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Biochemical evidence for conversion to milder form of hereditary mouse cataract by different genetic background
Exp. Eye Res. (1991) 52. 501-506
Biochemical Evidence for Conversion Hereditary Mouse Cataract by Different EIKO
WADAa,
HIROKO
KOYAMA-ITObAND
to Milder Genetic AK10
Form of Background
MATSUZAWA”
aDepartment of Cell Chemistry, b Cyclotron Laboratory and c Laboratory Animal Research Center, institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 708, Japan (Received
6 June
1989 and accepted
in revised form 13 August
1990)
Congenic hereditary cataract mice, BALB/c-net/n& were established by introducing the net genefrom Nakano into BALB/c mice. Thesemice developeda milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacifiedsometime later, and opacifiedlensesin the samegeneticand aging statuses.We comparedthe age-relatedchangesin water content and water-solubleand -insolublefractionsamongthesethree types of lenses.Congenicclear lensesand opaquelensesweremore similarto BALB/c normal clear lensesand Nakano opaqueones,respectively,in theseparameters.Theseresultssuggest,in addition to formation of aggregatedcrystallins and their accumulation in water-insolublefractions, that decreasedprotein synthesis,increasedprotein degradationand augmentedleakageof crystallin might have a significant role in the net-inducedlensopacification. Key words: hereditary cataract ; congenicnet mice: lenswater content; lensproteins. 1. Introduction
Hereditary diseases may be controlled by complicated interactions of the primary gene responsible for them with multiple background genes. We have developed such a situation in the mouse hereditary cataract by introducing the recessive autosomal cataract gene of Nakano mice (Nakano et al., 1960), net, into BALB/c mice using repeated backcrosses to elucidate the
effects of background genes on its expression (Matsuzawa and Wada, 1988). Interestingly, the resulting congenic mice, BALB/c-nct/nct, are different from the original Nakano mice in several respects. Cataract develops significantly later in life in congenic mice than in Nakano mice. In addition, the onset of lens opacification was earlier in females than in males in congenic mice, but similar in both sexes in Nakano mice. Moreover, the cataract disease process was different between both types of mice : in congenic mice lens opacification commenced in a diffuse form at the cortex, whereas in Nakano mice lens opacification commenced in a pin-head form at the core. These results have provided evidence that the n&-induced
hereditary cataract is influenced by background genes and is far milder in BALB/c than in Nakano mouse backgrounds. In the current study we compared the water content and water-soluble and -insoluble proteins of lenses among normal BALB/c, congenic BALB/c-nct/nct and Nakano mice in order to provide
biochemical
support for these findings.
previously (Wada et al., 1978), and normal BALB/c mice bred at the Laboratory Animal Research Center (Tanaka et al., 1987) were used in this study. All mice were allowed to eat MB-l pellets (Funabashi Nojo, Funabashi-city, Japan) and drink tap water freely in aluminium cages in a temperature and light-cycle controlled (12-hr light/l2-hr dark) room. Mice of various ages were killed by ether overdose, and normal and cataractous lenses were carefully enucleated with scissors, cleaned of adherent tissues and pooled. Several lenses were placed in a small testtube of known weight and weighed directly to obtain
the wet weight and again after lyophilization to obtain the dry weight. The water content of lenses was calculated by subtracting the dry weight from the wet weight and expressed in percent irrespective of side and sex in each group-wet lens weights have been shown not to differ between right and left sides or between males and females in the same age group (Matsuzawa and Wada, 1988). Lens proteins were separated by the procedure previously described (Geisler and Weber, 1981). Briefly, lenses were homogenized in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.005 M MgCl, and 0.01 M 2-ME with a Teflon-glass homogenizer and centrifuged at 36 000 g at 4°C for 20 min to separate water-
soluble protein in the supernatant. Gel chromatography of the water-soluble protein was carried out as described previously (Wada et al., 198 1) and crystallins were identified using the standards isolated from calf lenses.
2. Materials and Methods
Congenic BALB/c-nctlnct (Matsuzawa and Wada, 1988), Nakano cataract mice produced as described 00144835/91/050501+06 35
$03.00/O
The precipitate was washed with the same buffer solution as described above, ethanol and ether, dried and directly weighed to obtain the quantity of waterinsoluble protein. The precipitate was then suspended 0 1991 AcademicPressLimited EER
52
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502
in 8 M urea containing 0.05 M Tris-HCl (pH 7.4), O-05 M DTT, homogenized using a Teflon-glass homogenizer and centrifuged at 36 000 g for 30 min. The supernatant, designated the urea-soluble fraction of the water-insoluble protein. was separated into subfractions 14 by gel chromatography on Sepharose 6B column (180 x 15 mm ; flow rate, 0.05 ml min-’ ; O-72 ml per tube) using the homogenization solution for equilibration and elution (Fig. 3). The proportions of the subfractions were compared among normal and cataractous lenses from the three strains of mice. The total protein content of lenses was determined by the method of Lowry et al. (1951) using the alkaline digest of lens protein and BSA as a standard. The urea-insoluble fractions of lenses from 6-monthold mice were electrophoresed in SDS-polyacrylamide gels (Laemmli, 1970) and stained with Coomassie Brilliant Blue. 3. Results Water Content of Lenses Changes in the lens water content with age are depicted for normal BALB/c, cataract BALB/c-nct/nct and Nakano mice in Fig. 1. Noticeably, Nakano mice
ET AL.
were different from normal BALB/c mice in this parameter, and congenic mice were intermediated between them. In Nakano mice, the lens water content decreasedup to 10 days of age in the same course as that in normal BALB/c mice. However, it began to increase from about 18 days of age or just prior to commencement of lens opacification in a pin-head form and reached a plateau level of about 70% at 50 days of age (Iwata and Kinoshita. 1971). In contrast, the water content decreasedsteeply up to 30 days of age and then slowly to reach 5 3 Y0at 6 months of age in BALB/c mice. In clear lenses of BALB/c-nct/nct mice the water content decreasedin a similar course as in normal BALB/c lensesup to 100 days of age, although the initial decline was slightly slower in the former. Interestingly, the water content at 6 months of age was significantly higher in congenic clear lensesthan in normal ones.However, the water content of opaque lensesfrom congenic mice remained at about the 60 % level from 40 days of age, when lens opacillcation began to develop, to 70 days of age when 30% of male and 70% of female mice had visibly opacified lenses (Matsuzawa and Wada, 1988). The water content then increased slowly to reach the Nakano mouse level at 6 months of age. The water content of lenses at the end of the 6-month observation period was 53.4 % in normal eyes of BALB/c mice, 71.2 % in cataractous eyes of Nakano mice and 54.5 and 65.5 “/o in non-cataractous and cataractous eyes, respectively, of BALB/c-nctlnct mice. The wet weights were 7.7. 3.6. 6.6 and 5.4 mg, respectively. Water-solubleProtein
501
’ IO
0
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’ 30
’ 40
’ 50
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’ 80
’ 90
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Tk-zti
Aqe (days1
FIG. 1. Changesof water content with agein normal clear lensesof BALB/c (a), opaquelensesof Nakano (0 ), and clear (A) and opaque(A) lenses of BALB/c-nct/nct mice. Barsindicate k S.E.M.
I””
I
90 80
I
IO 0
I IO
I 20
I1 30
40
I 50
I 60
I 70
I 80
I, 90 100
Aqe (days)
Changesin percentageof water-solubleprotein with age in normal clear lenses of BALB/c (a), opaque lensesof Nakano (0). and clear (A) and opaque(A) lenses FIG.
2.
of BALB/c-nct/nct
mice. Bars indicate +
S&M.
In the three strains of mice the relative percentage of the water-soluble to the total lens protein altered with age as illustrated in Fig. 2. In normal BALB/c mice it decreasedslowly with age at an approximately constant rate: 90% at 10 days and 64% at 80 days of age. In Nakano mice, however, it dramatically decreasedfrom 90% at 10 days to 30% at 60 days of age during which time all mice developed mature cataract and then declined at the same rate as in normal mice: these results agree with our previous ones (Wada et al., 1981). BALB/c-nct/nct mice were intermediate between these two strains for this parameter. It was noticed, however, that the time course of the decrease in percent of water-soluble protein in the clear lenses of BALB/c-nctjnct mice were more similar to that in BALB/c lensesthan that in Nakano lensesand, further, that the time course in opaque lenses of BALBlc-nctlnct mice were more similar to that in Nakano lenses than in BALB/c lenses.The water-soluble protein levels at 6 months of age were 69.1 y0in BALB/c lenses,6 1.4 y0in clear and 30.3% in opaque BALB/c-nctlnct lenses, and 25.0% in Nakano mice lenses. The water-soluble protein was further separated
HEREDITARY
CATARACT
AND
BACKGROUND
503
GENES
TABLE
changes in fens protein
Age-dependent
Mouse strain
4% (months)
BALB/ct
1
1
BALB/c-
6 12 1
nctlnct
in normal and cataractous
Lens Water-soluble protein Conprotein (mg per lens) dition of (mg per lens lens) Total HMW a /I y
6 12 Nakanot
I
6 6 12 12
2.23 3.59 4.47 1.10 1.04 1.02 1.93 3.21 1.85 4.04 1.67
Clear Clear Clear Opaque Opaque Opaque Clear Clear Opaque
Clear Opaque
1.87 2.48 2.87 0.45 0.26 0.21 1.51 1.97 0.56 1.89 0.48
0.03 0.07 0.09 0.01 0.03 0.03 0.03 0.11 0.15 0.13 0.23
O+ll 0.73 0.88 0.12 0.08 0.05 0.41 0.50 0.08 0.45 0.05
0.57 0.86 1.03 0.15 0.09 0.06 045 0.62 0.16 0.68 0.10
0.70 0.74 0.77 0.11 0.01 0.01 0.57 0.67 0.13 0.56 0.05
mice
LMW
Water-insoluble protein urea-soluble* (mg per lens) ---.Total 1 2 3 4
0.06 0.08 0.10 0.06 0.05 0.06 0.05 0.07 0.04 0.08 0.07
0.31 0.92 0.23 1.34 0.54 0.72 0.12 0.75 0.37 1.09 0.25 1.19 0.06 1.99 1.10 0.06
0.34 0.36 0.42 0.77 0.50
0.18 017 0.04 0.20 0.20 0.22 015 0.21 0.18 0.36
Total ureainsoluble protein (mg per lens) 0.05
0.19 0.2 1 0.06 0.06 0.06 0.05 0.15 0.10 0.16 0.09
Abbreviations: wt. weight: HMW, high molecular weight protein: a, /3 and y, a-. /I- and y-crystalk, respectively: LMW. low molecular weight protein. * See the legend to Fie. 3 for definition of urea-soluble fraction (USFs) 1 to 4. ? From Wa;da et al. (i981).
2-l ll!!lcd
““i I.5
3
I.0
0.5
GL
Tube
numbel
FIG. 3. Separation by gel chromatography of the urea-soluble protein of the water-insoluble fraction in normal clear lenses (A) of BALB/c, and opaque lenses of Nakano (B) and BALB/c-nct/nct mice (C) at 6 months of age. The amounts of protein applied in (B) and (C) were approximately four times and twice that in (A), respectively. Urea-soluble fraction (USFs) 1 to 4 were determined by tube numbers as indicated.
into a-, ,& and y-crystallins, and high (HMW) and low molecular weight (LMW) proteins by chromatography in order to compare their proportions in transparent and opaque lenses from the three mice. The results are presented in Table I. The increase of HMW and marked decrease of y-crystallin in opacified lenses of Nakano mice, and the marked increase of HMW protein and decrease of all crystallins in opacified lenses of BALB/c-nct/nct mice were noticed especially when compared with BALB/c mice. The proportions of these protein fractions were similar in clear lenses from BALB/c and congenic mice at 1 month of age. Interestingly, however, the ol-crystallin content was relatively lower and the HMW protein content much higher in clear lenses from BALB/c-nct/nct mice than in those from BALB/c mice at 6 months of age.
Water-insolubleProtein
Opaque lenses from both Nakano
and BALB/cof waterinsoluble protein than clear lenses from age-matched normal and congenic BALB/c mice (Table I). The water-insoluble protein was mostly solubilized with urea. This solubilized protein was separated into four subfractions by gel chromatography (Fig. 3) designated urea-soluble fraction (USF’s) 14 in tube number order. At 6 months of age, the proportions of these four fractions were practically the same in the transparent lenses from BALB/c and congenic mice, while the opaque lenses were characterized by a larger proportion of USF 2 from congenic cataract and both LJSF 2 and 4 from Nakano cataract strain (Fig. 3). The nct/nct mice always had a higher proportion
35-2
504
E. WADA BALB/C Clear Lane
BALBhnct/nct Clear LOW
ET AL.
BALB/cnctlnct opaque Lane
FIG. 4. Polypeptide composition of USF 1 (lane 1). 2 (lane 2) and 4 (lane 3) from lenses of mice at 6 months of age. The samples (2 5 pg) were heated at 100°C for 2 min in incubation medium containing 2-5 % 2-mercaptoethanol and 2 % SDS in 60 mM TrisHCl, pH 6.8, subjected to electrophoresis in a 16% polyacrylamide slab gel. Gels were stained with Coomassie Brilliant Blue (Bio Rad).
FIG. 5. Photographs of cataractous lenses from a 2-month-old Nakano mouse (A) and a 4-month-old BALB/c-nctlncf mouse (B). Note the nuclear cataract in (A) and cortical cataract in (B). percentage of USF 1, 2, 3 and 4 in total lens protein at 6 months of age were 6.4, 9.5, 5.0 and 4.7 in BALB/c mice, 11.5, 34.6, 3.8 and 19.2 in Nakano mice, and 7.8, 13.1, 6.2 and 6.9 in clear lenses and 3.2, 41.6, 8.1 and 11.4 in opaque lenses of congenic mice, respectively. At 12 months of age. however, USF 2 decreased and USF 4 increased in opaque lenses of congenic mice (Table I). The amount of urea-insoluble protein was greatest in BALB/c lenses. The polypeptides presented in USF 1, 2 and 4 at 6 months of age were analysed by SDS/polyacrylamide gel electrophoresis (Fig. 4). The differences in the pattern of lens polypeptides between Nakano and congenic mouse opaque lenses were detected in USF 1 and 2. Most striking were the disappearance of band 2 in USF 1 and the appearance of clear band 1 in USF 2 in the congenic mice. Both Nakano and congenic mouse opaque lenses had several faintly staining bands under band 9 and showed the same pattern of USF 4. All USF 1, 2 and 4 gave practically the same
electrophoretic pattern in clear lenses from congenic and normal BALB/c mice. The difference in the age-dependent alteration in lens protein components between Nakano and BALB/ c-nct/nct mice might be associated with the distinct disease processes of cataract formation between both strains. In this respect, it should be noted that the former developed nuclear cataract [Fig. S(A)] while the latter had cortical cataract [Fig. S(B)]. 4. Discussion The congenic hereditary cataract mouse, BALB/cnct/nct, is characterized by sporadic and later development of lens opacity and a distinct disease process (cortical vs. nuclear cataract) compared with the original Nakano mice (Matsuzawa and Wada, 1988). These findings were confirmed by the biochemical data described here. Hydration and rapid decrease of the water-soluble fraction are characteristic changes
HEREDITARY
CATARACT
AND
BACKGROUND
GENES
observed in the lenses of Nakano mice (Iwata and Kinoshita, 1971; Wada et al., 1981). In the congenic strain, the water content of opacified lenses increased far more slowly than in the Nakano mouse (Fig. 1). More significantly, the water content of not-yetopacified lenses continued to decrease with age in the same course as did that of normal clear lenses of BALB/c mice. The relative percentage of water-soluble protein decreased with age at significantly higher rates in the following order: Nakano cataractous, congenic opaque, congenic clear, and BALB/c normal lenses. It is important to note, therefore, that normal lenses of BALB/c and clear lenses of BALB/c-nctlnct (due to undergo opacification later in life) had the same content, and that soluble protein content in BALB/cnct/nct mice was significantly lower throughout the 12-month observation period. This suggeststhat lens opacification in congenic mice may be preceded by a reduction in soluble protein and followed by hydration. It is not so easy to determine which of the two phenomena occurs earliest in Nakano mice, since cataract develops suddenly and very early in life (Nakano et al., 1960). In contrast, the newlyestablished model system has the advantage that opacified, not-yet-opacified and normal lensescan be compared in the same genetic background and in the same aging state. Such investigations at the age of 6 and 12 months provided the following significant results (Table I): (1) lens protein accumulation was reduced in both clear and opaque lensesof congenic mice : (2 ) the water-soluble HMW and water-insoluble protien contents were increased in the ordercongenic opacified, congenic not-yet-opacified lenses, and normal clear lenses; and, (3) the content of each crystallin decreased in the same order. In addition to the loss of total lens protein, further increases of the HMW and water-insoluble proteins and decreasesof all crystallins, especially cr-crystallin, were noted in opaque lensesof 12-month-old congenic mice. Consequently, the HMW protein was considered to be a precursor of the water-insoluble protein which scatters light (Spector, 1972). Taken together, these results suggest that the soluble HMW protein may first be formed mainly from ol-crystallin, aggregate with yand /3-crystallins. change into water-insoluble material and give rise to light scattering, which may be recognized as lens opacification. Generation of water-insoluble material at the expense of the water-soluble fraction in lensesseemsto be the most useful and consistent index for progression of cataract. This phenomenon was also observed with aging even in normal mice and accompanied conversion of sulfhydryl groups to disulfide (Anderson. Wright and Spector, 1979) or nonsulfide crosslinks (Buckingham, 1972 ; Roy et al., 1984). It should be noted with respect to this phenomenon, that the water-insoluble fraction was larger in clear lenses from congenic than in normal BALB/c mice in the same age groups (Table I). Thus, it is likely that the net
gene accelerates either the conversion of the watersoluble to insoluble material, or the aging of lenses. With regard to urea solubility of the insoluble fraction, the predominance of USF 2 was marked in opaque lenses of 6-month-old congenic mice, although it is unknown whether the aggregated HMW polypeptides present in it are the same as those found in Nakano mice (Roy et al., 1982) and human senile cataract lenses(Spector and Roy, 1978). By electrophoretic analysis, the large differences in polypeptide composition of USF 1 and 2 between opaque lensesfrom congenic and Nakano mice and clear lensesfrom congenic and normal BALB/c mice, both being similar in this respect, suggest that differential proteolysis or differential protein aggregation might have occurred in the congenic mouse lens. Acknowledgements This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. We thank Drs K. Hirosawa and S. Kanegasaki for their support.
References Anderson, E. J., Wright, D. D. and Spector, A. (1979). The state of sulphydryl groups in normal and cataractous human lens proteins. II. Cortical and nuclear regions. Exp. Eye Res. 29, 23343. Buckingham, R. (19 72). The behaviour of reduced proteins from normal and cataractous lenses in highly dissociating media : cross-linked protein in cataractous lenses. Exp. Eye Res. 14, 123-9. Garner, W. H. and Spector, A. (1978). Racemization of human lens : evidence of rapid insolubilization of specific polypeptides in cataract formation. Proc. Natl. Acad. Sci. U.S.A. 75, 3818-20. Geisler, N. and Weber. K. (1981). Isolation of polymeri-
zation-competentvimentin from porcineeyelenstissue. FEBSLett. 125, 253-6. Iwata, S. and development Ophthalmol. Laemmli. U. K. during the Nature
227,
Kinoshita. J. H. (19 71). Mechanism of of hereditary cataract in mice. Invest. 10, 504-12. (1970). Cleavage of structural proteins assembly of the head of bacteriophage T,. 680-S.
Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. Matsuzawa, A. and Wada, E. (1988). Retarded and distinct progress of lens opacification in congenic hereditary cataract mice, BALB/c-nctlnct. Exp. Eye Res. 47, 705-11.
Nakano, K., Yamamoto, S., Kutsukake, G., Ogawa, H., Nakajima, A. and Takano, E. (1960). Hereditary cataract in mice. ]pn. 1. Clin. Ophthalmol. 14, 196-200. Roy. D., Dillon, J., Wada, E.. Chaney, W. and Spector, A. (1984). No&sulfide polymerization of y- and /3crystallins in the human lens. Proc. Nutl. Acad. Sci. U.S.A. 81, 2378-81. Roy, D.. Garner, M. H., Spector, A., Carper, D. and Russell, P. (1982). Investigation of Nakano lens proteins. Exp. Eye Res. 34, 909-20. Spector, A. (1972). Aggregation of alpha crystallin and its possible relationship to cataract formation. Isr. 1. Med. Sci. 8, 1577-82.
E. WADA
Spector, A. and Roy, D. (1978). DisuIfide-linked high molecular weight protein associated with human cataract. Proc. Natl. Acad. Sci. U.S.A. 75. 3244-8. Tanaka, S., Matsuzawa, A., Kato. H., Sudo, K. and Yamanouchi, K. (1987). Inbred strains of mice maintained at the Institute of Medical Science, University of Tokyo. jpn. J. Exp. Med. 57, 241-S. Wada, E., Matsuzawa, A., Takenawa, T. and Tsumita, T.
ET AL.
(1978). myo-Inositol of the lens in hereditary cataract mice. Exp. Eye Res. 26, 119-22. Wada. E., Sugiura, T.. Nakamura, H. and Tsumita, T. (1981). Studies on lens proteins of mice with hereditary cataract. I. Comparative studies on the chemical and immunological properties of the soluble proteins of cataractous and normal mouse lenses. Biochim. Biophys. Acta 667, 251-9.
Biochemical Evidence for Conversion Hereditary Mouse Cataract by Different EIKO
WADAa,
HIROKO
KOYAMA-ITObAND
to Milder Genetic AK10
Form of Background
MATSUZAWA”
aDepartment of Cell Chemistry, b Cyclotron Laboratory and c Laboratory Animal Research Center, institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 708, Japan (Received
6 June
1989 and accepted
in revised form 13 August
1990)
Congenic hereditary cataract mice, BALB/c-net/n& were established by introducing the net genefrom Nakano into BALB/c mice. Thesemice developeda milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacifiedsometime later, and opacifiedlensesin the samegeneticand aging statuses.We comparedthe age-relatedchangesin water content and water-solubleand -insolublefractionsamongthesethree types of lenses.Congenicclear lensesand opaquelensesweremore similarto BALB/c normal clear lensesand Nakano opaqueones,respectively,in theseparameters.Theseresultssuggest,in addition to formation of aggregatedcrystallins and their accumulation in water-insolublefractions, that decreasedprotein synthesis,increasedprotein degradationand augmentedleakageof crystallin might have a significant role in the net-inducedlensopacification. Key words: hereditary cataract ; congenicnet mice: lenswater content; lensproteins. 1. Introduction
Hereditary diseases may be controlled by complicated interactions of the primary gene responsible for them with multiple background genes. We have developed such a situation in the mouse hereditary cataract by introducing the recessive autosomal cataract gene of Nakano mice (Nakano et al., 1960), net, into BALB/c mice using repeated backcrosses to elucidate the
effects of background genes on its expression (Matsuzawa and Wada, 1988). Interestingly, the resulting congenic mice, BALB/c-nct/nct, are different from the original Nakano mice in several respects. Cataract develops significantly later in life in congenic mice than in Nakano mice. In addition, the onset of lens opacification was earlier in females than in males in congenic mice, but similar in both sexes in Nakano mice. Moreover, the cataract disease process was different between both types of mice : in congenic mice lens opacification commenced in a diffuse form at the cortex, whereas in Nakano mice lens opacification commenced in a pin-head form at the core. These results have provided evidence that the n&-induced
hereditary cataract is influenced by background genes and is far milder in BALB/c than in Nakano mouse backgrounds. In the current study we compared the water content and water-soluble and -insoluble proteins of lenses among normal BALB/c, congenic BALB/c-nct/nct and Nakano mice in order to provide
biochemical
support for these findings.
previously (Wada et al., 1978), and normal BALB/c mice bred at the Laboratory Animal Research Center (Tanaka et al., 1987) were used in this study. All mice were allowed to eat MB-l pellets (Funabashi Nojo, Funabashi-city, Japan) and drink tap water freely in aluminium cages in a temperature and light-cycle controlled (12-hr light/l2-hr dark) room. Mice of various ages were killed by ether overdose, and normal and cataractous lenses were carefully enucleated with scissors, cleaned of adherent tissues and pooled. Several lenses were placed in a small testtube of known weight and weighed directly to obtain
the wet weight and again after lyophilization to obtain the dry weight. The water content of lenses was calculated by subtracting the dry weight from the wet weight and expressed in percent irrespective of side and sex in each group-wet lens weights have been shown not to differ between right and left sides or between males and females in the same age group (Matsuzawa and Wada, 1988). Lens proteins were separated by the procedure previously described (Geisler and Weber, 1981). Briefly, lenses were homogenized in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.005 M MgCl, and 0.01 M 2-ME with a Teflon-glass homogenizer and centrifuged at 36 000 g at 4°C for 20 min to separate water-
soluble protein in the supernatant. Gel chromatography of the water-soluble protein was carried out as described previously (Wada et al., 198 1) and crystallins were identified using the standards isolated from calf lenses.
2. Materials and Methods
Congenic BALB/c-nctlnct (Matsuzawa and Wada, 1988), Nakano cataract mice produced as described 00144835/91/050501+06 35
$03.00/O
The precipitate was washed with the same buffer solution as described above, ethanol and ether, dried and directly weighed to obtain the quantity of waterinsoluble protein. The precipitate was then suspended 0 1991 AcademicPressLimited EER
52
E. WADA
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in 8 M urea containing 0.05 M Tris-HCl (pH 7.4), O-05 M DTT, homogenized using a Teflon-glass homogenizer and centrifuged at 36 000 g for 30 min. The supernatant, designated the urea-soluble fraction of the water-insoluble protein. was separated into subfractions 14 by gel chromatography on Sepharose 6B column (180 x 15 mm ; flow rate, 0.05 ml min-’ ; O-72 ml per tube) using the homogenization solution for equilibration and elution (Fig. 3). The proportions of the subfractions were compared among normal and cataractous lenses from the three strains of mice. The total protein content of lenses was determined by the method of Lowry et al. (1951) using the alkaline digest of lens protein and BSA as a standard. The urea-insoluble fractions of lenses from 6-monthold mice were electrophoresed in SDS-polyacrylamide gels (Laemmli, 1970) and stained with Coomassie Brilliant Blue. 3. Results Water Content of Lenses Changes in the lens water content with age are depicted for normal BALB/c, cataract BALB/c-nct/nct and Nakano mice in Fig. 1. Noticeably, Nakano mice
ET AL.
were different from normal BALB/c mice in this parameter, and congenic mice were intermediated between them. In Nakano mice, the lens water content decreasedup to 10 days of age in the same course as that in normal BALB/c mice. However, it began to increase from about 18 days of age or just prior to commencement of lens opacification in a pin-head form and reached a plateau level of about 70% at 50 days of age (Iwata and Kinoshita. 1971). In contrast, the water content decreasedsteeply up to 30 days of age and then slowly to reach 5 3 Y0at 6 months of age in BALB/c mice. In clear lenses of BALB/c-nct/nct mice the water content decreasedin a similar course as in normal BALB/c lensesup to 100 days of age, although the initial decline was slightly slower in the former. Interestingly, the water content at 6 months of age was significantly higher in congenic clear lensesthan in normal ones.However, the water content of opaque lensesfrom congenic mice remained at about the 60 % level from 40 days of age, when lens opacillcation began to develop, to 70 days of age when 30% of male and 70% of female mice had visibly opacified lenses (Matsuzawa and Wada, 1988). The water content then increased slowly to reach the Nakano mouse level at 6 months of age. The water content of lenses at the end of the 6-month observation period was 53.4 % in normal eyes of BALB/c mice, 71.2 % in cataractous eyes of Nakano mice and 54.5 and 65.5 “/o in non-cataractous and cataractous eyes, respectively, of BALB/c-nctlnct mice. The wet weights were 7.7. 3.6. 6.6 and 5.4 mg, respectively. Water-solubleProtein
501
’ IO
0
’ 20
’ 30
’ 40
’ 50
’ 60
’ 70
’ 80
’ 90
’ 100
Tk-zti
Aqe (days1
FIG. 1. Changesof water content with agein normal clear lensesof BALB/c (a), opaquelensesof Nakano (0 ), and clear (A) and opaque(A) lenses of BALB/c-nct/nct mice. Barsindicate k S.E.M.
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I
90 80
I
IO 0
I IO
I 20
I1 30
40
I 50
I 60
I 70
I 80
I, 90 100
Aqe (days)
Changesin percentageof water-solubleprotein with age in normal clear lenses of BALB/c (a), opaque lensesof Nakano (0). and clear (A) and opaque(A) lenses FIG.
2.
of BALB/c-nct/nct
mice. Bars indicate +
S&M.
In the three strains of mice the relative percentage of the water-soluble to the total lens protein altered with age as illustrated in Fig. 2. In normal BALB/c mice it decreasedslowly with age at an approximately constant rate: 90% at 10 days and 64% at 80 days of age. In Nakano mice, however, it dramatically decreasedfrom 90% at 10 days to 30% at 60 days of age during which time all mice developed mature cataract and then declined at the same rate as in normal mice: these results agree with our previous ones (Wada et al., 1981). BALB/c-nct/nct mice were intermediate between these two strains for this parameter. It was noticed, however, that the time course of the decrease in percent of water-soluble protein in the clear lenses of BALB/c-nctjnct mice were more similar to that in BALB/c lensesthan that in Nakano lensesand, further, that the time course in opaque lenses of BALBlc-nctlnct mice were more similar to that in Nakano lenses than in BALB/c lenses.The water-soluble protein levels at 6 months of age were 69.1 y0in BALB/c lenses,6 1.4 y0in clear and 30.3% in opaque BALB/c-nctlnct lenses, and 25.0% in Nakano mice lenses. The water-soluble protein was further separated
HEREDITARY
CATARACT
AND
BACKGROUND
503
GENES
TABLE
changes in fens protein
Age-dependent
Mouse strain
4% (months)
BALB/ct
1
1
BALB/c-
6 12 1
nctlnct
in normal and cataractous
Lens Water-soluble protein Conprotein (mg per lens) dition of (mg per lens lens) Total HMW a /I y
6 12 Nakanot
I
6 6 12 12
2.23 3.59 4.47 1.10 1.04 1.02 1.93 3.21 1.85 4.04 1.67
Clear Clear Clear Opaque Opaque Opaque Clear Clear Opaque
Clear Opaque
1.87 2.48 2.87 0.45 0.26 0.21 1.51 1.97 0.56 1.89 0.48
0.03 0.07 0.09 0.01 0.03 0.03 0.03 0.11 0.15 0.13 0.23
O+ll 0.73 0.88 0.12 0.08 0.05 0.41 0.50 0.08 0.45 0.05
0.57 0.86 1.03 0.15 0.09 0.06 045 0.62 0.16 0.68 0.10
0.70 0.74 0.77 0.11 0.01 0.01 0.57 0.67 0.13 0.56 0.05
mice
LMW
Water-insoluble protein urea-soluble* (mg per lens) ---.Total 1 2 3 4
0.06 0.08 0.10 0.06 0.05 0.06 0.05 0.07 0.04 0.08 0.07
0.31 0.92 0.23 1.34 0.54 0.72 0.12 0.75 0.37 1.09 0.25 1.19 0.06 1.99 1.10 0.06
0.34 0.36 0.42 0.77 0.50
0.18 017 0.04 0.20 0.20 0.22 015 0.21 0.18 0.36
Total ureainsoluble protein (mg per lens) 0.05
0.19 0.2 1 0.06 0.06 0.06 0.05 0.15 0.10 0.16 0.09
Abbreviations: wt. weight: HMW, high molecular weight protein: a, /3 and y, a-. /I- and y-crystalk, respectively: LMW. low molecular weight protein. * See the legend to Fie. 3 for definition of urea-soluble fraction (USFs) 1 to 4. ? From Wa;da et al. (i981).
2-l ll!!lcd
““i I.5
3
I.0
0.5
GL
Tube
numbel
FIG. 3. Separation by gel chromatography of the urea-soluble protein of the water-insoluble fraction in normal clear lenses (A) of BALB/c, and opaque lenses of Nakano (B) and BALB/c-nct/nct mice (C) at 6 months of age. The amounts of protein applied in (B) and (C) were approximately four times and twice that in (A), respectively. Urea-soluble fraction (USFs) 1 to 4 were determined by tube numbers as indicated.
into a-, ,& and y-crystallins, and high (HMW) and low molecular weight (LMW) proteins by chromatography in order to compare their proportions in transparent and opaque lenses from the three mice. The results are presented in Table I. The increase of HMW and marked decrease of y-crystallin in opacified lenses of Nakano mice, and the marked increase of HMW protein and decrease of all crystallins in opacified lenses of BALB/c-nct/nct mice were noticed especially when compared with BALB/c mice. The proportions of these protein fractions were similar in clear lenses from BALB/c and congenic mice at 1 month of age. Interestingly, however, the ol-crystallin content was relatively lower and the HMW protein content much higher in clear lenses from BALB/c-nct/nct mice than in those from BALB/c mice at 6 months of age.
Water-insolubleProtein
Opaque lenses from both Nakano
and BALB/cof waterinsoluble protein than clear lenses from age-matched normal and congenic BALB/c mice (Table I). The water-insoluble protein was mostly solubilized with urea. This solubilized protein was separated into four subfractions by gel chromatography (Fig. 3) designated urea-soluble fraction (USF’s) 14 in tube number order. At 6 months of age, the proportions of these four fractions were practically the same in the transparent lenses from BALB/c and congenic mice, while the opaque lenses were characterized by a larger proportion of USF 2 from congenic cataract and both LJSF 2 and 4 from Nakano cataract strain (Fig. 3). The nct/nct mice always had a higher proportion
35-2
504
E. WADA BALB/C Clear Lane
BALBhnct/nct Clear LOW
ET AL.
BALB/cnctlnct opaque Lane
FIG. 4. Polypeptide composition of USF 1 (lane 1). 2 (lane 2) and 4 (lane 3) from lenses of mice at 6 months of age. The samples (2 5 pg) were heated at 100°C for 2 min in incubation medium containing 2-5 % 2-mercaptoethanol and 2 % SDS in 60 mM TrisHCl, pH 6.8, subjected to electrophoresis in a 16% polyacrylamide slab gel. Gels were stained with Coomassie Brilliant Blue (Bio Rad).
FIG. 5. Photographs of cataractous lenses from a 2-month-old Nakano mouse (A) and a 4-month-old BALB/c-nctlncf mouse (B). Note the nuclear cataract in (A) and cortical cataract in (B). percentage of USF 1, 2, 3 and 4 in total lens protein at 6 months of age were 6.4, 9.5, 5.0 and 4.7 in BALB/c mice, 11.5, 34.6, 3.8 and 19.2 in Nakano mice, and 7.8, 13.1, 6.2 and 6.9 in clear lenses and 3.2, 41.6, 8.1 and 11.4 in opaque lenses of congenic mice, respectively. At 12 months of age. however, USF 2 decreased and USF 4 increased in opaque lenses of congenic mice (Table I). The amount of urea-insoluble protein was greatest in BALB/c lenses. The polypeptides presented in USF 1, 2 and 4 at 6 months of age were analysed by SDS/polyacrylamide gel electrophoresis (Fig. 4). The differences in the pattern of lens polypeptides between Nakano and congenic mouse opaque lenses were detected in USF 1 and 2. Most striking were the disappearance of band 2 in USF 1 and the appearance of clear band 1 in USF 2 in the congenic mice. Both Nakano and congenic mouse opaque lenses had several faintly staining bands under band 9 and showed the same pattern of USF 4. All USF 1, 2 and 4 gave practically the same
electrophoretic pattern in clear lenses from congenic and normal BALB/c mice. The difference in the age-dependent alteration in lens protein components between Nakano and BALB/ c-nct/nct mice might be associated with the distinct disease processes of cataract formation between both strains. In this respect, it should be noted that the former developed nuclear cataract [Fig. S(A)] while the latter had cortical cataract [Fig. S(B)]. 4. Discussion The congenic hereditary cataract mouse, BALB/cnct/nct, is characterized by sporadic and later development of lens opacity and a distinct disease process (cortical vs. nuclear cataract) compared with the original Nakano mice (Matsuzawa and Wada, 1988). These findings were confirmed by the biochemical data described here. Hydration and rapid decrease of the water-soluble fraction are characteristic changes
HEREDITARY
CATARACT
AND
BACKGROUND
GENES
observed in the lenses of Nakano mice (Iwata and Kinoshita, 1971; Wada et al., 1981). In the congenic strain, the water content of opacified lenses increased far more slowly than in the Nakano mouse (Fig. 1). More significantly, the water content of not-yetopacified lenses continued to decrease with age in the same course as did that of normal clear lenses of BALB/c mice. The relative percentage of water-soluble protein decreased with age at significantly higher rates in the following order: Nakano cataractous, congenic opaque, congenic clear, and BALB/c normal lenses. It is important to note, therefore, that normal lenses of BALB/c and clear lenses of BALB/c-nctlnct (due to undergo opacification later in life) had the same content, and that soluble protein content in BALB/cnct/nct mice was significantly lower throughout the 12-month observation period. This suggeststhat lens opacification in congenic mice may be preceded by a reduction in soluble protein and followed by hydration. It is not so easy to determine which of the two phenomena occurs earliest in Nakano mice, since cataract develops suddenly and very early in life (Nakano et al., 1960). In contrast, the newlyestablished model system has the advantage that opacified, not-yet-opacified and normal lensescan be compared in the same genetic background and in the same aging state. Such investigations at the age of 6 and 12 months provided the following significant results (Table I): (1) lens protein accumulation was reduced in both clear and opaque lensesof congenic mice : (2 ) the water-soluble HMW and water-insoluble protien contents were increased in the ordercongenic opacified, congenic not-yet-opacified lenses, and normal clear lenses; and, (3) the content of each crystallin decreased in the same order. In addition to the loss of total lens protein, further increases of the HMW and water-insoluble proteins and decreasesof all crystallins, especially cr-crystallin, were noted in opaque lensesof 12-month-old congenic mice. Consequently, the HMW protein was considered to be a precursor of the water-insoluble protein which scatters light (Spector, 1972). Taken together, these results suggest that the soluble HMW protein may first be formed mainly from ol-crystallin, aggregate with yand /3-crystallins. change into water-insoluble material and give rise to light scattering, which may be recognized as lens opacification. Generation of water-insoluble material at the expense of the water-soluble fraction in lensesseemsto be the most useful and consistent index for progression of cataract. This phenomenon was also observed with aging even in normal mice and accompanied conversion of sulfhydryl groups to disulfide (Anderson. Wright and Spector, 1979) or nonsulfide crosslinks (Buckingham, 1972 ; Roy et al., 1984). It should be noted with respect to this phenomenon, that the water-insoluble fraction was larger in clear lenses from congenic than in normal BALB/c mice in the same age groups (Table I). Thus, it is likely that the net
gene accelerates either the conversion of the watersoluble to insoluble material, or the aging of lenses. With regard to urea solubility of the insoluble fraction, the predominance of USF 2 was marked in opaque lenses of 6-month-old congenic mice, although it is unknown whether the aggregated HMW polypeptides present in it are the same as those found in Nakano mice (Roy et al., 1982) and human senile cataract lenses(Spector and Roy, 1978). By electrophoretic analysis, the large differences in polypeptide composition of USF 1 and 2 between opaque lensesfrom congenic and Nakano mice and clear lensesfrom congenic and normal BALB/c mice, both being similar in this respect, suggest that differential proteolysis or differential protein aggregation might have occurred in the congenic mouse lens. Acknowledgements This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. We thank Drs K. Hirosawa and S. Kanegasaki for their support.
References Anderson, E. J., Wright, D. D. and Spector, A. (1979). The state of sulphydryl groups in normal and cataractous human lens proteins. II. Cortical and nuclear regions. Exp. Eye Res. 29, 23343. Buckingham, R. (19 72). The behaviour of reduced proteins from normal and cataractous lenses in highly dissociating media : cross-linked protein in cataractous lenses. Exp. Eye Res. 14, 123-9. Garner, W. H. and Spector, A. (1978). Racemization of human lens : evidence of rapid insolubilization of specific polypeptides in cataract formation. Proc. Natl. Acad. Sci. U.S.A. 75, 3818-20. Geisler, N. and Weber. K. (1981). Isolation of polymeri-
zation-competentvimentin from porcineeyelenstissue. FEBSLett. 125, 253-6. Iwata, S. and development Ophthalmol. Laemmli. U. K. during the Nature
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Kinoshita. J. H. (19 71). Mechanism of of hereditary cataract in mice. Invest. 10, 504-12. (1970). Cleavage of structural proteins assembly of the head of bacteriophage T,. 680-S.
Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. Matsuzawa, A. and Wada, E. (1988). Retarded and distinct progress of lens opacification in congenic hereditary cataract mice, BALB/c-nctlnct. Exp. Eye Res. 47, 705-11.
Nakano, K., Yamamoto, S., Kutsukake, G., Ogawa, H., Nakajima, A. and Takano, E. (1960). Hereditary cataract in mice. ]pn. 1. Clin. Ophthalmol. 14, 196-200. Roy. D., Dillon, J., Wada, E.. Chaney, W. and Spector, A. (1984). No&sulfide polymerization of y- and /3crystallins in the human lens. Proc. Nutl. Acad. Sci. U.S.A. 81, 2378-81. Roy, D.. Garner, M. H., Spector, A., Carper, D. and Russell, P. (1982). Investigation of Nakano lens proteins. Exp. Eye Res. 34, 909-20. Spector, A. (1972). Aggregation of alpha crystallin and its possible relationship to cataract formation. Isr. 1. Med. Sci. 8, 1577-82.
E. WADA
Spector, A. and Roy, D. (1978). DisuIfide-linked high molecular weight protein associated with human cataract. Proc. Natl. Acad. Sci. U.S.A. 75. 3244-8. Tanaka, S., Matsuzawa, A., Kato. H., Sudo, K. and Yamanouchi, K. (1987). Inbred strains of mice maintained at the Institute of Medical Science, University of Tokyo. jpn. J. Exp. Med. 57, 241-S. Wada, E., Matsuzawa, A., Takenawa, T. and Tsumita, T.
ET AL.
(1978). myo-Inositol of the lens in hereditary cataract mice. Exp. Eye Res. 26, 119-22. Wada. E., Sugiura, T.. Nakamura, H. and Tsumita, T. (1981). Studies on lens proteins of mice with hereditary cataract. I. Comparative studies on the chemical and immunological properties of the soluble proteins of cataractous and normal mouse lenses. Biochim. Biophys. Acta 667, 251-9.