Interaction of an altered β-crystallin with other proteins in the Philly mouse lens

Exp. Eye Res.(1990) 50, 683-687

Interaction

of an Altered

p-Crystallin Philly Mouse

PAUL

AND

National

RUSSELL Institutes

of Health,

CAROLYN Bethesda,

with Lens

Other

Proteins

in the

CHAMBERS MD 20892,

U.S.A.

An altered@B2-crystallinis synthesizedin the lens of the Philly mouse.This pB2 has a more acidic isoelectricpoint than the fl2 that isisolatedfrom normalmouselens.The alteredpB2 is immunologically reactive with antibody to the aminoterminal of the PBZ-crystallin.but appearsto be presentin only very small quantities in the Philly lens. When the solubleproteins are isolatedfrom the Philly lens and chromatographedby gel exclusion chromatography, the pB2 can be found primarily in the heavy molecular weight fraction. Some immunoreactive material was also found throughout the higher molecularweight ,&crystallin region,/3,,, and the lower molecularweight region,/&. Theseresultswould indicate that the altered,!?B2-crystaliinin the Philly lenscan interact with the other P-crystallinsin the lens: however, interactions of the @32-crystallinwith the other proteins of the lens may causerapid aggregationof the cellularproteinsleadingto the formation of the heavy molecularweight material.The increasednumber of theseaggregatesmay eventually leadto the cataract formation in the Philly mouse. Key words: lens: mouse: cataract : BBZ-crystallin: aggregation. 1. Introduction The Philly mouse, a strain derived from the SwissWebster mouse, has been studied because of the hereditary cataract which it develops (Kador et al., 1980). This mouse strain was first detected at the University of Pennsylvania, and has subsequently been developed in the laboratory of Jin Kinoshita. The cataract becomes prominent after 6 weeks, although studies have shown that morphological changes are apparent very shortly after birth (Uga, Kador and Kuwabara, 1980; Kador, Uga and Piatigorsky, 1980). The mouse lens is composed of three major groups of proteins: CI-, /3- and y-crystallins. The arrangement and interactions of these crystallin proteins are essential for transparency to visible light (Benedek, 1971). In the mouse, about 30% of the soluble proteins are ,!?-crystallins. It has recently been shown that a developmentally regulated protein which corresponds to /?B2-crystallin is altered in the Philly mouse lens (Carper, Smith-Gill and Kinoshita. 1984, 1986; Nakamura et al., 1988). The /3B2 protein, previously termed @p, is the principal basic beta crystallin in the lens of the mouse (Carper et al., 1984; Carper, Russell and Sanyal, 1985). This protein has a molecular weight of 27 kDa by SDS-PAGE and becomes part of aggregates with other /%crystallins in the lens (Zigler and Sidbury, 1976). /3B2-crystallin can be detected in the normal mouse lens about 5-10 days after birth and is associatedwith the elongating cells in the equatorial region of the lens rather than the epithelial cells on the anterior surface (Carper et al., 1986). Analysis of the pB2, based on its similarity to the y-crystallins, has indicated that the N-terminal region which is hydrophobic may be instrumental in interactions resulting in dimer formation (Berberset al., 1983). The carboxyl domain which is polar may be involved with surface 00144835/90/060683+05

$03.00/O

contacts leading to subunit aggregation (Slingsby et al., 1988). In the human lens there is an indication that the amino terminal is degraded during aging, although in the bovine lens this area appears to be far more resistant to proteolysis than the carboxyl terminal (Takemoto et al., 1987). One aspect of the PB2-crystallin that is unusual is its heat stability (Mostafapour and Schwartz, 1982, Horwitz et al., 1986). The /I’BZ will remain in solution when the soluble proteins of the lens are heated to 100°C. The protein will then renature and selfaggregate, and this ability is thought to be related to the absenceof core sulihydryls (Slingsby et al., 1988). The altered protein that is present in the Philly lens apparently does not have this property. The Philly protein has an amino terminal similar to the normal /?B2 but for an immunological difference in the carboxyl end of the protein (Nakamura et al., 1988). This difference may be responsiblefor the lack of heat stability. The altered protein in the Philly mouse lens may therefore prove to be of great interest, not only because it may be related to the cataract in this animal, but also it may give an indication of the importance of certain regions on the /3B2-crystallin to the aggregation of proteins in the lens. In addition, the altered fl2-crystallin may indicate what sequencesor structures are responsiblefor the property of heat stability in the normal fl2. To explore the properties of this altered protein further, the question of the extent to which this altered protein aggregates with other crystallins in the Philly lens was investigated. 2. Materials and Methods Philly and normal Swiss-Webster mouse lenses were dissectedfrom animals between 17 and 2 1 days of age. Lensesfrom both setsof animals appeared clear 0 1990 AcademicPressLimited

684

by visual inspection. The lenses were sonicated in 50 mM Tris (pH 7.1). 100 mM KCI, 1 mM EDTA and 1 mM dithiothreitol, using the method previously reported (King and Russell, 1988). Protein determinations were done according to the method of Bradford ( 1975). High performance liquid chromatography was done on a Pharmacia Superose-12 column by injecting 3 mg of soluble protein for each run (Pharmacia Inc., Piscataway, NJ). Fractions of O-5 ml were collected and the absorbance of the material eluting from the column was monitored at 280 nm. The elution buffer was identical to the sonication buffer. To concentrate fractions, Centriconprotein concentrators were used in accordance with the manufacturer’s directions (Amicon Corp., Lexington, MA). SDS gel sample buffer, consisting of 2 % SDS, 5 “/o /I-mercaptoethanol and 62.5 mM Tris (pH 6.8). was added to the insoluble pellet after sonication to obtain the ‘insoluble protein’ fraction. Most but not all of the pellet was soluble in the SDS buffer. SDS-PAGE was performed as previously reported: Western blots and dot blot analyses of the sampIes were done using an antibody to the first 12 amino acid residues of /3B2-crystallin (Nakamura et al., 1988). The antibody was the generous gift of Dr Larry J. Takemoto. Dot blot analyses were done by spotting one microliter samples of each fraction from the HPLC column onto nitrocellulose, allowing the paper to dry and after blocking the paper with a milk solution, incubating the paper as if it were a SDS-PAGE blot. Secondary antibody was biotinylated anti-rabbit IgG at a 1: 500 dilution and was incubated with the blot for ninety minutes at 37°C. Subsequent incubation with a biotinylated peroxidase and avidin complex was done for 1 hr at 37°C (Vector Labs, Burlingame, CA). The blots were developed with 4-chloro-1-naphthol and hydrogen peroxide. 3. Results

The HPLC profile for the soluble protein from the normal Iens was comparable to the pattern that has been reported using similar gel filtration matrixes (Russell et al., 1979). The Superose-12 did give a better resolution of the heavy molecular weight fraction (HMW) than other columns while still separating the lower molecular weight material very well (Fig. 1). Fractions 3 and 4 contain the HMW fraction: in 6-10, the cx-crystallin is present. The &crystallin is found in fractions 12-14 and did not completely resolve from the p,,, which ends at fraction 17. The low molecular weight fraction (y-crystallins) follows the p-crystallins. Dot blot analysis of one microliter samples from each fraction revealed that the immunoreactive /3B2crystallin was found primarily in the &crystallin fractions. In addition to this material, some the PB2 reactive material was also found in the HMW fraction. The Philly mouse soluble protein has a profile that was different from the normal mouse pattern (Fig. 2). One

P. RUSSELL

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L I

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I I 10 15 FRACTIONS

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FIG. 1. HPLC chromatograph of normal mouse lens soluble protein. A Superose-12 column was used to separate 3 mg of the soluble protein. Fractions (0.5 ml) were collected and the absorbance at 280 nm was monitored. Dot blot analysis of one microliter samples of each fraction using antibody to /IB2-crystallin residues l-12 is shown (A). Immunoreactive /IB2 is present primarily in the /+crystallin fractions.

difference that is immediately apparent is the decreased amount of the P-crystallin fractions, particularly the /3* fraction around fractions 12-14. The p,-crystallins were 15.9 Y0of the soluble protein in the normal lens but only 11.1% of the Philly lens at this age (17-2 1 days). Conversely, the amount of the HMW protein was higher in the Phiiiy mouse. This fraction represented about 9.5% of the total, whereas in the normal lens the HMW fraction was about 6.9%. Interestingly, the dot blot analysis of Philly soluble fractions indicated that immunoreactive material was present in the HMW fractions. The immunoreactivity was more evident after the fractions from the HMW fraction and the P-crystallin region were concentrated about sevenfold. Dot blots [Fig. 2(B)] showed the immunoreactive material evident in the HMW fraction but only weakly visible in the P-crystallin fractions. Although the amount of immunoreactive material was small, p-crystallin fractions 13-l 7 had protein that reacted with the /3B2 antibody, suggesting that at least some of the altered /3B2 in the Philly lens was present throughout the entire fi-crystallin region. SDS-PAGE was run on the fractions that were obtained from the normal and the Philly mouse after

INTERACTIONS

OF AN

ALTERED

P-CRYSTALLIN

685

the HPLC run (Fig. 3). Fractions shown in Fig. 3 are representative of the HMW fraction (3), a-crystallin (8), the /3-crystallin region (13-17) and the start of the low molecular weight region containing the ycrystallins (18). The PBZ-crystallin was prominent in

the normal mouse in the P-crystallin but was absent as expected in the Some of the P-crystallin bands in the be more strongly staining than those is not

clear

whether

there

region (arrow) Philly fractions. Philly appear to in the normal. It

is an increase

in these

polypeptides in the Philly lens or whether the decrease in intensity of these bands in the normal pattern is the result of the large amount of the pB2. As has been shown before with the dot blots, the Ievei of altered pB2 in the Philly fractions was not sufficient to be

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3 8131415161718

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3 8131415161718

FRACTIONS I

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46KD-

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10 15 FRACTIONS

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FIG. 2. HPLC chromatograph of Philly mouse lens soluble protein. The conditions were identical with the normal mouse soluble in Fig. 1. Dot blot analysis indicated that immunoreactive material to the pB2,_,, antibody was present in the HMW fractions (A). Concentration of the fractions indicated that a small amount of reactive material was also present throughout the ,&crystallin region (B).

2OKD-

FIG. 3. SDS-PAGE (A, B) and Western blots (C, D) of the main fractions isolated from the HPLC columns of Philly (A, C) and normal mouse (B, D) soluble protein. Fraction numbers correspond to the material from the fractions isolated as shown in Figs 1 and 2. Immunoreactive /X32-crystallin is present in the p-crystallin region of the normal mouse lens from fractions 13-l 7. but the antibody was unable to detect immunoreactive material at this concentration in the Philly soluble fractions. The positions of the molecular weight markers are shown.

B

FIN. 4. SDS-PAGE (A) and Western blot (B) of the HMW fractions and the insoluble material from the Philly and the normal mouse lens. The HMW fractions were isolated by gel filtration and concentrated sevenfold. The insoluble material was solubilized by SDS from the buffer insoluble pellets. Immunoreactive material to the antibody for the first 12 residues of /J’BZcrystallin was found around 2 7 kDa in all of the samples. (1) Insoluble material from the normal lens. (2) Insoluble material from the Philly lens. (3) Molecular weight standards. (4) HMW fraction from the normal lens. (5) HMW fraction from the Philly lens.

686

detected at these protein concentrations, On the Western blots of the normal mouse lens with fractions at the same concentrations, the pB2 was very evident in all the fractions in the /$crystallin region, Repeated attempts were not successful in demonstrating immunoreactive /1B2 in the Philly fractions at similar concentrations with the normal lens fractions; however. concentrated samples did show some slight reactivity with the antibody. Since the dot blots to the concentrated fractions from the HPLC column suggested that the pB2 might be found primarily in the HMW fraction, this fraction was further investigated. The concentrated HMW fraction from the normal and Philly lens, as well as the SDS solubilized material from the insoluble pellet from both sets of lenses, were run on SDS-PAGE and a Western blot was performed (Fig. 4). The Western blot showed that there was an immunoreactive band at around 27 kDa in both the normal and the Philly HMW fractions and this band was also present in the material from the insoluble pellets. The stained pattern of the HMW fraction from the Philly lens showed several bands between 25 kDa and 30 kDa in the /Icrystallin region of the gel. These bands were more prominent than those bands in the normal lens HMW fraction. The most heavily stained bands in the normal pattern appear to correspond to z-crystallin. The insoluble fractions from both the Philly and the normal lens were more similar and were composed primarily of bands around 20 kDa. 4. Discussion The Philly mouse may be an extremely useful animal model of cataract since there is an alteration in one of the crystallin polypeptides, and this change may play a major role in the opacification of the lens. The polypeptide which is altered in the Philly may be a particularly important one since this p-crystal&n normally interacts with other P-crystallins. These interactions may be essential in maintaining lens clarity. The data would indicate that the Philly /3B2crystallin can associate with the other P-crystallins in the Philly lens, since immunoreactive /3B2 is found in all of the /&crystallin fractions; however, the largest amount of the altered ,6B2 is found in the heavy molecular weight material. These results would suggest that the altered pS2 is capable of some types of interactions with other crystallins and non-crystalIins in the lens, and these interactions may quickly lead to HMW or insoluble material. The HMW material is thought to be the precursor of insoluble protein which may be responsible for lens opacity (Spector, Li and Sigelman, 1974 ; Liem-The and Hoenders, 1974). In other mouse lens cataracts, there is an increase in the HMW fraction during the process of lens opacification (Russell et al., 1979). In the Philly mouse, this also appears to be the case. One possible hypothesis is that the altered /1B2 forms some

P. RUSSELL

AND

C. CHAMBERS

associations with other /r’-crystallins in the fiber cells which are unstable because the proper protein-protein interactions are not formed with the altered /1B2. The unstable P-crystallins would then rapidly interact with other crystallin and non-crystallin components in the cytoplasm of the cell and form the HMW material. The more rapid formation of this material or perhaps the inherent instability of the aggregate might cause an acceleration in the formation of insoluble protein. The resulting changes in the protein matrix within the fiber cell could then trigger cataract formation. The presence of /j’B2-crystallin in the normal lens HMW and insoluble fractions indicates that this crystallin is one component of the large aggregates in the normal lens; however, most of the altered pB2 in the Philly is found in the aggregated fractions whereas the bulk of the normal ,8B2 is in the soluble fraction. Thus, the rate or the process of aggregation may be different in the two lenses. An alteration of the structure of the pB2 molecule may also affect its susceptibility to proteolytic attack. Data with the human lens suggests that the amino terminal is degraded in vivo in preference to the carboxyl end of the molecule (Takemoto et al.. 198 7). Any proteoIysis at the amino terminal of the /,‘B2crystallin would probably render the protein undetectable by the antibody used in this study. Although there may be increased proteolysis at the amino terminal of the Philly /3B2, additional antibodies would have to be developed in order to detect this type of change in the protein. The amount of modified PB2-crystallin in the Philly lens appears to be much lower than the level of the pB2-crystallin in the Swiss-Webster lens. Previous data has indicated that the mRNA from the Philly lens which hybridized to the bB2-crystallin probe was at about the same level as the mRNA found in the normal lens. Decreased protein synthesis in the Philly lens has also been reported (Piatigorsky, Kador and Kinoshita, 1980). In addition to the proteolysis and insolubility, a net decrease in synthesis may contribute to the very low accumulation of the altered /J’BZ in the soluble protein of the Philly lens (Carper et al. 1982). An intriguing question is the role that the altered protein might have on protein synthesis. The interactions that are necessary for the fiber cells to elongate and differentiate correctly may be severely affected by the modified bB2. Without the correct communication between the fiber cells in the lens, the resulting ion imbalances in the fiber cells may influence the synthesis of new proteins and cause the rapid cataract formation. It is therefore possible that the alteration of the /j’B2 crystallin in the Philly lens is by itself directly responsible for the cataract formation. The resolution of many of these questions will have to await the sequencing of both the normal and the Philly /?B2-crystallin. An understanding of the differences between these two proteins may give insight into the associations which occur with the I{-

INTERACTIONS

OF AN ALTERED

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crystallins. It may also be possible to obtain sufficient protein through expression of the altered /B2crystallin DNA to be able to study the types of interactions that are formed with this protein and to reveal how the alteration in the protein affects the stability of the large protein complexes in the lens. References Benedek, G. B. ( 1971). Theory of transparency of the eye. Appl. Optics IO. 459-73. Berbers. G. A. M.. Brans. A. M. M.. Hoekman. W. A., Slingsby. C., Bloemendal. H. and DeJong, W. W. (1983). Aggregation behavior of the bovine ,&crystallin Bp chain studied by limited proteolysis. Biochim. Biophys. Acta 748, 213-Y. Bradford, M. M. (1975). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biorhern. 72, 248-54. Carper, D. A., Russell, P. and Sanyal, S. (1985). Comparison of the lens crystallin proteins from normal, rd, and rds mutant mice utilizing specific monoclonal antibodies. Esp E!je Res. 40. 757-61. Carper. 13. A., Shinohara, T., Piatigorsky. J. and Kinoshita, J. H. ( I Y 82 ). Deficiency of functional messenger RNA for a developmentally regulated b-crystallin polypeptide in a hereditary cataract. Science 217. 463-4. Carper. D. A.. Smith-Gill. S. J. and Kinoshita, J. H. (1984). Production and characterization of a monoclonal antibody to bovine /j-crystallin. Curr. Eye Res. 3, 501-7. Carper. I). A.. Smith-Gill. S. J. and Kinoshita, J. H. (1986). lmmunocytochemical localization of the 27K /I-crystallin polypeptide in the mouse lens during development using a specific monoclonal antibody: implications for cataract formation in the Philly mouse. Dev. Biol. 113. 104-9. Horwitz, J.. McFall-Ngai. M., Ding. L.-L., and Yaron. 0. (IY86). Thermal stability of lens crystallins. In The Lens: Transparency and Cataract (Ed. Duncan, G.). Pp. 227-39. Eurage: Rijswijk, the Netherlands. Kador. P. F.. Fukui, H. N.. Fukushi. S., Jernigan, H. M. and Kinoshita, J. H. I 1980). Philly mouse: a new model of hereditary cataract. Erp. Ege Res. 30. 59-68.

Kador, P., Uga, S. and Piatigorsky, J. (1980). The Philly mouse hereditary cataract. In Aging of the Lens (Eds

Regnault, F.. Hockwin, 0. and Courtois, Y.). Pp. 15 7-70.

Elsevier/North-Holland

Biomedical

Press :

Amsterdam. King,

L. and Russell, P. (1988). The EDTA extractable protein is glycosylated in the bovine lens. Cum. Eye Res. 7. 837-40. Liem-The, K. N. and Hoenders, H. J. (19 74). HM-crystallin as an intermediate in the conversion of water soluble into water insoluble rabbit lens protein. Exp. Eye Res. 19, 549-58. Mostafapour. M. K. and Schwartz, C. A. ( 1982). Purification of a heat-stable beta-crystallin polypeptide of the bovine lens. Curr. Ege Res. 1. 517-22. Nakamura, M.. Russell. P.. Carper. D. A., Inana. G. and Kinoshita, J. H. (1988). Alteration of a developmentally regulated. heat-stable polypeptide in the lens of the Philly mouse. 1. Biol. Chem. 263, 19218-21. Piatigorsky, J.. Kador. P. F. and Kinoshita. J. H. (1980). Differential synthesis and degradation of protein in the hereditary Philly mouse cataract. Exp. Eyr? Res. 30. hY-78. Russell, P.. Smith. S. G.. Carper, D. A. and Kinoshita, J. H. ( I 9 79). Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens. Exp. Eye Res. 29. 245-55. Slingsby, C.. Driessen. H. P. C.. Mahadevan. D.. Bax. B. and Blundell, T. L. (1988). Evolutionary and functional relationships between the basic and acidic /I-crystallins. l:l~p. Eye Res. 46, 375-403. Spector, A., Li, S. and Sigelman, J. (1974). Age dependent changes in the molecular size of human lens proteins and their relationship to light scatter. Invest. @phthaZmol. 13. 795-8. Takemoto. L.. Takemoto, D., Brown, G.. Takehana. M., Smith, J. and Horwitz, J. (1987). Cleavage from the Nterminal region of /j’Bp crystallin during aging of the human lens. Exp. Eye Res. 45, 385-92. Uga. S., Kador. P. F. and Kuwabara. T. (I 980). Cytological study of Philly mouse cataract. Exp. Eye Res. 30. 79-90. Zigler. J. S., Jr. and Sidbury , J. B.. Jr. (1976). Studies on /jcrystallin from primate lens. Invest. Ophthalmol. 15, 673-7.