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Lens Proteomics: The Accumulation of Crystallin Modifications in the Mouse Lens with Age

¹ From the Department of Animal Sciences, Oregon State University, Corvallis, Oregon; the ³ Department of Biological Sciences, The University of Delaware, Newark, Delaware; and the ⁴ Departments of Oral Molecular Biology and ⁵ Ophthalmology, Schools of Dentistry and Medicine, Oregon Health and Science University, Portland, Oregon.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
PURPOSE. To identify modified crystallins associated with aging of lens and produce two-dimensional electrophoresis (2-DE) proteome maps of crystallins in mouse lens.

METHODS. Lens proteins from mice of increasing age or different strains were separated by either chromatography or 2-DE. Masses of whole proteins or tryptic peptides were analyzed by mass spectrometry. Changes in the abundance of individual crystallins were determined by image analysis of 2-DE gels.

RESULTS. The measured masses of all known mouse crystallins, with the exception of D and F, matched the masses calculated from their reported sequences. Analysis by 2-DE indicated that most posttranslational modifications took place in mice after 6 weeks of age. Partially degraded crystallins, including ßB1, ßB2, ßB3, ßA3, A, and B, were found in greater proportion in the insoluble fractions. -Crystallins A through F also became insoluble during aging. However, insolubilization of -crystallins was associated with a decrease in isoelectric point (pI). Aging was also associated with increased phosphorylation of soluble A- and B-crystallins, confirmed by mass measurements of these proteins eluted from 2-DE gels. Comparison of protein profiles between several strains of mice used to produce transgenic or knockout models of cataract indicated few differences, except for an additional acidic form of a -crystallin, possibly due to a polymorphism.

CONCLUSIONS. These results suggest that partial degradation of - and ß-crystallins and increased acidity of -crystallins may cause insolubilization during aging. The 2-DE proteome maps of mouse lens proteins created in this study, using immobilized pH gradients, will be useful for comparison with maps of lens proteins of mice with cataracts so that cataract-specific modifications may be identified.

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The transparency of eye lens is largely determined by the properties of crystallins, the structural proteins of the lens. Aging of normal lens leads to the accumulation of posttranslationally modified proteins, because crystallins undergo very little turnover after synthesis.¹ We hypothesize that these modifications may contribute to cataract by causing aggregation and insolubilization of crystallins. However, studying the role of crystallin modification in lens is complex, because many modifications are part of the normal maturation process. For example, site-specific proteolysis of ß-crystallins in young lens may serve to initiate tighter packing of crystallins during lens maturation.^{2 3 4} Evidence from experimental cataracts in young rats also suggests that opacity may result when this process becomes unregulated.² In contrast, cataracts in mature lenses may result from unique modifications that are not found in normal age-matched lenses.

The mouse is an especially useful species in which to test the role of crystallin modification in cataracts, because a large number of transgenic and knockout strains have been produced in which cataract develops. These include the A-crystallin,⁵ SPARC,⁶ 3 connexin,⁷ knockout mice and transgenic mice overexpressing a large variety of proteins including truncated fibroblast growth factor receptors,⁸ transcription factors such as PAX6 (5a),⁹ or fosB,¹⁰ HIV protease,¹¹ and structural proteins.¹² In addition, a number of spontaneous mutant mouse strains have been identified in which cataract develops. See the recent review by Graw¹³ for further information on murine models of congenital cataract.

Many groups have used two-dimensional electrophoresis (2-DE) to study protein modifications occurring in the mouse lens during aging and/or during cataractogenesis.^{11 14 15 16} However, the interpretation of the available data are hampered by the unavailability of standardized 2-DE protein maps comparing mice of different ages. Furthermore, the precise protein modifications that occur with normal aging have not been systematically explored. Thus, in this study, reproducible mouse lens 2-DE maps were created for lenses of increasing age and of different strains to serve as reference maps. Further, the molecular identity of the separated proteins was confirmed by mass spectrometry (LC-MS/MS), and a number of posttranslational modifications were identified. In future studies, these data will facilitate the identification of specific modifications in cataractous lenses of mice.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Preparation of Mouse Lens
FVB/N mice¹⁷ were generated in-house from a breeding stock obtained from Taconic Laboratories (Germantown, NY). C57BL/6 and ICR mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN). CB6F1 mice were obtained from Charles River Laboratories (Wilmington, MA). The FVB/N and C57BL/6 strains were analyzed because they are derived from mice with widely different genetic backgrounds,¹⁸ and cells from these strains are commonly used to produce transgenic mice to study lens biology.^{8 19 20} Descriptions of these and other inbred strains of mice can be obtained at a Web site maintained by the Jackson Laboratory (Bar Harbor, ME).²¹ Mice were killed, and the lenses were dissected and flash frozen until use. All experiments using animals were approved by the University of Delaware’s institutional review board and conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Isolation of Lens Crystallins and Mass Determination
Six lenses of 6-week-old FVB/N mice were homogenized in 200 µL of lysis buffer, containing 20 mM sodium phosphate (pH 7.0), 1 mM EGTA, 100 mM NaCl, and 1 tablet of protease inhibitor (Complete Mini Protease Inhibitor Cocktail; Roche Molecular Biochemicals, Indianapolis, IN) dissolved at 10 mL lysis buffer per tablet. Lens homogenates were centrifuged at 20,000g for 45 minutes at 4°C and supernatants were removed for further crystallin purification.

Crystallin aggregates and monomers were fractionated by gel filtration on a 10x 250-mm chromatography column (Superose 6HR 10/30; Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated with lysis buffer at a flow rate of 0.2 mL/min. This resolved the , ßH, and ßL aggregates of approximately 600, 150, and 60 kDa, respectively, and the -crystallin monomers of approximately 20 kDa molecular weight, which were further separated by ion exchange or reversed phase chromatography. Beta heavy aggregates, containing a complete complement of individual ß-subunits, were deaggregated in 6 M urea and individual subunits isolated by diethylaminoethyl (DEAE) chromatography, as previously described.²² -Crystallin monomers were separated by sulfopropyl (SP) chromatography, as previously described,²³ except using a 7.5 x 75-mm column (SP 5-PW; TosHaas, Montgomeryville, PA), 20 mM histidine (pH 6.0), 1.0 mM EGTA, 2 mM dithiothreitol (DTT) mobile phase, and 0.1 M NaCl gradient over 60 minutes.

Approximate 5-µg samples of whole -crystallin aggregate or isolated ß- and -crystallin subunits were injected onto a 1.0 x 250-mm C4 column (214 LC-MS/MS C4; Vydac, Hesperia, CA) and masses determined by online analysis of eluents by electrospray ionization-mass spectrometry (ESIMS) on an iontrap system (model LCQ; ThermoFinnigan, San Jose, CA). The column used a 25-µL/min flow rate and linear gradient of 10% to 50% acetonitrile over 30 minutes in a mobile phase containing 0.1% acetic acid and 0.025% trifluoroacetic acid. Crystallin masses were then calculated as previously described.²²

2-DE and Identification of Lens Proteins
Four lenses from identically aged mice were homogenized in 200 µL lysis solution containing protease inhibitors, followed by centrifugation, as described earlier. The supernatant containing the soluble protein was removed, and the pellet (insoluble protein) was washed once. The insoluble protein was then resuspended by sonication, and the protein contents in both the soluble and insoluble fractions were measured by the bicinchoninic acid (BCA) assay (Pierce Chemical Co., Rockford, IL), using bovine serum albumin as a standard. Both fractions of lens proteins were aliquoted into 400-µg portions and stored at -70°C.

Isoelectric focusing was performed using immobilized pH gradient (IPG) gel strips (18 cm, pH 5–9), followed by molecular weight separation, using 12% SDS-PAGE gels, as previously described.²² The Coomassie blue–stained gel images were captured and image analysis performed on computer (Melanie 3 software; Geneva Bioinformatics, Geneva, Switzerland) to determine the percent that each spot contributed to the total protein on the gel.²² The isoelectric points (pIs) of the modified crystallin species were extrapolated by the software, using the calculated pIs and positions of unmodified A, ßA2, and B as reference. Calculation of these crystallin pIs and validation of pI assignments for the other crystallins was performed as previously described.²²

Protein identification and mapping on 2-DE gels was performed using soluble protein from pooled lenses of 6-week-old mice (FVB/N strain). Similarly, mapping of identities of insoluble proteins was performed by pooling protein from 6- to 51-week-old mice. Regions from negatively stained gels containing protein spots were washed, dried, and digested within excised gel spots with trypsin, as described by Courchesne and Patterson.²⁴ Proteins in gel spots were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of digests to determine the amino acid sequences of peptides, as previously described.²²

Mass Measurement of Proteins Isolated from 2-DE Gels
Protein masses were determined after recovery from 2-DE gel spots, either by elution using a custom electroelution device or by passive diffusion. Two-DE gels of soluble protein from 10-week-old mice were prepared as described earlier, and then negatively stained.²⁵ Excised spots from three gels were preincubated twice for 15 minutes at room temperature by rotation in 1 mL of elution buffer (25 mM Tris, 192 mM glycine, and 1 mM thioglycolic acid [pH 8.8]) supplemented with 0.1% SDS. For electroelution, the gel piece was minced into approximate 2-mm cubes and placed into a 250-µL disposable pipette tip plugged with 4 µL 4% polyacrylamide gel, and the tip was filled with 150 µL elution buffer. The pipette tip was placed into a 200-µL microtiter plate well (catalog number 3690; Costar, Cambridge, MA) filled with 130 µL elution buffer and the protein electroeluted into the microtiter plate well. Current was applied by placing platinum wires into both the elution buffer contained in the upper 250-µL pipette tip and the lower microtiter plate well. The protein was eluted for 90 minutes at 100 V inside a 4°C cold room.

Alternately, proteins were recovered by passive diffusion using a modification of the method of Castellanos-Serra et al.²⁶ After preincubation as described earlier, excised protein spots were dispersed into 20-µM particles by forcing them through a 20-µm porous metal frit (catalog number A-120X; Upchurch Scientific, Oak Harbor, WA) placed at the bottom of a 500-µL airtight glass syringe. This required removal of the plastic ring surrounding the frit. The gel particles remaining in the syringe were collected by passing 100 µL elution buffer containing 0.1% SDS through the syringe. Proteins were then allowed to diffuse from the gel particles by incubation for 30 minutes at 37°C in an ultrasonic bath. The slurry was then filtered using a 0.22-µm microcentrifuge filter (Micropure-0.22; Millipore, Bedford, MA).

Masses of proteins recovered by both of these methods were then determined by online LC-MS/MS, as described earlier for mass measurement of intact crystallins. The approximately 100 µg SDS present in each sample did not interfere with chromatography or mass measurement of the eluted protein.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Crystallin Mass Determination
To compare the actual masses of mouse crystallins and calculated masses based on the reported cDNA sequences, the masses of individual HPLC-separated or gel-eluted crystallins from 6-week-old mice were measured by ESIMS (Table 1) . The measured masses of all HPLC-purified - and ß-crystallins matched the theoretical masses derived from published cDNA sequences within an instrumental error of three mass units. This suggested that the reported sequences of these crystallins matched the sequences found in FVB/N mice. ßA1-crystallin was not obtained in sufficient purity by HPLC to determine its mass. However, the mass of ßA1 eluted from 2-DE gels differed by only 0.8 mass units from the theoretical mass (Table 1) , again suggesting that the reported sequence was identical.

Composition of Soluble Proteins in Young Mouse Lens
To determine the identities and relative abundance of crystallin subunits, the time course of posttranslational modifications, and variation between the water-soluble and -insoluble fractions in mouse lens, 2-DE was performed and spots identified by in-gel trypsin digestion and LC-MS/MS.

A 2-DE gel of lens soluble protein from 1.5-week-old FVB/N mice is shown in Figure 1 , with the identities of major proteins indicated. All crystallins previously reported in a 2-DE map of 33- to 51-week-old mouse lens were identified,¹⁶ in addition, we were able to confirm the presence of F-crystallin by identifying a F-specific peptide within the digest of the comigrating E and F spots. Similarly, B and C did not separate from one another, but peptides unique to each protein were identified in the digest. The majority of crystallins in the lenses of these young animals migrated to their expected relative pIs, except E-crystallin, which, based on its expected pI of 7.7, should have been the most basic crystallin subunit. Because it was unlikely that E was modified in lenses from these very young animals, the result suggested that the reported sequence (Swiss Prot accession no. P26999) was different from the E sequence in the FVB/N strain. This result was unexpected, because a -crystallin was found with a mass only 2.7 U different from the expected mass of E (Table 1) .

View larger version (40K): [in this window] [in a new window]   Figure 1. 2-DE map showing the identities of the major soluble proteins in whole lenses of 1.5-week-old mice. Protein spots were detected and quantified within circled regions by 2-DE image analysis software and identified by MS/MS analysis of in-gel tryptic digests. Approximate molecular weight and pH range of the gel were determined by reference to molecular weight markers and calculated pIs of selected crystallins. Only the lower molecular weight region of the gel is shown, which contained all crystallin subunits and fatty-acid–binding protein. In all gels shown in the figures, staining was performed using Coomassie blue, and 400 µg of protein was applied.

Developmental Changes in Crystallin Composition
To determine developmental changes in crystallin composition, water-soluble lens proteins from newborn to 6-week-old mice were separated by 2-DE (Fig. 2) , the percentage abundance of each crystallin determined, and the percentage change in abundance from newborn to 6 weeks of age calculated (Table 2) . The observations of developmental changes in crystallin abundance were limited to the first 6 weeks of life, because posttranslational modifications and protein insolubilization in older lenses prevented accurate quantification. The most profound changes occurred in the content of ßB2-, S-, and B-crystallins. These proteins increased approximately 50-, 5-, and 2-fold, respectively, during the first 6 weeks of life. The fatty acid binding protein also increased 12-fold during this period. In contrast, by 6 weeks of age, ßB1-, ßB3-, and A-crystallins decreased to approximately one half the amounts found in newborn lens. Although quantities of A_insert also decreased, inconsistencies in the quantity of this protein between gels prevented any conclusions regarding changes in its abundance with lens growth.

View larger version (35K): [in this window] [in a new window]   Figure 2. Changes in relative abundance of the major proteins of mouse lens during maturation. 2-DE gels of soluble proteins of (a) newborn, (b) 1.5-week-old, and (c) 6-week-old mouse lenses are shown. Protein spots undergoing changes in relative abundance during lens maturation are labeled. Unlabeled spots can be identified by reference to Figure 1 . The relative abundance of each protein spot was determined by image analysis, as shown in Table 2 . FABP, fatty-acid–binding protein.

View larger version (112K): [in this window] [in a new window]   Figure 3. Accumulation of modified crystallins in mouse lenses during aging. Lens proteins from 6-, 10-, 31-, and 51-week-old mice were separated into (a) soluble and (b) insoluble fractions and analyzed by 2-DE. The appearance of modified crystallins with age can be followed by reference to the identities of unmodified crystallins indicated in the 2-DE gels of proteins from 6-week-old lens. Age-induced changes in the abundance of unmodified crystallins was determined by image analysis as shown in Table 4 . The identities of the major modified crystallin subunits appearing with age are shown in Figure 4 .

View larger version (104K): [in this window] [in a new window]   Figure 4. Identification of modified crystallins appearing with age in mouse lens. 2-DE gels of soluble (a) and insoluble protein (b) from lenses of 51-week-old mice, as shown in Figure 3 , are enlarged so that labels on protein spots can be seen. The major modified crystallin species appearing with age are numbered, and identities are indicated by reference to Table 3 . The position of unmodified crystallin subunits (underscored labels) are circled in the soluble fraction (a) and marked (+) in the insoluble fraction (b). Note that whereas many modified crystallins appear in both soluble and insoluble fractions, for clarity they are labeled with a number only in the fraction where they are most abundant.

Age-Related Changes in -Crystallins
-Crystallin aggregates are composed of A, B, and A_insert subunits. A and A_insert are identical, except for the insertion of 17 extra amino acids in A_insert, because of differential mRNA splicing.²⁹ The concentrations of unmodified -crystallin subunits did not significantly change in the soluble fraction of the adult mouse lens during aging (Table 4) . The major modification of both A and B in the soluble fraction was the age-dependent appearance of acidic forms (Fig. 4A , spots 1 and 11). The isolation of these acidic forms from 2-DE gels and measurement of their whole masses yielded unit masses of 19,970.5, and 20,190.6 (Fig. 5) . These masses were each, respectively, approximately 80 mass units greater than the expected masses of alkylated A and B. This indicated that the increased acidity of these species was due to single phosphorylations. The phosphorylated form of A increased to 38% of unmodified soluble A by 51 weeks of age. In contrast, the content of phosphorylated B peaked by 10 weeks of age, and the proportion of phosphorylated B was less than that of phosphorylated A.

View larger version (20K): [in this window] [in a new window]   Figure 5. Masses of phosphorylated -crystallins appearing with maturation and increasing age in mouse lens. Deconvoluted mass spectra of phosphorylated A (a) and phosphorylated B (b). These proteins were isolated for mass analysis by elution from the soluble fraction of 2-DE gels. The calculated masses of alkylated nonphosphorylated A- and B-crystallins are 19,891.2 and 20,110.8, respectively. A single phosphorylation increased the mass of proteins by 80 units.

Several pieces of evidence suggest that C-terminal truncation of A and B led to their insolubilization. A truncated form of A (Fig. 4B , spot 2), just below intact A, was isolated from 2-DE gels. Its 21,436.7-unit mass indicated that it was missing five residues from its C terminus. Similarly, MS/MS analysis of a tryptic digest from spot 3 yielded peptide 146-151 of A, indicating that this species was missing 22 residues from its C terminus. These results suggest that the other numerous species of truncated A and B may result from the progressive removal of C-terminal residues. This hypothesis was supported by LC-MS/MS analysis of spots in similar positions from 2-DE gels of insoluble protein from adult rat lens. These modified forms of A and B were all C-terminally truncated (Ueda Y., unpublished results, 2001). The similar relative molecular weights of spots 12–14 in Figure 4B also suggested that a single truncated species of B may be progressively phosphorylated. This suggestion was also supported by mass spectral analysis of a similar species of B in rat lens, which was both C terminally truncated and phosphorylated (Ueda Y., unpublished results, 2001).

Age-Related Changes in ß-Crystallins
During aging, soluble forms of ßB1 and ßB3 continued to decrease, so that each comprised less than 0.5% of the total soluble lens protein by 51 weeks of age (Table 4) . This loss was due to insolubilization after truncation of ßB1 (Fig. 4B , spots 21–24) and ßB3 (Fig. 4B , spots 27–30). ßB2 and ßA3-crystallins also underwent truncation and insolubilization with increasing age (Fig. 4B , spots 25–26, and 31–33). However, the accumulation of these insoluble truncated forms did not deplete the soluble fraction of the intact forms of these proteins. In fact, ßB2 continued to accumulate in the soluble fraction and became the major species by 51 weeks of age. Unlike other ß-crystallin subunits, ßA2 and ßA4, and possibly ßA1, were not truncated and did not become selectively insolubilized with age. The stability of these subunits was likely related to their shorter and therefore protease-resistant N-terminal extensions.

The truncation of ß-crystallins differed from -crystallin truncation in that N-terminal regions were removed. Although specific sites of truncation were not assigned for all modified ß-species, truncated ßA3, missing either 11 or 22 residues (spots 31 and 33), ßB2 missing 7 residues (spot 26), and ßB3 missing 17 residues from its N terminus (spot 27) were identified by LC-MS/MS analysis of peptide digests (Table 3) . These N-terminal truncation sites were identical with ones previously described in rat lenses and attributed to activation of a class of calcium-activated proteases called calpains.²

The analysis of spots with similar positions in 2-DE gels of both soluble and insoluble fractions suggested that caution must be used when identifying proteins based on similar positions. The spot identified as A_insert on the 2-DE gels of soluble protein had a position identical with a spot on the 2-DE gel of insoluble protein identified as a truncated ßA3 (Fig. 4B , spot 32).

Although partial degradation was the major modification to ß-crystallins in mouse crystallins with age, there was also evidence for either deamidation or phosphorylation. Soluble acidic forms of ßB2, ßA3, and ßA4 appeared with age and underwent no alteration in apparent molecular weight on the 2-DE gels (Fig. 4A , spots 17–20).

Age-Related Changes in -Crystallins
In contrast to - and ß-crystallins, there was no evidence of -crystallin’s proteolysis during lens maturation. However, large shifts in the relative abundance in these proteins occurred. After the developmentally related decrease in A-crystallin (Table 1) , this protein underwent insolubilization and was entirely lost from the soluble fraction by 51 weeks of age (Fig. 4 , Table 4 ). E and F also decreased approximately 80% in the soluble fraction from 6 to 51 weeks of age. Because A through F (A–F) all accumulated in the insoluble fraction during aging, the selective loss of soluble A, E, and F was probably due to decreased synthesis of these proteins.

The age-related insolubilization of A–F crystallins was associated with progressive acidification, so that a duplicate pattern of four spots appeared for these six proteins at identical apparent molecular weights, but shifted an average of 0.47 pH units more acidic than the unmodified species (Fig. 4B , spots 34–37, Table 3 ). These acidified forms of A–F were almost exclusively found in the insoluble fraction. The modification causing this acidification was not determined, but could be due to either deamidation, phosphorylation, or very limited proteolysis. The acidification and insolubilization of A–F was very specific, because the closely related protein S underwent no acidification and remained for the most part soluble with increasing age.

Protein Profile Comparison of Different Strains of Mice
To determine whether lens proteins from different strains of mice varied in abundance or position on 2-DE gels, proteins from the FVB/N strain used in the current studies were compared with those in C57BL/6, ICR, and CB6F1 strains. In general, all four strains of mice had identical protein profiles, including crystallin modifications in both soluble and insoluble fractions. However, an additional spot was found in the -region in the strains C57BL/6 and its hybrid CB6F1. Gels comparing the soluble protein profiles of 10- and 12-week-old FVB/N and C57BL/6 strains, respectively, are shown in Figure 6 . The new spot in the C57BL/6 strain (Fig. 6 ; filled arrow) was identified by LC-MS/MS analysis of tryptic peptides as either B or C. The region in the position of B and C in the FVB/N strain was also less abundant (Fig. 6 ; open arrow). This suggested that a polymorphism may exist in these strains resulting in the presence of a more acidic B- and/or C-crystallin. This result was not unexpected, because the FVB/N and C57BL/6 inbred stains are genetically dissimilar.²¹

View larger version (49K): [in this window] [in a new window]   Figure 6. Comparison of soluble lens protein profiles from two mouse strains by 2-DE. (a) A 10-week old FVB/N mouse strain used throughout this study and (b) a 12-week-old C57BL/6 strain. The profiles were very similar, except for altered migration of a B or C-crystallin found in the C57BL/6 strain (filled arrow) that was largely absent in the FVB/N strain. Open arrow: normal position of B- and C-crystallins, which were decreased in abundance in the C57BL/6 strain.

	Discussion

Top Abstract Introduction Methods Results Discussion References

The masses and measured pIs of mouse crystallins suggest that the previously reported sequences deduced from cDNA are similar to sequences in the FVB/N strain, except for D, F, and possibly E. Similar inconsistencies between the measured masses of rat -crystallins²² and deduced sequences from cDNAs suggest that many polymorphisms exist in rodent -crystallins. This suggestion was also supported in the present study by the finding of B or C in the C57BL/6 strain with an altered pI. Whereas mutations altering critical residues or introducing stop codons in -crystallins lead to spontaneous cataracts,^{30 31} polymorphisms, such as in the C57BL/6 stain, may be relatively silent but cause an altered susceptibility to cataract. LC-MS/MS may provide a rapid method to further investigate heterogeneity of crystallin sequences in mice.

The relative composition of crystallin subunits in 12-day-old rats, measured in the accompanying study,²² were similar to the relative composition of crystallins in 1.5-week-old mice. Quantification of the relative amounts of each crystallin subunit in lenses from newborn to 6-week-old mice allowed estimation of changes in the relative rates at which these proteins are synthesized during lens maturation. Generally, the changes in abundance of various crystallin subunits in mice matched previously reported measurements of gene expression.

During development of the mouse lens, transcription from the B gene occurs before transcription from the A gene.³² However, by embryonic day 12.5, transcripts of A become very abundant and localize to the newly developed primary fibers, whereas B expression remains localized in the lens epithelium. Once secondary fibers form, this pattern changes, and the site of greatest B expression shifts from epithelium to secondary fibers. The rapid increase in B protein content measured in this study from birth to 11 days of age was likely the result of the delayed onset of B expression in secondary fibers.

The reported postnatal increase in rat ßB2 and decrease in rat ßB3 gene expression³³ also closely followed changes in ßB2 and ßB3 proteins in mice. The postnatal accumulation of ßB2 was especially dramatic, going from the least to most abundant ß-crystallin during the first 6 weeks of life. The age-related increase in mouse lens ßB2-crystallin synthesis has also been demonstrated immunohistochemically with a monoclonal antibody against ßB2.³⁴ Although changes in ßB1 gene expression have not been determined in postnatal rodent lenses, the similar 50% decline in both mouse ßB1 and ßB3 by 6 weeks of age suggests that the expression of genes coding for both proteins rapidly declines in mice after birth. Human lenses also undergo a rapid loss of ßB3, because this protein is only detected in lenses less than 3 years of age.³⁵ Unlike decreases in ßB3, the decrease in ßB1 may be restricted to mouse lenses. Similar rates of ßB1 protein synthesis have been observed in 1-, 2-, and 4-month-old rats.³⁶

The increase in S, B, and C proteins during the first 6 weeks of life also paralleled the reported increases in mouse lens S, B, and C gene expression.^{37 38} In contrast, expressions of A, E, and F genes were all reported to decrease during maturation.³⁸ These decreases in gene expression did not match the changes in protein levels, in that E and F were maintained in 6-week-old lenses. Only A decreased by 50% from birth to 6 weeks. However, this rapid loss of soluble A may be more related to its greater tendency to undergo insolubilization during lens maturation than its decreased level of gene expression (see Fig. 3 , 10 weeks).

The changes in the relative abundance of crystallin subunits would be expected to significantly alter the properties of lens fibers from the center to the periphery of the growing lens. The lower concentrations of B, ßB2, and B, C, and S and higher concentrations of ßB1, ßB3, and A in the older fibers in the lens nucleus may favor dehydration, insolubilization, and a cytosol with a higher index of refraction.

Major posttranslational modifications occurred in mice after 6 weeks of age. Although numerous modified crystallins were observed in the insoluble fraction in younger lenses, the quantity of insoluble protein did not significantly increase until after 6 weeks. The major modifications were phosphorylation of -crystallins and altered pIs and/or relative molecular weights of -, ß-, and -crystallins in the insoluble fraction, due to proteolysis and/or possibly deamidation.

A-crystallin became progressively phosphorylated with increasing age in the lens soluble fraction, with the phosphorylated form comprising more than one third of the total A by 51 weeks. Phosphorylation of -crystallins has been well documented in human,^{39 40} bovine,⁴¹ and rat⁴² lenses. The observed pattern of phosphorylation of -crystallins in mouse lens is consistent with the hypothesis that each subunit is phosphorylated by a different kinase activity. A became progressively phosphorylated with increasing age, whereas B phosphorylation was essentially complete by 10 weeks of age. In this regard, B phosphorylation in mice is more like phosphorylation of A in humans, where it is a maturationally related rather than age related.³⁹ Whereas cAMP-dependent kinases are likely responsible for at least a portion of -crystallin phosphorylations, these proteins also exhibit autokinase activity.⁴³ Further studies are required to determine which mechanism of -crystallin phosphorylation predominates in mice. These studies are important, because phosphorylation of -crystallin may cause dissociation of -oligomers and reduction of chaperone-like activity.⁴⁴

-Crystallins also became progressively truncated with increasing age, and these truncated -crystallins were selectively found in the insoluble fraction. The truncation of -crystallins occurs mainly at the C terminus (summarized by Groenen et al.⁴⁵ ). Although the specific sites of all C-terminal truncations in mouse -crystallins were not determined in this study, identification of A missing 5 and 22 residues from its C terminus was consistent with similar forms of these and other C-terminally truncated -crystallins characterized in rat lens (Ueda Y., unpublished observations, 2001). The loss of C-terminal residues in -crystallins is physiologically significant, because it reduces chaperone activity.^{46 47} The flexible C-terminal extensions of -crystallins are probably required to maintain the solubility of complexes between -crystallin and the substrate proteins being chaperoned.⁴⁸ If truncated -crystallins become insoluble in mouse lenses during maturation due to the binding of chaperoned proteins, the resultant complexes are compatible with transparency. Perhaps the greatest significance of the C-terminal truncation and insolubilization of -crystallin during maturation is that it consumes soluble -crystallins that then become unavailable to chaperone proteins in stressed lenses.

Recent evidence suggests that the proteases causing truncation of -crystallins during maturation and aging in rodent lenses are a combination of the calpain class proteases m-calpain and Lp 82.⁴⁹ The species of A-crystallin missing five residues from its C terminus in rat lenses is specifically produced by Lp82 (Ueda Y., unpublished results, 2001). However, the protease(s) removing five amino acids from the C terminus of A-crystallin in human lenses^{50 51} remains unknown, because human lenses contain no Lp82.⁵²

Mouse lenses also underwent extensive truncation of ß-crystallin N-terminal extensions during maturation. Similar to truncated -crystallins, these truncated ß-crystallins were also selectively found in the insoluble fraction of the lens. Calpain-induced truncations of ß-crystallin N-terminal extensions were previously reported in the insoluble fraction of the rat lens nucleus during maturation.² Furthermore, analysis of crystallins from both mouse and rat lenses suggested that calpain-induced proteolysis was accelerated during formation of experimental cataracts.^{2 53} Lenses from transgenic and knockout mice with cataracts probably would exhibit similar accelerated truncation of - and ß-crystallins secondary to the primary genetic defect that initiates cataract.

Analysis of insolubilized -crystallins suggested that increased acidification of all six A- through F-subunits may partially contribute to their insolubilization. The nature of the modification causing the decreased pI was not investigated but could be deamidation, phosphorylation, or very limited proteolysis. Proteolysis could decrease the pI of -crystallins without significantly altering their relative migration by SDS-PAGE, because residue 2 in all -crystallins is lysine.

In conclusion, this study of mouse lenses and the accompanying study of rat lenses²² provide baseline data that will facilitate the analysis of modified crystallins appearing in cataractous rodent lens. The 2-DE maps produced by identification of proteins by LC-MS/MS analysis can be directly compared with similar 2-DE gels run in other laboratories. Two-DE gel separation coupled with LC-MS/MS analysis of in-gel digests or eluted whole proteins was also demonstrated as a useful technique to identify crystallin modifications. A comprehensive analysis of these modifications in cataractous rodent lenses may provide the necessary information to model how these alterations contribute to insolubilization and light scatter. The information is particularly useful when compared with similar analysis of human crystallins.

	Footnotes

 
² Present affiliation: TRA Urology Research Center, Bayer Yakuhin Ltd., Kyoto Japan.

Supported by National Eye Institute Grants EY-07755 and EY-12016 (LLD) and EY-12221 (MKD).

Submitted for publication May 18, 2001; accepted July 3, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Larry L. David, School of Dentistry, Department of Oral Molecular Biology, Oregon Health & Science University, 611 SW Campus Drive, Portland, OR 97201; davidl{at}ohsu.edu.

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