the required refractive index necessary for their function. The protein concentration is not uniform throughout a particular lens, it is highest in the central regions although the concentration gradient varies among species (Waley, 1969; Philipson, 1969; van Heyningen, 1972; Rink, 1978). A major component of the lens soluble crystallins is y-crystallin, a protein synthesized in early development and thus occurring predominantly in the central regions of adult lenses (Bjijrk, 1961, 1964; Papaconstantinou, 1965; Waley, 1969; Slingsby and Croft, 1973; Harding and Dilley, 1976; Bloemendal, 1981). In those animals which contain a steep gradient of refractive index and hence a relatively dehydrated lens core region, the y-crystallin must be associated with other erystallins and cell components in an environment of limited hydration. One approach to studying the macromolecular interactions responsible for maintaining a high refractive index is to investigate the structure of crystals of y-crystallin. An investigation of the proteins in their crystalline state not only allows the determination of their three-dimensional structure, but also yields information relating to their potential for interaction with additional protein and water molecules at a protein concentration similar to the lens core region. Single crystal X-ray diffraction analyses are now underway (Blundell et al., 1981; Chirgadze, Sergeev, Fomenkova and Oreshin, 1981). Crystallization of y-crystallins requires a large scale isolation and purification syst’em for the many closely related members of this family (Bjiirk, 1964; Croft, 1972; Slingsby & Croft, 1978). In this paper purification and crystallization methods of several bovine y-crystallins are described. As there is little protein turnover in the eye lens cells of the bovine core region (Wannemacher and Spector, 1968), the y-crystallin laid down in early development must perform its structural role as one of the components maintaining eye lens transparency in the adult core yet be able to adapt’ to an environment of increasing dehydration as the lens matures. The crystallins may become modified on ageing in 00144835/83/0105i7+13
$OS.OO/O
0
1983 Academic
Press Inc. (London)
Limited
C. SL,INGBBU
518
AND
L. R. MILLER
a manner which could effect their interactions with other molecules. In this paper an ageing effect is reported which could be attributed to chemical modification of specific y-crystallin fractions. Differences in the relative proportions of crystallins along the lens optical axis are probably related to the refractive index gradient. In the bovine lens monomeric y-crystallin is replaced by monomeric /&-crystallin (van Dam, 1966) in adult lens cortex. A further investigation of the differences in the relative proportions of the two monomeric crystallin classes was undertaken for adult cortical and calf lens extracts. Similarly the variation in the relative proportions of different members of the y-crystallin family between young and old lenses was explored. In order to explore further the variation in structural properties associated with y-crystallins, purification procedures have been developed for a different species, rabbit, together with observations of an ageing effect of one of the rabbit y-crystallin components. 2. Materials Puri$cation
of calf
lens
Calf lenses, weighing
am
y-esystallins
removed from the animal shortly after death decapsulated and stirred at 4“C in twice their volume of 005 M Tris-HCI buffer pH 7.2,0.02 o/0 NaN, and 0005 o/o (v/v) 2-mercaptoethanol. Lenses were completely disintegrated into a homogenate which was centrifuged at 17000 g for 1 hr at 1O’C. The resulting supernatant was subjected, in 40 ml portions, to gel filtration on Sephadex G-75 (Pharmacia), column size 1000 x 90 mm (diam) from which the low molecular weight protein fraction was isolated as described by Bjijrk (1961). Low molecular weight crystallins were concentrated from a volume of 70&20 ml by ultrafiltration in an Amicon cell equipped with a UMlO membrane. Separation of /3s-crystallin from y-crystallin was achieved using ion-exchange chromatography based on the method of van Dam (1966). DEAE-Sephadex A-50 (Pharmacia), equilibrated with 0.005 M sodium hydrogen phosphate buffer, pH 8.2, 601% NaN,, 0.005 y0 (v/v) 2- mercaptoethanol, was poured into a column 100 x 50 mm (diam). The protein solution, 500 mg in 26 ml, equilibrated in the same buffer was applied to the column and the y-crysta.llin was rapidly eluted. Alternatively, DEAEcellulose ion exchanger was used, equilibration and first elution being performed with 0.025 M Tris-HCl buffer pH 8.3 followed by elution of ps-crystallin with 605 M Tris-IX1 pH 8.0. y-Crystallin after concentration to 20 ml was immediately equilibrated with sodium acetate Separation buffer pH 50, ionic strength 02,902 y0 NaN,, 6005 ‘% (v/v) 2- mercaptoethanol. of the mixture of y-crystallins into fractions I, II, III, and IV was achieved on a cation exchanger, sulphopropyl-(SP)-Sephadex C-50 (Pharmacia), column size 500 x 35 mm (diam) using a linear salt gradient, ionic strength 0.2 --t 95, pH 50 according to the method of Bjiirk (1964). This size column will adequately resolve y-components up to a load of 1 g. y-Crystallin fraction III was resolved into two components, TIIa and IIIb, using ion-exchange chromatography on DEAE-cellulose. DE52 (Whatman) was equilibrated in O-05 M Tris-HCl buffer pH 9.0 containing 002 y0 NaN, and 0605 y0 (v/v) 2- mercaptoethanol and made into a column 500 x 13 mm (diam). y-Crystallin III (100 mg), previously equilibrated by dialysis against the same buffer, was applied to the column in a volume of 10 ml. Fraction IIIb elutes with this buffer. Fraction IIIa is eluted with 91 M Tris-HCl bullFer, pH 9.0. Alternatively the separation can be carried out with sodium hydrogen phosphate buffer pH 8.5, fraction IIIb being eluted with 0.05 M phosphate buffer with fraction IIIa subsequently eluted with 0075 x phosphate buffer, pH 8.5, containing 0.005% mercaptoethanol. Each protein fraction was dialysed against distilled water at 4°C and lyophilized. Fraction IV was chromatographed on a similar DE52 column, employing a linear gradient of 605 M Tris-HCl buffer pH 90 + @I M Tris-DC1 buffer pH 9.0, containing 002 y0 NaN, and9005 % (v/v) 2-mercaptoethanol. Fraction IV was also subjected to further purification using chromatofocusing. Fraction IV (50 mg) in a volume of 1 ml was equilibrated in 0.025 M Tris-acetate buffer pH 8.3, 002°/0 NaN, and 0.005 o/0 (v/v) 2-mercaptoethanol. Polybuffer exchanger PBETM94 (Pharmacia) was equiliand stored
below
- 20%.
about I g each, were Lenses were thawed,
yCRYSTALLIN
519
brated with the same buffer and poured to form a column 200 x 15 mm (diam.). Elution was TM96 (Pharmacia) diluted x 13 with distilled water and titrated performed with Polybuffer to pH 6 with acetic acid. y-Crystalline IVb is eluted first and incompletely resolved from IVa. Removal of the protein from the polybuffer was achieved by precipitation of the proteins in 80 o/o saturated ammonium sulphate. The precipitate was centrifuged, washed in saturated ammonium sulphate and redissolved in distilled water followed by dialysis into distilled water and lyophilization. Ageing
ejj’ect on c.alf y-crystal&n
fraction
IZI
Eight samples of calf fraction III were prepared as follows : (i) Fraction III, after sulphopropyl Sephadex chromatography, was left in acetate buffer, pH 5.0. (ii) Fraction III, in acetate buffer pH 50, was dialyzed for 24 hr in 605 A~ phosphate buffer, pH 8.5, 0095 y0 (v/v) 2-mercaptoethanol. (iii) Fraction IIIb, after isolation by DEAE-cellulose chromatography, was in phosphate buffer, pH 8.5; 0.005 o/0 (v/v) 2-mercaptoethanol. (iv) Fraction IIIb (150 nmol) from (iii) was treated with dithiothreitol (200 nmol). (v) Fraction IIIb from (iv) was dialyzed into 500 ml phosphate buffer, pH 7.0, 05 rnM in dit)hiothreitol. (vi) Fraction IIIa after isolation by DEAF-cellulose chromatography was in 0.075 M phosphate buffer, pH 85, 0.005 y0 (v/v) 2-mercaptoethanol. (vii) Fraction IIIa (75 nmol) from (vi) was treated with dithiothreitol (100 nmol). (viii) Sample (vii) was dialyzed into 005 M phosphate buffer, pH 7, 1 mM in dithiothreitol. All samples (i-viii) were immediately subjected to polyacrylamide gel electrophoresis and then stored at -20%. Electrophoresis of all samples was repeated three weeks later, whereupon the samples were returned to -2O’C and subjected to electrophoresis three months later. Crystallization
of calf
y-crystallins
Calf y-crystallin II, 60 mg, after isolation by sulphopropyl Sephadex, was concentrated by ultra-filtration, dialyzed against distilled water at 4”C, then dialyzed against 905 RI Tris-acetate buffer pH 8.5 at 4OC for 16 hr. The y-11 solution was removed from the dialysis tubing, brought up to 20% and 15 mg dithiothreitol added. The reduction was allowed to proceed for 30 min with occasional stirring after which the reduction mixture was dialyzed against distilled water at 4% for 24 hr followed by lyophilization. The freeze-dried material was dissolved in 1.0 ml 0.05 M sodium hydrogen phosphate buffer, pH 7,0.02 o/0 NaN,, 1 mM in dithiothreitol. The solution was filtered using a millipore filter, placed in a small, stoppered test-t.ube and left standing at O°C for a few days after which it was transferred to 4%. Calf fractions IIIa and b, after chromatography on DEAE-cellulose, were reduced, dialyzed and lyophilized as described for y-11. y-IIIa was dissolved in phosphate buffer as described for fraction II except that the concentration was 10 y0 and the solution was left at 20%. Fraction IIIb was treated similarly except that the phosphate buffer was pH 7.5, the protein concentration 8, 9, or lo%, and the solution was left at O°C for three days before being transferred to 4%. Fraction IV, after separation on sulphopropyl Sephadex, was reduced, dialyzed and lyophilized as described for fraction II. Fraction IV after chromatography on DEAE-cellulose was similarly treated. Solutions were made 2. 3 and 4% in protein concentration using phosphate buffer, pH 7, as solvent as described for fraction II and left to stand for several months at 20°C. Following purification using chromatofocusing, the subfraction of fraction IV, designated fraction IVa: was made 3 o/0 in protein concentration with phosphate buffer pH 7.4, 3 mM in dithiothreitol, 0.02 O/v NaN, and left to stand at 20% X-ray
diffraction
photography
of fraction
IVa
Crystals were mounted at 2O’C in quartz capillary tubes containing a drop of motherliquor and sealed. A diffraction photograph was obtained using a precession camera following exposure of the crystal to 18 hr of X-ray irradiation (ACuKcr = 1.5418 A) on a rotating anode generator. at 2O’C.
520
Purification
6. SLINGSBY
of adult
bovine
BND
L. R. MILLER
y-crystallins
Adult bovine lenses weighing at least 2 g each were removed from the animal shortly after death and stored at -20%. The lenses were decapsulated and separated into a cortical fraction representing two-thirds of the lens wet weight by stirring rapidly at 4“C, in Tris-HCl buffer pH 72 as described for calf lenses, for 30 min. The supernatant was decanted and then the nuclear core region was further disintegrated in the same buffer with the assistance of pestle and mortar. Centrifugation of both cortical and nuclear fractions, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephadex were all performed as described for calf lens. Adult bovine y-crystallin from cortical and nuclear regions was separately chromatographed on sulphopropyl Sephadex at pH 50 as described for calf lens. Adult bovine nuclear y-crystallin fraction III was further separated into fraction IIIa and b as described for calf fraction III. Adult bovine cortical y-crystallin was subjected to ion-exchange chromatography on sulphopropyl Sephadex, pH 50. The major fraction was collected, concentrated, reduced, dialyzed, lyophilized, as described for calf fraction II, dissolved in phosphate buffer, pH 7, 002% NaN,, 1 mM dithiothreitol to make a protein concentration of 6 o/0 and left to stand at 4’C for crystallization. Puri$eation
of
rabbit lens y-crystallins
Adult rabbit lenses were removed from the animal immediately after death and shortly afterwards were decapsulated and completely disintegrated in 0.05 M Tris-HCl buffer, pH 7.2, The supernatant was collected by centrifuga602 o/o NaN,, 6005 o/o (v/v) 2- mercaptoethanol. tion and subjected to gel filtration on Sephadex G-75, and ion-exchange chromatography on DEAE-Sephadex, as was described for calf lens extracts. Rabbit y-crystallin was fractionated on sulphopropyl Sephadex under exactly the same conditions as calf y-crystallin. Further purification of rabbit y-fractions was achieved by chromatography on DEAE-cellulose employing a linear gradient to elute fractions, from 0.05 M Tris-HCl buffer, pH 9.0 -+ 61 ,n Tris-HCl buffer, pH QO, containing 0.02 y0 NaN,, 6005 y0 (v/v) 2-mercaptoethanol. The molecular weight of a rabbit y-fraction was estimated using gel filtration. A Sephadex G-75 column (700 x 17 mm diam) was poured and equilibrated in 605 M sodium hydrogen phosphate buffer, pH 68. The load was 10 mg in a volume of 65 ml, 25 ml fractions were collected. The void volume was determined with Blue Dextran (12 mg/ml). The position of elution of normal y-crystallin was estimated using a freshly isolated sample of calf y-11. A solution of rabbit fraction IV(2), concentrated as for fraction II, was dissolved in the phosphate buffer containing 1 mM dithiothreitol and 602 y0 NaN, and filtered through a millipore filter before incubating at room temperature for 6 months. It was then subjected to gel filtration and the volume of elution of the peak fraction recorded. Fractions comprising the main peak were pooled, concentrated by ultrafiltration and subjected to polyacrylamide gel electrophoresis. Attewbpted
crystallization
of rabbit
y-II
(1)
Rabbit y-11 (1) (33 mg) was concentrated to a volume of 1 ml after purification on DEAEcellulose, and then reduced with 5 mg dithiothreitol at pH 8.5, 20% for 1 hr followed by dialysis into distilled water at 4°C. The protein was lyophilized followed by dissolution in 64 ml 005 M sodium hydrogen phosphate buffer, pH 6.86, 602 y0 NaN,, 3 mM in dithiothreitol. The solution was filtered using a millipore filter, placed in a small, stoppered test-tube and left at O°C for 2 hr. It was then left at 4OC for one week. An equal volume of 0.05 M disodium hydrogen phosphate solution was added and the solution left at room temperature for several days. Polyacrylamide
gel electrophoresis
Gel electrophoresis was performed in 10 y0 polyacrylamide gels with a Trigglycine running buffer (6 g Tris, 28% g glycine/l), pH 8.3 in which case samples were applied at the cathode or with a fi-alanine (31 g/l), glacial acetic acid buffer, pH 4.5 in which case the samples were applied at the anode (Shuster, 1971). Samples were run for approximately 2 hr with a current of -4 mA/gel. Gels were fixed in 12 y0 trichloroacetic acid followed by staining in Coomassie brilliant blue.
y-CRYSTALLIN
3. Results
521
and Discussion
The distribution of crystallins varies between young and old lenses due to different levels of crystallin synthesis in developing and mature lenses concomitant with the gradual incorporation of the young lens into the nuclear region of the adult lens (Piatigorsky, 1981). y-Crystallin is the predominant low molecular weight protein of calf lens whereas a greater synthesis of ps-crystallin in the adult lens results in the relative diminution of y-crystallin (Slingsby and Croft, 1973). The separation of the two monomeric crystallin classes, /?s and y, from the oligomeric crystallins was achieved using gel filtration on Sephadex G-75 (Bjiirk, 1961). The crystallin components are eluted with characteristic volumes which depend both on the molecular weight of the protein and the bed volumes of the column. The large scale Sephadex G-75 column described here is able to achieve separation of /3s and y-crystallin at elution volumes of peak fractions corresponding to 3660 and 4000 ml respectively. Estimates of the amounts of the two types of monomeric crystallins can be calculated from the total volume and extinction coefficients (Bjork, 1964; van Dam, 1966) of combined fractions from each of the protein peaks. In this study it was shown that adult bovine cortical extract contained /3s- and y-crystallin in the ratio 10 : 3 whereas a total calf lens soluble extract contained ps and y in the ratio 3: 8. y-Crystallin is thus considerably reduced in the adult bovine cortical regions. These differences in crystallin location within the mature lens, may well be related to the level of hydration and hence the refractive index value along the increasing gradient from cortex to nucleus. Complete separation of ps- from y-crystallin is achieved on ion-exchange chromatography on DEAE-Sephadex (van Dam, 1966) or DEAE-cellulose. Further purification of the closely related y-crystallins is performed on sulphopropyl Sephadex using the method of Bjijrk (1964). Four fractions, I, II; III and IV are clearly resolved [Fig. 1 (a)]. Furthermore, the extraction procedure used results in recovery of different proportions of the fractions in a way which is related to the age of the lens cells. The amount, for example, of IV extracted from whole calf lens [(Fig. i(a)] compared with the other y-fractions is greater than that recovered from adult bovine nuclear extract [Fig. l(b)] which reflects either that fraction IV is selectively degraded in the adult lens or that it becomes insoluble with ageing due to closer interaction with other crystallins or cell components. After chromatography on sulphopropyl Sephadex, fraction II is predominantly one component as judged by polyacrylamide gel eleetrophoresis whereas fraction III contains a mixture of two proteins [Fig. i(a)]. These two proteins called Illa and lllb present in fraction III can be well separated by ion-exchange chromatography on DEAE-cellulose (Fig. 2). Fraction IV, after isolation by sulphopropyl Sephadex chromatography also appears as one component on gel electrophoresis [Fig. i(a)]. Polyacrylamide gel electrophoresis clearly indicates the differences in mobility of fractions IIIa and Illb whereas fractions II, IIIb and IV all have very similar mobilities. Alkaline gel electrophoresis is therefore not a useful analytical method for discriminating these closely related proteins whereas the elution profiles of the separation on sulphopropyl Sephadex are both qualitively superior as well as being quantitative. Although fraction IV is known from amino acid sequence studies to be comprised of at least two related polypeptide chains in a ratio of about 3 :2 (Slingsby and Croft, 1978), chromatography on DEAE-cellulose failed to resolve these components further, supporting the results of gel electrophoresis that the components must be of a very
522
6’. SLINGSBY
AND
L. R. MILLER
n
( (a)
I
I 5 (b)
20
40
60
80 Fraction
1. Purification salt gradient at pH and IV. Fractions (i) calf lens low chromatography; Fractions collected co!leoted every 20 FIG.
100
120
140
160
180
i 2 0 ‘0
number
of y-crystallins using sulphopropyl Sephadex chromatography employing a linear 5.0. (a) Whole calf lens y-crystallins showing the separation into fractions I, II, III collected every 20 min, flow rate 30 ml/hr. The inset shows the electrophoretic gels molecular proteins after gel filtration; (ii) y-crystallin after DEBE-Sephadex (iii) y-1; (iv) y-11; (iv) y-111; (vi) y-11’. (b) Adult bovine lens nuclear y-crystallins. every 20 min; flow rate, 30 ml/hr. (c) adult bovine cortical y-crystallins. Fractions min: Aow rate. 25 ml/hr.
y-CRYSTALLIN
Fraction FIG. 2. Fractionation arrow indicates the (i) ~-111; (ii) y-IIIb;
&art (iii)
of calf y-crystallin of elution with y-IIIa.
number
the
III
on DEAE-cellulose second buffer. The
IV:
chromatofocusing
Fraction FIG. 3. Purification using a pH gradient
of calf y-crystallin from 8.3 to 6.
523
using
stepwise
elution.
The
verticai
inset shows the electrophoretic gels of
number of y-IV
on polybuffer
exchanger
PBE94
similar overall charge. However, using the higher resolving technique of chromatofocusing, a partial separation of fraction IV was achieved, the two components being called fraction IVa and IVb (Fig. 3). 0 ne complication of the method is the removal of the higher molecular weight polybuffer: this was achieved for fractions IVa and HVb by precipitation of the proteins with 80 y’ saturated ammonium sulphate. Although calf y-11; -III, -1Va and -1Vb are all translation products of different structural genes as they have different yet closely related amino acid sequences (Croft, 1972, Slingsby and Croft, 1978), the acidic componenl;, y-IIIa appears to be a post-translational modification of y-IIIb, the modification being accelerat,ed at moderate alkaline pH. This was indicated by the calf y-111 ageing experiment (Fig. 4) whereby y-111, and the purified components IIIa and IIIb, were exposed to conditions of varying pH, namely pH 5, 7 and 8.5, both with and without excess dithiothreitol, before being subjected to electrophoresis at various time intervals
6. SLINGSBY
524
AND
L. R. MILLER
i
i
ii
iii
i
v
iV
vi
vii
ii
iii
iv
M
viii f
-i-
i
ii
iii
IV
v -I-
vi
vii
viii
i
ii
iii
iV
-c
FIG. 4. Effect of pH and dithiothreitol on storage of calf y-111. Eleetrophoretic gels (a, b, c) of(i) calf y-III, pH 50; (ii) y-111 pH 8.5; (iii) y-IIIb, pH 85; (iv) y-IIIb, pH 85+ DTT; (v) y-IIIb, pH 7+ DTT; (vi) y-IIIa, pH 85; (vii) y-IIIa, pH %5+DTT; (viii) y-IIIa, pH 7,O+DTT. Samples were subjected to electrophoresis (a) immediately; (b) after storage at -2O’C for three weeks; (c) after storage at -20°C for three months; (d) (i) old y-IIIb solution, (ii) old y-IIIb solution+DTT, (iii) old y-IIIb dissolved crystal, (iv) old y-IIIb dissolved crystal + DTT.
following storage at -20°C. y-111 appears stable at pH 5 [Fig. 4(a)i, (b)i and (c)i] whereas at pH 8.5 an additional acidic component appears on storage [Fig. 4(c)ii]. Purified y-IIIb clearly shows a partial transformation on storage at pH 8.5 to an acidic component [Fig. 4(a)iii, (b)iii and (c)iii] which comigrates with y-IIIa [Fig. 4(a)vi]. However, this transformation is markedly diminished if excess dithiothreitol is present inthebuffer[Fig.4(a)iv, (b)ivand(c)iv],andthetransformationcontinuestobeinhibited at pH 7 in the presence of dithiothreitol [Fig. 4(a)v, (b)v and (c)v]. In a similar way isolated y-IIIa at pH 8.5 is unstable in the sense that a more acidic component is generated during storage [Fig. 4(a)vi, (b)vi and (c)vi] yet differs from y-IIIb in that the presence of excess dithiothreitol does not inhibit its formation [Fig. 4(c)vii] whereas a change of pH from 8.5 to 7 completely inhibits the modification [Fig, 4(e)viii]. Thus, even within this one subfraction of calf y-crystallin there are at least two types
y-CRYSTALLIR’
FIG. 5. Light
micrographs
525
of calf y-crystallin
crystals.
of chemical modification which can occur resulting in discrete electrophoretic forms as a result of acquisition of additional negative charges. It was of interest to determine the effects of ageing on long-term storage of y-IIIb at room temperature both in solution and in t5he crystalline state at pH 7.5. Fig. 4(d) shows that y-IIIb both in solution (i) and in the crystal (iii) comprises nearly equal proportions of three electrophoret’ie forms of y-IIIb after standing at room temperature for six months. It was also demonstrated that once formed the reaction is not reversed with excess dithiothreitol [Fig. 4(d)ii and iv]. Further investigation would be necessary in order to delineate the nature of these changes and whether or not they occur in the ageing lens. Purified calf y-crystallins can be induced to grow single crystals large enough for X-ray diffraction experiments. The conditions of pH and ionic strength for growth of micro-crystals were initially discovered by Bjijrk (1964). Since then large crystals of y-11, y-IIIb and y-IV have been produced such that three-dimensional X-ray diffraction studies have been initiated (Garber and Reshetnikova, 1976; Carlisle,
526
FIG.
generator
C. SLINGSBY
AND
L. R. MILLER
6. X-ray diffraction precession photograph of the Ml zone of calf y-IVa exposed for 18 hr, crystal to film distance, 60 mm.
taken
Cd)
on a rotating
anode
IV( 1) IVY
1
2.0 IA
I
I 0 40 Fraction
60
80
100
number
FIG. 7. (a) Separation of rabbit y-crystallin on sulphopropyl gradient. (b) (c) and (d) Show further chromatography of rabbit DEAE-cellulose, pH 9.0 using a linear salt gradient
Sephadex, y-II, y-IIIh
pH 5.0 using a linear and y-IV respectively
salt on
Lindley, Moss, and Slingsby, 1977; Chirgadze, Nikonov, Garber and Reshetnikova, 1977; Blundell et al., 1978; Blundell et al., 1981; Chirgadze et al., 1981; Wistow et a.l., 1983). Crystals of 7II and yIIIb are the easiest to grow as single crystals (Fig. 5). y-IIIa Crystals were once grown but tended to grow as polycrystalline spherulites although occasionally single crystals were observed (Fig. 5). Fraction IV purified by sulphopropyl Sephadex chromatography tended to grow in smectic clusters and more rarely as single crystals, whereas after purification using chromatofocusing, the same crystals grew only from y-IVa yet they grew more quickly and
y-CRYSTALLIN
527
C bl
i
ii
iv
iii
ii
i
iii
iv
+ 04 02
1765 ml
(cl
r
‘,,,,,A
1
20
40
60
80
Fractlan
100
i
ii
iii
iv
4-
120 140 160 180 2(
number
FIG. 8. Poiyacrylamide gel electrophoresis of rabbit y-crystallins. (a) Electrophoresis performed at pH 8.3, (i) unfractionated rabbit y-crystallin, (ii) y-IIIa, (iii) y-IIIb, (iv) y-IV. (b) Electrophoresis performed at pH 4.5. (i) y-IIIa; (ii) y-IIIb, (iii) IV(i), (iv) IV(2). (c) Electrophoresis at pH 8.3 and molecular weight estimation of ‘aged ’ rabbit y-IV(S). The elution profile is that of y-IV(B) on Sephadex G-75 gel filtration. The inset shows the electrophoretic gels of ‘aged’ rabbit y-IV(S) before, (iii) and (iv), and after, (i) and (ii), passage through the Sephadex column.
more consistently as single crystals (Fig. 5), although the crystals still have a tendency to grow in microlayers. The diffraction pattern of y-IVa (Fig. 6) shows that it is possible to grow excellent? well ordered, highly diffracting crystals of this early foetal protein. The predominant component of adult bovine y-crystallin, purified from lens cortex by gel filtration on Sephadex G-75 followed by DEAE-Sephadex and sulphopropyl Sephadex chromatography [Fig. 1 (c)l, was treated to identical crystallization conditions as calf y-II. The solution grew many small crystals of typical y-11 habit. Although the crystals were not big enough to obtain X-ray diffraction photographs, the evidence would so far suggest that the predominant adult y-crystallin is the same protein as calf y-II. It was considered of interest to compare y-crystallins from a different species of mammal and so a purification system for rabbit y-crystallins was devised. The young adult lenses which were used in this study did not easily separate into a cortical and nuclear region, a property which may be related to their uniquely simple suture system
528
c.
SLINGSBY
AI-CD
L. Rm. MILLER
(Harding, Susan and Murphy, 1976) and hence only a whole lens extraction was performed. y-Crystallin was separated from oligomeric crystallins by gel filtration, and from @crystallin by ion-exchange chromatography exactly as described for calf lens y-crystallin. Rabbit y-crystallin on sulphopropyl Sephadex chromatography revealed four components which have been labelled II, IIIa, IIIb and IV [Fig. 7(a)]. This nomenclature is provisional and is based on calling the most predominant component y-11. A proper classification must await further amino acid sequencing studies or gene sequenciug studies whereby y-crystallins can be assigned to a family name which survives species divergence. Amino acid composition and C-terminal analyses of rabbit y-crystallins have demonstrated that at least three components, capable of resolution on carboxymethyl-cellulose chromatography, are related to bovine y-crystallins (Hines and Olive, 1970). However, this same study also demonstrated that rabbit y-crystallins clearly exhibited variation from calf y-crystallins such as the absence of alanine and threonine from their amino acid compositions. The component fractions II, IIIb and IV were further chromatographed on DEAE-cellulose using a linear salt gradient at pH 9. Although IIIb appeared largely homogeneous [Fig. 7(c)], fraction II resolved into two components which have been called II(l) and 11(2) [Fig. 7(b)], and fraction IV likewise separates into two components, IV(l) and IV(2) [Fig. 7(d)]. Gel electrophoresis at pH Q*q -.vas performed on rabbit y-crystallins after their purification from oligomeric crystallins on gel filtration and ion exchange chromatography on DEAE-Sephadex. Electrophoresis revealed proteins of predominantly two electrophoretic mobilities [Fig. S(a)i]. Fractions IIIa, IIIb and IV were chromatographed on sulphopropyl Sephadex and then subjected to gel electrophoresis at pH 8.3 which revealed that the more basic band is IIIa [Fig. B(a)ii] and the slightly more acidic band is comprised of IIIb and IV [Fig. 8( a )’iii ., and iv]. Fraction II does not enter the gel at this pH. Fractions IIIa and IIIb however corn&rate on gel electrophoresis at pH 45 [Fig. B(b)i and ii] whereas fractions IV(l) and W(2), separated from each other by DEAE-cellulose chromatography, display different mobilities at the acidic pH [Fig. B(b)iii and iv]. Attempts were made to crystallize all of the rabbit y-fractions but the only promising result was with y-II(l). Small, plate-like hexagonal crystals appeared in the test-tube but failed to grow to a size large enough for X-ray diffraction photography and hence the definitive proof that the crystals were protein was not obtained. One of the components of rabbit y-crystallin was selected for further investigation of possible ageing effects. Rabbit fraction IV(2) which had been left in solution at 20% for six months was subjected to electrophoresis at pH %3 whereupon it was observed that in addition to the original basic band typical of the freshly isolated preparation, a further four bands appeared, each apparently possessing consecutive additional negative charges [Fig. 8(c)iv]. The approximate molecular weight of this ‘aged’ rabbit fraction IV(2) was determined by passage through a gel filtration column previously calibrated with freshly prepared calf y-II. The rabbit sample eluted predominantly as a molecule the size of calf y-II with a small amount of higher molecular weight material. The fractions corresponding to the major peak, after pooling and concentrating, were subjected to electrophoresis. It can be seen from Fig. S(c)i that a similar collection of electrophoretic forms is present in the monomeric fraction and hence it can be concluded that this ageing effect of rabbit Y-IV(~) does not lead to obligatory self-oligomer formation. Although lens cytoplasm is a complex system of polydisperse crystallins interacting
y-CRPSTALLIN
529
with each other and cell components, it is possible to extract and purify many of the low molecular weight proteins. Moreover, it is also possible to control the conditions such that most of the calf y-crystallins self-associate in the crystalline state back to a protein concentration equal to that of the lens. Some of the y-crystallins are labile in that under certain conditions not far removed from physiological, they became more negatively charged. Calf y-crystallin IIIa and IIIb stored in the deep freeze under mildly alkaline conditions will generate electrophoretic variants. It is important to differentiate these modified crystallins from gene product proteins of similar electrophoretic mobility. The possibility that similar modifications may occur ‘in vivo’ remains to be investigated. Nevertheless y-crystallins with varying amino acid sequences and their modified fractions provide an ideal system for comparing their relative potentials for self-association and other crystallin interactions. ACKNOWLEDGMENTS The support of the Medical Research Council is gratefully acknowledged. Rabbit Company for the gift of rabbit lenses. We thank all of our Crystallography Department.
We thank colleagues
Buxted in the
REFERENCES Bjork,
I. (1961). Studies on y-crystallin from calf lens I. Isolation by gel filtration. Exp. Eye Res. I, 145-54. Rjbrk, I. (1964). Studies on y-crystallin from calf lens II. Purification and some properties of the main protein components. Exp. Eye Res. 3, 254-61. Bloemendal, H. (1981). In Molecular and Cellular Biologyofthe Eye Lens (Ed. H. Bloemendal). Pp. l-47. John Wiley, New York. Blundell, T. L., Lindley, P. F., Ness, D. S., Slingsby, C., Tickle, I. J. and Turnell, W. G. (1978). The low resolution structure analysis of the lens protein y-crystallin. Acta Cry&. 34, 3653-7. ell, T. L.; Lindley, P. F., Miller, L., Moss, D. S., Slingsby, C., Tickle, I.; Turnell, B. and Wistow, G. (1981). The molecular structure and stability of the eye-lens: X-ray ana.lysis of y-crystallin II. Nature 289, 771-7. Carlisle, C. H., Lindley, P. F., Moss, D. S. and Slingsby, C. (1977). Preliminary X-ray crystallographic study of the bovine lens protein, y-crystallin II. J. Mol Biol. 110, 417-19. Chirgadze, Y. N., Nikonov, S. V., Garber, RI. B. and Reshetnikova, L. S. (1977). Crystallographio study of y-crystallins from calf lens. J. Mol. Biol. 110, 619-24. Chirgadze, Y. N.. Sergeev, Y. V., Fomenkova, N. P. and Oreshin, V. D. (1981). Polypeptide chain pathway in y-crystallin IIIb from calf lens at 3 A resolution. FEBS Lett. 131,81-4. no acid sequence of y-crystallin (Fraction II) from calf lens. Garber, M. B. and Reshetnikova, L. S. (1976). Growing single crystals of calf y-crystallin from the calf crystalline lens. Doklady Akademii Nauk SSSR 226, 1452-4. Warding, J. J. and Dilley, K. J. (197.6). Structural proteins of the mammalian lens: a review with emphasis on changes in development, ageing and cataract. Exp. Eye Res. 22, l-75. Fiarding, C. V.: Susan, S. and Murphy, H. (1976). Scanning electron microscopy of the adult rabbit lens. Ophthalmic. Res. 8, 443-55. Hines. NM. C. and Olive, J. A. (1970). On the components of rabbit lens y-crystallin. &+e Sciences 9, 1101-10. Papaconstantinou, J. (1965). Biochemistry of bovine lens proteins II. The y-crystallins of adult bovine, calf and embryonic lenses. Biochim. Biophys. Acta 107, 81-90. Philipson; B. (1969). Distribution of protein within the normal rat lens. Invest. Opht/&mol. 8, 258-70. Piatigorsky, J. (1981). Lens differentiation in vertebrates. A review of cellular and molecular
features. Difleerentiation
19, 134-53.
530
Rink,
C. SLINGSBY
H. (1978). The water Geront.
content
AND
L. R. MILLER
in bovine lenses during
ageing. Interdiscipl.
Topics
12, 271-7. (1971). In Methods
Shuster, L. in Enzynzology, vol. XXII. (Ed. Jakoby, W. B.). Pp. 412-33. Academic Press, London. Slingsby, C. and Croft, L. R. (1973). Developmental changes in the low molecular weight proteins of the bovine lens. Exp. Eye Res. 17, 369-76. Slingsby, C. and Croft, L. R. (1978). Structural studies on calf lens y-crystallin fraction IV: a comparison of the cysteine-containing tryptic peptides with the corresponding amino acid sequence of y-erystallin fraction II. Exp. Eye Res. 26, 291-304. van Dam, A. F. (1966). Purification and composition studies of /&+crystallin. Exp. Eye Res. 5, 255-66. Van Heyningen, R. (1972). The human lens III. Some observations on the post-mortem lens. Exp.
Eye
Res.
13, 155-60.
Waley, S. G. (1969). In Tke Eye, vol. 1 (2nd edn). (Ed. Davson, H.). Pp. 291-379. Academic Press, London. Wannemacher, C. F. and Spector, A. Protein synthesis in the core of calf lens. Exp. Eye Res. 7, 623-25. Wistow, G., Lindley, P. F., Miller, L., Moss, D., Slingsby, C., Turnell, B. and Blundell, T. L. (1983). X-ray analysis of the eye-lens protein y-crystallin II at 1.9 A resolution. J. Mol. Biol. (In press).
Eye
Res.
(1983)
37,
urification
517-530
and Crystallization y-Crystallins C. SLINCSBY
Department
of Crystallography,
(Received 23 March
AND
Birkbeck London,
of Mammalian
Lens
L. R. MILLER
College, University WClE 7HX
of London,
Malet
Street,
1983 and accepted 25 May 1983, London)
Monomeric crystallins, ps and several members of the y-crystallin family, occw in different relative proportions in a way which is related to the age of the lens cells. Methods of purification of large amounts of these different low molecular weight protein components from young and old bovine lenses are described along with details of crystallization of several y-fractions. Purification procedures have been developed for rabbit y-crystallins. The chromatographic methods achieve separation of several bovine and rabbit y-crystallins which have very similar electrophoretic mobilities. However, on storage, many electrophoretic variants are generated from some of these fractions. Key words: y-crystallin; /Is-wystallin; crystals; ageing of y-crystallin.
1. Introduction Eye lenses contain high concentrations of proteins in order to achieve the required refractive index necessary for their function. The protein concentration is not uniform throughout a particular lens, it is highest in the central regions although the concentration gradient varies among species (Waley, 1969; Philipson, 1969; van Heyningen, 1972; Rink, 1978). A major component of the lens soluble crystallins is y-crystallin, a protein synthesized in early development and thus occurring predominantly in the central regions of adult lenses (Bjijrk, 1961, 1964; Papaconstantinou, 1965; Waley, 1969; Slingsby and Croft, 1973; Harding and Dilley, 1976; Bloemendal, 1981). In those animals which contain a steep gradient of refractive index and hence a relatively dehydrated lens core region, the y-crystallin must be associated with other erystallins and cell components in an environment of limited hydration. One approach to studying the macromolecular interactions responsible for maintaining a high refractive index is to investigate the structure of crystals of y-crystallin. An investigation of the proteins in their crystalline state not only allows the determination of their three-dimensional structure, but also yields information relating to their potential for interaction with additional protein and water molecules at a protein concentration similar to the lens core region. Single crystal X-ray diffraction analyses are now underway (Blundell et al., 1981; Chirgadze, Sergeev, Fomenkova and Oreshin, 1981). Crystallization of y-crystallins requires a large scale isolation and purification syst’em for the many closely related members of this family (Bjiirk, 1964; Croft, 1972; Slingsby & Croft, 1978). In this paper purification and crystallization methods of several bovine y-crystallins are described. As there is little protein turnover in the eye lens cells of the bovine core region (Wannemacher and Spector, 1968), the y-crystallin laid down in early development must perform its structural role as one of the components maintaining eye lens transparency in the adult core yet be able to adapt’ to an environment of increasing dehydration as the lens matures. The crystallins may become modified on ageing in 00144835/83/0105i7+13
$OS.OO/O
0
1983 Academic
Press Inc. (London)
Limited
C. SL,INGBBU
518
AND
L. R. MILLER
a manner which could effect their interactions with other molecules. In this paper an ageing effect is reported which could be attributed to chemical modification of specific y-crystallin fractions. Differences in the relative proportions of crystallins along the lens optical axis are probably related to the refractive index gradient. In the bovine lens monomeric y-crystallin is replaced by monomeric /&-crystallin (van Dam, 1966) in adult lens cortex. A further investigation of the differences in the relative proportions of the two monomeric crystallin classes was undertaken for adult cortical and calf lens extracts. Similarly the variation in the relative proportions of different members of the y-crystallin family between young and old lenses was explored. In order to explore further the variation in structural properties associated with y-crystallins, purification procedures have been developed for a different species, rabbit, together with observations of an ageing effect of one of the rabbit y-crystallin components. 2. Materials Puri$cation
of calf
lens
Calf lenses, weighing
am
y-esystallins
removed from the animal shortly after death decapsulated and stirred at 4“C in twice their volume of 005 M Tris-HCI buffer pH 7.2,0.02 o/0 NaN, and 0005 o/o (v/v) 2-mercaptoethanol. Lenses were completely disintegrated into a homogenate which was centrifuged at 17000 g for 1 hr at 1O’C. The resulting supernatant was subjected, in 40 ml portions, to gel filtration on Sephadex G-75 (Pharmacia), column size 1000 x 90 mm (diam) from which the low molecular weight protein fraction was isolated as described by Bjijrk (1961). Low molecular weight crystallins were concentrated from a volume of 70&20 ml by ultrafiltration in an Amicon cell equipped with a UMlO membrane. Separation of /3s-crystallin from y-crystallin was achieved using ion-exchange chromatography based on the method of van Dam (1966). DEAE-Sephadex A-50 (Pharmacia), equilibrated with 0.005 M sodium hydrogen phosphate buffer, pH 8.2, 601% NaN,, 0.005 y0 (v/v) 2- mercaptoethanol, was poured into a column 100 x 50 mm (diam). The protein solution, 500 mg in 26 ml, equilibrated in the same buffer was applied to the column and the y-crysta.llin was rapidly eluted. Alternatively, DEAEcellulose ion exchanger was used, equilibration and first elution being performed with 0.025 M Tris-HCl buffer pH 8.3 followed by elution of ps-crystallin with 605 M Tris-IX1 pH 8.0. y-Crystallin after concentration to 20 ml was immediately equilibrated with sodium acetate Separation buffer pH 50, ionic strength 02,902 y0 NaN,, 6005 ‘% (v/v) 2- mercaptoethanol. of the mixture of y-crystallins into fractions I, II, III, and IV was achieved on a cation exchanger, sulphopropyl-(SP)-Sephadex C-50 (Pharmacia), column size 500 x 35 mm (diam) using a linear salt gradient, ionic strength 0.2 --t 95, pH 50 according to the method of Bjiirk (1964). This size column will adequately resolve y-components up to a load of 1 g. y-Crystallin fraction III was resolved into two components, TIIa and IIIb, using ion-exchange chromatography on DEAE-cellulose. DE52 (Whatman) was equilibrated in O-05 M Tris-HCl buffer pH 9.0 containing 002 y0 NaN, and 0605 y0 (v/v) 2- mercaptoethanol and made into a column 500 x 13 mm (diam). y-Crystallin III (100 mg), previously equilibrated by dialysis against the same buffer, was applied to the column in a volume of 10 ml. Fraction IIIb elutes with this buffer. Fraction IIIa is eluted with 91 M Tris-HCl bullFer, pH 9.0. Alternatively the separation can be carried out with sodium hydrogen phosphate buffer pH 8.5, fraction IIIb being eluted with 0.05 M phosphate buffer with fraction IIIa subsequently eluted with 0075 x phosphate buffer, pH 8.5, containing 0.005% mercaptoethanol. Each protein fraction was dialysed against distilled water at 4°C and lyophilized. Fraction IV was chromatographed on a similar DE52 column, employing a linear gradient of 605 M Tris-HCl buffer pH 90 + @I M Tris-DC1 buffer pH 9.0, containing 002 y0 NaN, and9005 % (v/v) 2-mercaptoethanol. Fraction IV was also subjected to further purification using chromatofocusing. Fraction IV (50 mg) in a volume of 1 ml was equilibrated in 0.025 M Tris-acetate buffer pH 8.3, 002°/0 NaN, and 0.005 o/0 (v/v) 2-mercaptoethanol. Polybuffer exchanger PBETM94 (Pharmacia) was equiliand stored
below
- 20%.
about I g each, were Lenses were thawed,
yCRYSTALLIN
519
brated with the same buffer and poured to form a column 200 x 15 mm (diam.). Elution was TM96 (Pharmacia) diluted x 13 with distilled water and titrated performed with Polybuffer to pH 6 with acetic acid. y-Crystalline IVb is eluted first and incompletely resolved from IVa. Removal of the protein from the polybuffer was achieved by precipitation of the proteins in 80 o/o saturated ammonium sulphate. The precipitate was centrifuged, washed in saturated ammonium sulphate and redissolved in distilled water followed by dialysis into distilled water and lyophilization. Ageing
ejj’ect on c.alf y-crystal&n
fraction
IZI
Eight samples of calf fraction III were prepared as follows : (i) Fraction III, after sulphopropyl Sephadex chromatography, was left in acetate buffer, pH 5.0. (ii) Fraction III, in acetate buffer pH 50, was dialyzed for 24 hr in 605 A~ phosphate buffer, pH 8.5, 0095 y0 (v/v) 2-mercaptoethanol. (iii) Fraction IIIb, after isolation by DEAE-cellulose chromatography, was in phosphate buffer, pH 8.5; 0.005 o/0 (v/v) 2-mercaptoethanol. (iv) Fraction IIIb (150 nmol) from (iii) was treated with dithiothreitol (200 nmol). (v) Fraction IIIb from (iv) was dialyzed into 500 ml phosphate buffer, pH 7.0, 05 rnM in dit)hiothreitol. (vi) Fraction IIIa after isolation by DEAF-cellulose chromatography was in 0.075 M phosphate buffer, pH 85, 0.005 y0 (v/v) 2-mercaptoethanol. (vii) Fraction IIIa (75 nmol) from (vi) was treated with dithiothreitol (100 nmol). (viii) Sample (vii) was dialyzed into 005 M phosphate buffer, pH 7, 1 mM in dithiothreitol. All samples (i-viii) were immediately subjected to polyacrylamide gel electrophoresis and then stored at -20%. Electrophoresis of all samples was repeated three weeks later, whereupon the samples were returned to -2O’C and subjected to electrophoresis three months later. Crystallization
of calf
y-crystallins
Calf y-crystallin II, 60 mg, after isolation by sulphopropyl Sephadex, was concentrated by ultra-filtration, dialyzed against distilled water at 4”C, then dialyzed against 905 RI Tris-acetate buffer pH 8.5 at 4OC for 16 hr. The y-11 solution was removed from the dialysis tubing, brought up to 20% and 15 mg dithiothreitol added. The reduction was allowed to proceed for 30 min with occasional stirring after which the reduction mixture was dialyzed against distilled water at 4% for 24 hr followed by lyophilization. The freeze-dried material was dissolved in 1.0 ml 0.05 M sodium hydrogen phosphate buffer, pH 7,0.02 o/0 NaN,, 1 mM in dithiothreitol. The solution was filtered using a millipore filter, placed in a small, stoppered test-t.ube and left standing at O°C for a few days after which it was transferred to 4%. Calf fractions IIIa and b, after chromatography on DEAE-cellulose, were reduced, dialyzed and lyophilized as described for y-11. y-IIIa was dissolved in phosphate buffer as described for fraction II except that the concentration was 10 y0 and the solution was left at 20%. Fraction IIIb was treated similarly except that the phosphate buffer was pH 7.5, the protein concentration 8, 9, or lo%, and the solution was left at O°C for three days before being transferred to 4%. Fraction IV, after separation on sulphopropyl Sephadex, was reduced, dialyzed and lyophilized as described for fraction II. Fraction IV after chromatography on DEAE-cellulose was similarly treated. Solutions were made 2. 3 and 4% in protein concentration using phosphate buffer, pH 7, as solvent as described for fraction II and left to stand for several months at 20°C. Following purification using chromatofocusing, the subfraction of fraction IV, designated fraction IVa: was made 3 o/0 in protein concentration with phosphate buffer pH 7.4, 3 mM in dithiothreitol, 0.02 O/v NaN, and left to stand at 20% X-ray
diffraction
photography
of fraction
IVa
Crystals were mounted at 2O’C in quartz capillary tubes containing a drop of motherliquor and sealed. A diffraction photograph was obtained using a precession camera following exposure of the crystal to 18 hr of X-ray irradiation (ACuKcr = 1.5418 A) on a rotating anode generator. at 2O’C.
520
Purification
6. SLINGSBY
of adult
bovine
BND
L. R. MILLER
y-crystallins
Adult bovine lenses weighing at least 2 g each were removed from the animal shortly after death and stored at -20%. The lenses were decapsulated and separated into a cortical fraction representing two-thirds of the lens wet weight by stirring rapidly at 4“C, in Tris-HCl buffer pH 72 as described for calf lenses, for 30 min. The supernatant was decanted and then the nuclear core region was further disintegrated in the same buffer with the assistance of pestle and mortar. Centrifugation of both cortical and nuclear fractions, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephadex were all performed as described for calf lens. Adult bovine y-crystallin from cortical and nuclear regions was separately chromatographed on sulphopropyl Sephadex at pH 50 as described for calf lens. Adult bovine nuclear y-crystallin fraction III was further separated into fraction IIIa and b as described for calf fraction III. Adult bovine cortical y-crystallin was subjected to ion-exchange chromatography on sulphopropyl Sephadex, pH 50. The major fraction was collected, concentrated, reduced, dialyzed, lyophilized, as described for calf fraction II, dissolved in phosphate buffer, pH 7, 002% NaN,, 1 mM dithiothreitol to make a protein concentration of 6 o/0 and left to stand at 4’C for crystallization. Puri$eation
of
rabbit lens y-crystallins
Adult rabbit lenses were removed from the animal immediately after death and shortly afterwards were decapsulated and completely disintegrated in 0.05 M Tris-HCl buffer, pH 7.2, The supernatant was collected by centrifuga602 o/o NaN,, 6005 o/o (v/v) 2- mercaptoethanol. tion and subjected to gel filtration on Sephadex G-75, and ion-exchange chromatography on DEAE-Sephadex, as was described for calf lens extracts. Rabbit y-crystallin was fractionated on sulphopropyl Sephadex under exactly the same conditions as calf y-crystallin. Further purification of rabbit y-fractions was achieved by chromatography on DEAE-cellulose employing a linear gradient to elute fractions, from 0.05 M Tris-HCl buffer, pH 9.0 -+ 61 ,n Tris-HCl buffer, pH QO, containing 0.02 y0 NaN,, 6005 y0 (v/v) 2-mercaptoethanol. The molecular weight of a rabbit y-fraction was estimated using gel filtration. A Sephadex G-75 column (700 x 17 mm diam) was poured and equilibrated in 605 M sodium hydrogen phosphate buffer, pH 68. The load was 10 mg in a volume of 65 ml, 25 ml fractions were collected. The void volume was determined with Blue Dextran (12 mg/ml). The position of elution of normal y-crystallin was estimated using a freshly isolated sample of calf y-11. A solution of rabbit fraction IV(2), concentrated as for fraction II, was dissolved in the phosphate buffer containing 1 mM dithiothreitol and 602 y0 NaN, and filtered through a millipore filter before incubating at room temperature for 6 months. It was then subjected to gel filtration and the volume of elution of the peak fraction recorded. Fractions comprising the main peak were pooled, concentrated by ultrafiltration and subjected to polyacrylamide gel electrophoresis. Attewbpted
crystallization
of rabbit
y-II
(1)
Rabbit y-11 (1) (33 mg) was concentrated to a volume of 1 ml after purification on DEAEcellulose, and then reduced with 5 mg dithiothreitol at pH 8.5, 20% for 1 hr followed by dialysis into distilled water at 4°C. The protein was lyophilized followed by dissolution in 64 ml 005 M sodium hydrogen phosphate buffer, pH 6.86, 602 y0 NaN,, 3 mM in dithiothreitol. The solution was filtered using a millipore filter, placed in a small, stoppered test-tube and left at O°C for 2 hr. It was then left at 4OC for one week. An equal volume of 0.05 M disodium hydrogen phosphate solution was added and the solution left at room temperature for several days. Polyacrylamide
gel electrophoresis
Gel electrophoresis was performed in 10 y0 polyacrylamide gels with a Trigglycine running buffer (6 g Tris, 28% g glycine/l), pH 8.3 in which case samples were applied at the cathode or with a fi-alanine (31 g/l), glacial acetic acid buffer, pH 4.5 in which case the samples were applied at the anode (Shuster, 1971). Samples were run for approximately 2 hr with a current of -4 mA/gel. Gels were fixed in 12 y0 trichloroacetic acid followed by staining in Coomassie brilliant blue.
y-CRYSTALLIN
3. Results
521
and Discussion
The distribution of crystallins varies between young and old lenses due to different levels of crystallin synthesis in developing and mature lenses concomitant with the gradual incorporation of the young lens into the nuclear region of the adult lens (Piatigorsky, 1981). y-Crystallin is the predominant low molecular weight protein of calf lens whereas a greater synthesis of ps-crystallin in the adult lens results in the relative diminution of y-crystallin (Slingsby and Croft, 1973). The separation of the two monomeric crystallin classes, /?s and y, from the oligomeric crystallins was achieved using gel filtration on Sephadex G-75 (Bjiirk, 1961). The crystallin components are eluted with characteristic volumes which depend both on the molecular weight of the protein and the bed volumes of the column. The large scale Sephadex G-75 column described here is able to achieve separation of /3s and y-crystallin at elution volumes of peak fractions corresponding to 3660 and 4000 ml respectively. Estimates of the amounts of the two types of monomeric crystallins can be calculated from the total volume and extinction coefficients (Bjork, 1964; van Dam, 1966) of combined fractions from each of the protein peaks. In this study it was shown that adult bovine cortical extract contained /3s- and y-crystallin in the ratio 10 : 3 whereas a total calf lens soluble extract contained ps and y in the ratio 3: 8. y-Crystallin is thus considerably reduced in the adult bovine cortical regions. These differences in crystallin location within the mature lens, may well be related to the level of hydration and hence the refractive index value along the increasing gradient from cortex to nucleus. Complete separation of ps- from y-crystallin is achieved on ion-exchange chromatography on DEAE-Sephadex (van Dam, 1966) or DEAE-cellulose. Further purification of the closely related y-crystallins is performed on sulphopropyl Sephadex using the method of Bjijrk (1964). Four fractions, I, II; III and IV are clearly resolved [Fig. 1 (a)]. Furthermore, the extraction procedure used results in recovery of different proportions of the fractions in a way which is related to the age of the lens cells. The amount, for example, of IV extracted from whole calf lens [(Fig. i(a)] compared with the other y-fractions is greater than that recovered from adult bovine nuclear extract [Fig. l(b)] which reflects either that fraction IV is selectively degraded in the adult lens or that it becomes insoluble with ageing due to closer interaction with other crystallins or cell components. After chromatography on sulphopropyl Sephadex, fraction II is predominantly one component as judged by polyacrylamide gel eleetrophoresis whereas fraction III contains a mixture of two proteins [Fig. i(a)]. These two proteins called Illa and lllb present in fraction III can be well separated by ion-exchange chromatography on DEAE-cellulose (Fig. 2). Fraction IV, after isolation by sulphopropyl Sephadex chromatography also appears as one component on gel electrophoresis [Fig. i(a)]. Polyacrylamide gel electrophoresis clearly indicates the differences in mobility of fractions IIIa and Illb whereas fractions II, IIIb and IV all have very similar mobilities. Alkaline gel electrophoresis is therefore not a useful analytical method for discriminating these closely related proteins whereas the elution profiles of the separation on sulphopropyl Sephadex are both qualitively superior as well as being quantitative. Although fraction IV is known from amino acid sequence studies to be comprised of at least two related polypeptide chains in a ratio of about 3 :2 (Slingsby and Croft, 1978), chromatography on DEAE-cellulose failed to resolve these components further, supporting the results of gel electrophoresis that the components must be of a very
522
6’. SLINGSBY
AND
L. R. MILLER
n
( (a)
I
I 5 (b)
20
40
60
80 Fraction
1. Purification salt gradient at pH and IV. Fractions (i) calf lens low chromatography; Fractions collected co!leoted every 20 FIG.
100
120
140
160
180
i 2 0 ‘0
number
of y-crystallins using sulphopropyl Sephadex chromatography employing a linear 5.0. (a) Whole calf lens y-crystallins showing the separation into fractions I, II, III collected every 20 min, flow rate 30 ml/hr. The inset shows the electrophoretic gels molecular proteins after gel filtration; (ii) y-crystallin after DEBE-Sephadex (iii) y-1; (iv) y-11; (iv) y-111; (vi) y-11’. (b) Adult bovine lens nuclear y-crystallins. every 20 min; flow rate, 30 ml/hr. (c) adult bovine cortical y-crystallins. Fractions min: Aow rate. 25 ml/hr.
y-CRYSTALLIN
Fraction FIG. 2. Fractionation arrow indicates the (i) ~-111; (ii) y-IIIb;
&art (iii)
of calf y-crystallin of elution with y-IIIa.
number
the
III
on DEAE-cellulose second buffer. The
IV:
chromatofocusing
Fraction FIG. 3. Purification using a pH gradient
of calf y-crystallin from 8.3 to 6.
523
using
stepwise
elution.
The
verticai
inset shows the electrophoretic gels of
number of y-IV
on polybuffer
exchanger
PBE94
similar overall charge. However, using the higher resolving technique of chromatofocusing, a partial separation of fraction IV was achieved, the two components being called fraction IVa and IVb (Fig. 3). 0 ne complication of the method is the removal of the higher molecular weight polybuffer: this was achieved for fractions IVa and HVb by precipitation of the proteins with 80 y’ saturated ammonium sulphate. Although calf y-11; -III, -1Va and -1Vb are all translation products of different structural genes as they have different yet closely related amino acid sequences (Croft, 1972, Slingsby and Croft, 1978), the acidic componenl;, y-IIIa appears to be a post-translational modification of y-IIIb, the modification being accelerat,ed at moderate alkaline pH. This was indicated by the calf y-111 ageing experiment (Fig. 4) whereby y-111, and the purified components IIIa and IIIb, were exposed to conditions of varying pH, namely pH 5, 7 and 8.5, both with and without excess dithiothreitol, before being subjected to electrophoresis at various time intervals
6. SLINGSBY
524
AND
L. R. MILLER
i
i
ii
iii
i
v
iV
vi
vii
ii
iii
iv
M
viii f
-i-
i
ii
iii
IV
v -I-
vi
vii
viii
i
ii
iii
iV
-c
FIG. 4. Effect of pH and dithiothreitol on storage of calf y-111. Eleetrophoretic gels (a, b, c) of(i) calf y-III, pH 50; (ii) y-111 pH 8.5; (iii) y-IIIb, pH 85; (iv) y-IIIb, pH 85+ DTT; (v) y-IIIb, pH 7+ DTT; (vi) y-IIIa, pH 85; (vii) y-IIIa, pH %5+DTT; (viii) y-IIIa, pH 7,O+DTT. Samples were subjected to electrophoresis (a) immediately; (b) after storage at -2O’C for three weeks; (c) after storage at -20°C for three months; (d) (i) old y-IIIb solution, (ii) old y-IIIb solution+DTT, (iii) old y-IIIb dissolved crystal, (iv) old y-IIIb dissolved crystal + DTT.
following storage at -20°C. y-111 appears stable at pH 5 [Fig. 4(a)i, (b)i and (c)i] whereas at pH 8.5 an additional acidic component appears on storage [Fig. 4(c)ii]. Purified y-IIIb clearly shows a partial transformation on storage at pH 8.5 to an acidic component [Fig. 4(a)iii, (b)iii and (c)iii] which comigrates with y-IIIa [Fig. 4(a)vi]. However, this transformation is markedly diminished if excess dithiothreitol is present inthebuffer[Fig.4(a)iv, (b)ivand(c)iv],andthetransformationcontinuestobeinhibited at pH 7 in the presence of dithiothreitol [Fig. 4(a)v, (b)v and (c)v]. In a similar way isolated y-IIIa at pH 8.5 is unstable in the sense that a more acidic component is generated during storage [Fig. 4(a)vi, (b)vi and (c)vi] yet differs from y-IIIb in that the presence of excess dithiothreitol does not inhibit its formation [Fig. 4(c)vii] whereas a change of pH from 8.5 to 7 completely inhibits the modification [Fig, 4(e)viii]. Thus, even within this one subfraction of calf y-crystallin there are at least two types
y-CRYSTALLIR’
FIG. 5. Light
micrographs
525
of calf y-crystallin
crystals.
of chemical modification which can occur resulting in discrete electrophoretic forms as a result of acquisition of additional negative charges. It was of interest to determine the effects of ageing on long-term storage of y-IIIb at room temperature both in solution and in t5he crystalline state at pH 7.5. Fig. 4(d) shows that y-IIIb both in solution (i) and in the crystal (iii) comprises nearly equal proportions of three electrophoret’ie forms of y-IIIb after standing at room temperature for six months. It was also demonstrated that once formed the reaction is not reversed with excess dithiothreitol [Fig. 4(d)ii and iv]. Further investigation would be necessary in order to delineate the nature of these changes and whether or not they occur in the ageing lens. Purified calf y-crystallins can be induced to grow single crystals large enough for X-ray diffraction experiments. The conditions of pH and ionic strength for growth of micro-crystals were initially discovered by Bjijrk (1964). Since then large crystals of y-11, y-IIIb and y-IV have been produced such that three-dimensional X-ray diffraction studies have been initiated (Garber and Reshetnikova, 1976; Carlisle,
526
FIG.
generator
C. SLINGSBY
AND
L. R. MILLER
6. X-ray diffraction precession photograph of the Ml zone of calf y-IVa exposed for 18 hr, crystal to film distance, 60 mm.
taken
Cd)
on a rotating
anode
IV( 1) IVY
1
2.0 IA
I
I 0 40 Fraction
60
80
100
number
FIG. 7. (a) Separation of rabbit y-crystallin on sulphopropyl gradient. (b) (c) and (d) Show further chromatography of rabbit DEAE-cellulose, pH 9.0 using a linear salt gradient
Sephadex, y-II, y-IIIh
pH 5.0 using a linear and y-IV respectively
salt on
Lindley, Moss, and Slingsby, 1977; Chirgadze, Nikonov, Garber and Reshetnikova, 1977; Blundell et al., 1978; Blundell et al., 1981; Chirgadze et al., 1981; Wistow et a.l., 1983). Crystals of 7II and yIIIb are the easiest to grow as single crystals (Fig. 5). y-IIIa Crystals were once grown but tended to grow as polycrystalline spherulites although occasionally single crystals were observed (Fig. 5). Fraction IV purified by sulphopropyl Sephadex chromatography tended to grow in smectic clusters and more rarely as single crystals, whereas after purification using chromatofocusing, the same crystals grew only from y-IVa yet they grew more quickly and
y-CRYSTALLIN
527
C bl
i
ii
iv
iii
ii
i
iii
iv
+ 04 02
1765 ml
(cl
r
‘,,,,,A
1
20
40
60
80
Fractlan
100
i
ii
iii
iv
4-
120 140 160 180 2(
number
FIG. 8. Poiyacrylamide gel electrophoresis of rabbit y-crystallins. (a) Electrophoresis performed at pH 8.3, (i) unfractionated rabbit y-crystallin, (ii) y-IIIa, (iii) y-IIIb, (iv) y-IV. (b) Electrophoresis performed at pH 4.5. (i) y-IIIa; (ii) y-IIIb, (iii) IV(i), (iv) IV(2). (c) Electrophoresis at pH 8.3 and molecular weight estimation of ‘aged ’ rabbit y-IV(S). The elution profile is that of y-IV(B) on Sephadex G-75 gel filtration. The inset shows the electrophoretic gels of ‘aged’ rabbit y-IV(S) before, (iii) and (iv), and after, (i) and (ii), passage through the Sephadex column.
more consistently as single crystals (Fig. 5), although the crystals still have a tendency to grow in microlayers. The diffraction pattern of y-IVa (Fig. 6) shows that it is possible to grow excellent? well ordered, highly diffracting crystals of this early foetal protein. The predominant component of adult bovine y-crystallin, purified from lens cortex by gel filtration on Sephadex G-75 followed by DEAE-Sephadex and sulphopropyl Sephadex chromatography [Fig. 1 (c)l, was treated to identical crystallization conditions as calf y-II. The solution grew many small crystals of typical y-11 habit. Although the crystals were not big enough to obtain X-ray diffraction photographs, the evidence would so far suggest that the predominant adult y-crystallin is the same protein as calf y-II. It was considered of interest to compare y-crystallins from a different species of mammal and so a purification system for rabbit y-crystallins was devised. The young adult lenses which were used in this study did not easily separate into a cortical and nuclear region, a property which may be related to their uniquely simple suture system
528
c.
SLINGSBY
AI-CD
L. Rm. MILLER
(Harding, Susan and Murphy, 1976) and hence only a whole lens extraction was performed. y-Crystallin was separated from oligomeric crystallins by gel filtration, and from @crystallin by ion-exchange chromatography exactly as described for calf lens y-crystallin. Rabbit y-crystallin on sulphopropyl Sephadex chromatography revealed four components which have been labelled II, IIIa, IIIb and IV [Fig. 7(a)]. This nomenclature is provisional and is based on calling the most predominant component y-11. A proper classification must await further amino acid sequencing studies or gene sequenciug studies whereby y-crystallins can be assigned to a family name which survives species divergence. Amino acid composition and C-terminal analyses of rabbit y-crystallins have demonstrated that at least three components, capable of resolution on carboxymethyl-cellulose chromatography, are related to bovine y-crystallins (Hines and Olive, 1970). However, this same study also demonstrated that rabbit y-crystallins clearly exhibited variation from calf y-crystallins such as the absence of alanine and threonine from their amino acid compositions. The component fractions II, IIIb and IV were further chromatographed on DEAE-cellulose using a linear salt gradient at pH 9. Although IIIb appeared largely homogeneous [Fig. 7(c)], fraction II resolved into two components which have been called II(l) and 11(2) [Fig. 7(b)], and fraction IV likewise separates into two components, IV(l) and IV(2) [Fig. 7(d)]. Gel electrophoresis at pH Q*q -.vas performed on rabbit y-crystallins after their purification from oligomeric crystallins on gel filtration and ion exchange chromatography on DEAE-Sephadex. Electrophoresis revealed proteins of predominantly two electrophoretic mobilities [Fig. S(a)i]. Fractions IIIa, IIIb and IV were chromatographed on sulphopropyl Sephadex and then subjected to gel electrophoresis at pH 8.3 which revealed that the more basic band is IIIa [Fig. B(a)ii] and the slightly more acidic band is comprised of IIIb and IV [Fig. 8( a )’iii ., and iv]. Fraction II does not enter the gel at this pH. Fractions IIIa and IIIb however corn&rate on gel electrophoresis at pH 45 [Fig. B(b)i and ii] whereas fractions IV(l) and W(2), separated from each other by DEAE-cellulose chromatography, display different mobilities at the acidic pH [Fig. B(b)iii and iv]. Attempts were made to crystallize all of the rabbit y-fractions but the only promising result was with y-II(l). Small, plate-like hexagonal crystals appeared in the test-tube but failed to grow to a size large enough for X-ray diffraction photography and hence the definitive proof that the crystals were protein was not obtained. One of the components of rabbit y-crystallin was selected for further investigation of possible ageing effects. Rabbit fraction IV(2) which had been left in solution at 20% for six months was subjected to electrophoresis at pH %3 whereupon it was observed that in addition to the original basic band typical of the freshly isolated preparation, a further four bands appeared, each apparently possessing consecutive additional negative charges [Fig. 8(c)iv]. The approximate molecular weight of this ‘aged’ rabbit fraction IV(2) was determined by passage through a gel filtration column previously calibrated with freshly prepared calf y-II. The rabbit sample eluted predominantly as a molecule the size of calf y-II with a small amount of higher molecular weight material. The fractions corresponding to the major peak, after pooling and concentrating, were subjected to electrophoresis. It can be seen from Fig. S(c)i that a similar collection of electrophoretic forms is present in the monomeric fraction and hence it can be concluded that this ageing effect of rabbit Y-IV(~) does not lead to obligatory self-oligomer formation. Although lens cytoplasm is a complex system of polydisperse crystallins interacting
y-CRPSTALLIN
529
with each other and cell components, it is possible to extract and purify many of the low molecular weight proteins. Moreover, it is also possible to control the conditions such that most of the calf y-crystallins self-associate in the crystalline state back to a protein concentration equal to that of the lens. Some of the y-crystallins are labile in that under certain conditions not far removed from physiological, they became more negatively charged. Calf y-crystallin IIIa and IIIb stored in the deep freeze under mildly alkaline conditions will generate electrophoretic variants. It is important to differentiate these modified crystallins from gene product proteins of similar electrophoretic mobility. The possibility that similar modifications may occur ‘in vivo’ remains to be investigated. Nevertheless y-crystallins with varying amino acid sequences and their modified fractions provide an ideal system for comparing their relative potentials for self-association and other crystallin interactions. ACKNOWLEDGMENTS The support of the Medical Research Council is gratefully acknowledged. Rabbit Company for the gift of rabbit lenses. We thank all of our Crystallography Department.
We thank colleagues
Buxted in the
REFERENCES Bjork,
I. (1961). Studies on y-crystallin from calf lens I. Isolation by gel filtration. Exp. Eye Res. I, 145-54. Rjbrk, I. (1964). Studies on y-crystallin from calf lens II. Purification and some properties of the main protein components. Exp. Eye Res. 3, 254-61. Bloemendal, H. (1981). In Molecular and Cellular Biologyofthe Eye Lens (Ed. H. Bloemendal). Pp. l-47. John Wiley, New York. Blundell, T. L., Lindley, P. F., Ness, D. S., Slingsby, C., Tickle, I. J. and Turnell, W. G. (1978). The low resolution structure analysis of the lens protein y-crystallin. Acta Cry&. 34, 3653-7. ell, T. L.; Lindley, P. F., Miller, L., Moss, D. S., Slingsby, C., Tickle, I.; Turnell, B. and Wistow, G. (1981). The molecular structure and stability of the eye-lens: X-ray ana.lysis of y-crystallin II. Nature 289, 771-7. Carlisle, C. H., Lindley, P. F., Moss, D. S. and Slingsby, C. (1977). Preliminary X-ray crystallographic study of the bovine lens protein, y-crystallin II. J. Mol Biol. 110, 417-19. Chirgadze, Y. N., Nikonov, S. V., Garber, RI. B. and Reshetnikova, L. S. (1977). Crystallographio study of y-crystallins from calf lens. J. Mol. Biol. 110, 619-24. Chirgadze, Y. N.. Sergeev, Y. V., Fomenkova, N. P. and Oreshin, V. D. (1981). Polypeptide chain pathway in y-crystallin IIIb from calf lens at 3 A resolution. FEBS Lett. 131,81-4. no acid sequence of y-crystallin (Fraction II) from calf lens. Garber, M. B. and Reshetnikova, L. S. (1976). Growing single crystals of calf y-crystallin from the calf crystalline lens. Doklady Akademii Nauk SSSR 226, 1452-4. Warding, J. J. and Dilley, K. J. (197.6). Structural proteins of the mammalian lens: a review with emphasis on changes in development, ageing and cataract. Exp. Eye Res. 22, l-75. Fiarding, C. V.: Susan, S. and Murphy, H. (1976). Scanning electron microscopy of the adult rabbit lens. Ophthalmic. Res. 8, 443-55. Hines. NM. C. and Olive, J. A. (1970). On the components of rabbit lens y-crystallin. &+e Sciences 9, 1101-10. Papaconstantinou, J. (1965). Biochemistry of bovine lens proteins II. The y-crystallins of adult bovine, calf and embryonic lenses. Biochim. Biophys. Acta 107, 81-90. Philipson; B. (1969). Distribution of protein within the normal rat lens. Invest. Opht/&mol. 8, 258-70. Piatigorsky, J. (1981). Lens differentiation in vertebrates. A review of cellular and molecular
features. Difleerentiation
19, 134-53.
530
Rink,
C. SLINGSBY
H. (1978). The water Geront.
content
AND
L. R. MILLER
in bovine lenses during
ageing. Interdiscipl.
Topics
12, 271-7. (1971). In Methods
Shuster, L. in Enzynzology, vol. XXII. (Ed. Jakoby, W. B.). Pp. 412-33. Academic Press, London. Slingsby, C. and Croft, L. R. (1973). Developmental changes in the low molecular weight proteins of the bovine lens. Exp. Eye Res. 17, 369-76. Slingsby, C. and Croft, L. R. (1978). Structural studies on calf lens y-crystallin fraction IV: a comparison of the cysteine-containing tryptic peptides with the corresponding amino acid sequence of y-erystallin fraction II. Exp. Eye Res. 26, 291-304. van Dam, A. F. (1966). Purification and composition studies of /&+crystallin. Exp. Eye Res. 5, 255-66. Van Heyningen, R. (1972). The human lens III. Some observations on the post-mortem lens. Exp.
Eye
Res.
13, 155-60.
Waley, S. G. (1969). In Tke Eye, vol. 1 (2nd edn). (Ed. Davson, H.). Pp. 291-379. Academic Press, London. Wannemacher, C. F. and Spector, A. Protein synthesis in the core of calf lens. Exp. Eye Res. 7, 623-25. Wistow, G., Lindley, P. F., Miller, L., Moss, D., Slingsby, C., Turnell, B. and Blundell, T. L. (1983). X-ray analysis of the eye-lens protein y-crystallin II at 1.9 A resolution. J. Mol. Biol. (In press).