Department of Oral Molecular Biology and Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97201
¶ Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Beaverton, Oregon 97006
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ABSTRACT |
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Truncation of lens crystallins is a common feature of both aging and cataract formation in rodents. A number of experiments revealed that calcium-activated proteases (calpains) were involved in formation of cataracts induced by selenite (4), galactose (5), diamide (6), and the hypocholesterolemic drug U18666A (7) and in hereditary rat Shumiya cataract (8). One of the ubiquitous calpains, m-calpain, has been credited with the proteolysis of - and ß-crystallins during cataractogenesis in rodents. Incubation of
A-crystallin with m-calpain reduced chaperone activity and produced truncated forms of
A that migrated during two-dimensional electrophoresis (2-DE)1 to positions similar to truncated forms of
A observed in cataractous lenses (9). A recently discovered lens-specific calpain, a splice variant of calpain 3 termed Lp82, may also be activated during formation of rodent cataracts (10).
The primary truncation sites on ß-crystallins in rodent lenses in vivo are at the N terminus where the cleavage sites can be determined by Edman sequencing (11, 12). In contrast, determinations of the exact -crystallin truncation sites have not been performed because of the lack of a convenient C-terminal sequencing method. To solve this problem, mass spectral analysis of peptides from in-gel digests (13) and accurate mass measurement of whole proteins eluted from gels (14) have become powerful tools to determine C-terminal truncation sites. Therefore, the purposes of the present report were to use mass spectrometry to 1) determine in vitro cleavage sites produced by m-calpain and Lp82, 2) compare the C-terminal cleavage sites to those produced in vivo during aging and cataract formation in rats, and 3) use this information to determine whether the activity of one rat lens calpain isoform is dominant in vivo.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of Recombinant Lp82
The Lp82 cDNA from rat was cloned into a pFASTBAC HTb vector (Invitrogen) with a His tag on the N terminus. This plasmid was used to transform DH10Bac competent cells containing a bacmid and helper plasmid (Invitrogen). Recombinant bacmid DNA containing Lp82 was isolated and used to transfect insect cells. Transfection of Spodoptera frugiperda insect cells (Sf9 cells; Invitrogen) was performed with amplified recombinant Lp82 baculovirus (108 plaque forming units/ml). After three days of culture, cells were sonicated in buffer containing 20 mM Tris (pH 7.5), 0.5 mM EGTA, and 2 mM dithioerythritol. The soluble protein was obtained by centrifugation at 13000 x g for 30 min, and rLp82 was purified by nickel-nitrilotriacetic acid (Qiagen Inc., Valencia, CA) metal-affinity chromatography according to the manufacturers protocol. Eluted rLp82 was then purified further by HPLC using a 7.5 x 75-mm DEAE 5PW column (TosoHaas, Montgomery, PA) with a linear 00.5 M NaCl gradient in Buffer A containing 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 2 mM dithioerythritol at a 1 ml min-1 flow rate. Enzyme-linked immunosorbent assay for Lp82 was performed by absorbing 50 µl of each column fraction in 0.1 M NaHCO3 buffer (pH 9.3) overnight onto 96-well plates. The wells were blocked with 5% non-fat dry milk and incubated with Lp82 antibody (1:1000 dilution) for 1 h (16). rLp82 antigen was visualized using goat anti-rabbit alkaline phosphatase-conjugated secondary antibody and an alkaline phosphatase substrate kit (Bio-Rad). The rLp82 peak from DEAE fractions was concentrated by ultrafiltration (Microcon 10; Millipore Corporation, Bedford, MA) and then used in the experiments described below.
Incubation of Lens Proteins with Calpains
Soluble proteins from lens cortex of 12-day-old rats were diluted to 50 mg/ml in 20 mM Tris (pH 7.4) buffer. Endogenous cysteine protease activity was inactivated by 5 mM iodoacetamide for 30 min at 37 °C, followed by addition of excess dithioerythritol to quench unreacted iodoacetamide. After the inactivation, 400 µg of protein was diluted further to 2.3 mg/ml in Tris buffer, and either 2.3 µg of recombinant rat m-calpain (Calbiochem-Novabiochem) or recombinant Lp82 of equal caseinolytic activity were added (17). m-Calpain and Lp82 were then activated by adding CaCl2 at a final 2 mM concentration, followed by incubation at 37 °C for 3 h. A control incubation containing soluble lens protein, CaCl2, and no added protease was also included. The reaction was stopped by addition of 10 mM EDTA, and the mixture was dried by vacuum centrifugation.
2-DE of Lens Protein
Proteins from each of the six experimental groups, each derived from lenses pooled from 512 animals, were separated in duplicate by 2-DE using immobilized pH gradient gel strips (18 cm, pH 59) in the first dimension by application of 400 µg of protein per strip, followed by 12% SDS-PAGE in the second dimension as described previously (18). The gels were stained negatively by the imidazole-zinc procedure (19). Immediately after staining, images were captured using a flat bed scanner at a resolution of 200 dots per inch in 12-bit gray scale. Images were analyzed, and protein spots were detected using Melanie 3 software (GeneBio, Geneva, Switzerland). Protein spots from two gels were then excised manually and pooled for analysis by mass spectrometry.
Protein Elution from 2-DE Gels
For whole mass determination, proteins were eluted directly from spots excised from duplicate 2-DE gels by passive diffusion. Excised gel pieces pooled from two gels were pre-incubated twice for 15 min at room temperature by shaking in 1.7-ml microcentrifuge tubes containing 1 ml of elution buffer (25 mM Tris base, 192 mM glycine (pH 8.8), 1 mM thioglycolic acid, and 0.1% SDS). The protein was then eluted by finely crushing the gel pieces using a modification of the method described by Castellanos-Serra et al. (20). Briefly, pre-incubated gel pieces were crushed through a 20-µm frit (part number A-120X; Upchurch Scientific, Oak Harbor, WA) by removing the plastic ring surrounding the frit, placing it at the bottom of a 500-µl glass air-tight syringe, and forcing the gel pieces through the frit using the syringe plunger. The gel particles left in the needle were collected by washing the syringe with an additional 50100 µl of elution buffer. After brief vortexing, the gel particles were sonicated in a 37 °C water bath for 30 min. The slurry was then filtered through a 0.22-µm microcentrifuge filter (Micropure-0.22; Millipore) at 13,000 x g for 15 min, and the filtrate containing the eluted protein was analyzed by mass spectrometry. The use of elution buffer containing 0.1% SDS was required to extract sufficient protein for mass analysis.
Determination of Eluted Protein Masses
The eluted protein was injected onto a 1.0 x 250-mm C4 column (214 MS C4; Vydac, Hesperia, CA), and masses were determined on-line by electrospray ionization mass spectrometry on a model LCQ iontrap (ThermoFinnigan, San Jose, CA). The flow rate was 25 µl/min with a linear gradient of 1850% acetonitrile over 40 min in a mobile phase containing 0.1% acetic acid and 0.05% trifluoroacetic acid. Samples were autoinjected and concentrated/purified using a microprotein trap cartridge (Michrom Bioresources, Inc., Auburn, CA). Mass spectra of proteins eluted from the C4 column were deconvoluted using Xcalibur software with BioWorks (ThermoFinnigan). Mass accuracy of better than 0.01% was confirmed using horse myoglobin. Experiments using carbonic anhydrase eluted from SDS-PAGE gels estimated that a minimum of 3.5 pmol of protein was required to obtain an accurate mass using this procedure. Use of a smaller 0.5 x 150-mm column with identical packing material and a 10 µl/min flow rate was unsuccessful in increasing sensitivity because of overloading with SDS and elution of SDS·protein complexes.
Assignment of Protein Cleavage Sites and Calculation of Isoelectric Points
After mass determination, the truncation sites in proteins were determined using PAWS software (prowl.rockefeller.edu/software/contents.htm) to match measured masses with calculated masses of truncated species. For further confirmation of truncation sites, the isoelectric points of identified truncated species were calculated using GeneWorks 2.5 software (Accelrys, San Diego, CA).
Assay of m-Calpain and Lp82 Activity
Activity of recombinant m-calpain and Lp82 was determined by a fluorescence assay (EnzChek protease assay kit E-6638; Molecular Probes, Eugene, OR) as described previously (17). The calcium activation requirements of the two enzymes were determined by varying the final concentration of free calcium in the incubation mixture from 0 to 1000 µM.
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RESULTS |
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The remaining partially truncated -crystallins produced by m-calpain and Lp82 were identical.
A-crystallin missing 8, 10, 16, 17, and 22 residues from its C terminus and
B-crystallin missing 5 and 12 residues from its C terminus were produced by both enzymes (Fig. 1, b and c, spots 37, 10, 11). However, m-calpain and Lp82 differed in their preferred cleavage sites. The major truncation product of m-calpain was
A1163, followed by
A1162 and
A1157. In contrast,
A1168 was the dominant truncation product produced by Lp82, and
A1163 was a relatively minor species.
In general, the measured isoelectric points of the partially degraded -crystallins matched their calculated isoelectric points. The two exceptions were the fragment identified in spot 8 discussed above and the fragment in spot 4 matching the mass of
A1165. This fragment was expected to migrate to a pI nearly identical to intact
A but migrated to a more acidic position. The cause of the pI shifts of these truncated species is unknown. However, minor species of
A were found in the undigested control sample (Fig. 1a) with shifts in pI to both basic and acidic sides of the major
A species. This suggested that the truncated
A species in spots 4 and 8 were derived from the pI-shifted
A species that existed before incubation. It was also interesting to note that the relative molecular weights estimated for
A1163 and
A1162 by SDS-PAGE did not agree with their actual molecular weights determined by mass spectrometry.
A1163 migrated faster than
A1162 during SDS-PAGE. This result illustrated the inherent inaccuracy of molecular weight estimation by SDS-PAGE.
Fragmentation of -Crystallin in Vivo
Analysis of water-insoluble lens proteins from the nucleus of normal 16-day-old rats by 2-DE indicated that -crystallins became fragmented at an early age (Fig. 3a). This fragmentation was largely absent from the water-soluble fraction (data not shown). Cataract produced by an overdose of selenite in age-matched lenses increased dramatically the concentration of fragmented
-crystallins. Intact
A was almost absent in the insoluble fraction of the nucleus following cataract formation (Fig. 3b, circle), the density of most
-fragments increased (Fig. 3b, spots 117), and the % of total protein that was insoluble increased 2.5-fold (Fig. 3d). Aging from 16 days to 6 weeks in normal lenses also increased the abundance of
-crystallin fragments (compare Fig. 3, a and c). However, the fragmentation occurring with age was not as extensive as in cataract. Intact
A was still observed in the insoluble fraction of the nucleus of 6-week-old rats, and the amount of insoluble protein only increased by 35% from 16 days to 6 weeks of age (Fig. 3d). The correlation between fragmentation of ß-crystallins and formation of insoluble protein has been documented in both cataractous lenses from young rats and mature normal rat lens (11). The present results indicated that breakdown of
A-crystallin was also associated with accelerated crystallin insolubilization.
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The mass of spot 18 did not match the mass of any possible -crystallin fragment. Therefore, this protein was identified by MS/MS analysis of its peptide fragments as the recently characterized protein GRIFIN (galectin-related inter-fiber protein) (22).
Calcium Requirement for m-Calpain and Lp82
Calcium requirements of recombinant Lp82 and m-calpain were tested. Activity of m-calpain and Lp82 reached of maximum at
120 and 20 µM calcium, respectively (Fig. 5). Maximum activity of Lp82 was reached at 50 µM calcium, whereas m-calpain required 500 µM calcium for maximum activity.
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DISCUSSION |
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Previous studies in our laboratory did not analyze fragmented -crystallins in detail, because, unlike ß-crystallins, which all undergo cleavage within their N-terminal extensions, fragmented
-crystallins in rats remain intact at their N terminus (9). This complicates the analysis of their cleavage sites by Edman sequencing. The experiments in the current study avoided these limitations by isolating whole proteins from 2-DE gels and accurately measuring their masses. The methodology, based on dispersion of gel pieces into 20-µm particles, allowed more rapid extraction and analysis of proteins than previous methods using passive elution (14, 23) and represents an improvement in the analysis of proteolytically modified proteins. The technique should also be useful to detect other post-translational modifications in 2-DE-separated proteins. Future studies will determine the utility of the method to isolate and measure masses of a wider variety of 2-DE-separated proteins.
m-Calpain has long been implicated as the major protease responsible for the processing of rodent crystallins, during both lens maturation and cataract formation (11, 24). However, because of its abundance and stability, the more recently discovered lens protease, Lp82, may also play an important role (25). Because there was no previous direct evidence that Lp82 was responsible for degradation of endogenous substrates in rat lens, the first goal in the present study was to identify biological markers that could estimate the relative activities of m-calpain and Lp82 in vivo. The A-fragment,
A1168, missing five residues from its C terminus, was shown to be an Lp82-specific product, whereas the
A-fragment,
A1162, missing 11 residues from its C terminus, was shown to be an m-calpain-specific product. This finding agrees with the studies of Yoshida et al. (26), who showed that purified bovine lens m-calpain could remove 10 and 11 residues, but not five residues, from the C terminus of
A-crystallin. Earlier studies using purified Lp82 and m-calpain from rat and bovine lens also confirmed these m-calpain cleavage sites and showed that Lp82 removed uniquely five residues from the C terminus of
A (10, 17). In the present study, using 2-DE gel separation to isolate the various truncated forms of
-crystallins, we were able to demonstrate for the first time that
A1168 appeared in lens before
A1162. This suggested that Lp82 is activated much earlier than m-calpain during lens maturation and that Lp82 may be responsible for the majority of excess crystallin proteolysis associated with experimental cataract formation. Evidence for m-calpain activity was only found in the lens nucleus of normal 6-week-old rats. m-Calpain may exhibit relatively greater activity in mature lenses, because, unlike Lp82, enzymatic activity and m-RNA for m-calpain are maintained in lens with age (27). m-Calpain may function in mature lens to provide very slow but sustained proteolytic activity, whereas Lp82 may function only during the period of rapid postnatal growth.
Three recent findings may explain the greater activity of Lp82 compared with m-calpain in young rat lens. Unlike m-calpain, Lp82 is relatively insensitive to calpastatin, the endogenous inhibitor of calpains (28). Lp82 also undergoes autolytic inactivation more slowly than does Lp82 (17). Finally, the calcium requirement for activation of Lp82 is much lower than the calcium requirement of m-calpain. The lower calcium requirement of Lp82 compared with m-calpain was demonstrated for both enzymes purified from bovine lens (17) and the recombinant forms of rat Lp82 and m-calpain used in the present study. Although the 108 µM free calcium concentration in the nucleus of young rats developing selenite-induced cataracts is high enough to activate Lp82, the mechanism for Lp82 activation in normal rat lens containing <1 µM free calcium is unknown (29). One possibility is that Lp82 is activated following association of the enzyme with lens membranes. This hypothesis is supported by the preferential association of Lp82 with the insoluble fraction of the lens (27).
The C-terminal truncation of -crystallins may have great biological significance in lens. Loss of C-terminal regions of
-crystallins has been shown previously to diminish the chaperone-like properties of
-crystallins (3, 9). The present study also demonstrated that truncated
-crystallins were insolubilized selectively. Similar selective insolubilization of truncated
-crystallins was also observed during analysis of mouse lens proteins by 2-DE (30). Carver and Lindner (31) have postulated that the flexible, solvent-exposed, hydrophilic C-terminal extensions of
-crystallins may function to keep
-crystallin·denatured protein complexes in solution. Selenite cataract may form because of insolubilization of truncated
·ß-crystallin complexes that are unable to stay in solution because of a loss of the solubilizing C-terminal extensions of
-crystallins. Similar complexes of truncated
- and ß-crystallins may not form opacities during normal aging of lens, because their slower accumulation could allow a more ordered arrangement within the lens cytosol.
The increased rate of proteolysis observed during formation of selenite-induced cataract in the present study is a common response of rodent lenses to stress. For example, similar accelerated truncation of -crystallins have been reported following cataract formation induced by galactose feeding (5), treatment with an inhibitor of cholesterol synthesis (7), and inherited mutations (8, 32). In contrast,
-crystallins in human lenses undergo less proteolysis. The majority of
A and
B examined from the water-insoluble fraction of normal aged human lenses remained intact (33). Additionally, unlike rodent lenses, the major
A-crystallin truncation product found in human and bovine lenses is only missing the C-terminal serine residue (33, 34). Whereas small quantities of
A1168, missing five residues from its C terminus, have also been detected in human lens (35, 36), the protease causing this cleavage is unknown, because no Lp82 activity is present in human lens (37). Thus, until the truncation states of
-crystallins are examined more closely in cataractous human lenses, or additional calpain isoforms are discovered, caution should be used in extrapolating the current findings in rodents to cataracts in man.
During the course of this study, we identified unexpectedly the recently discovered lens-specific protein GRIFIN (galectin-related inter-fiber protein). Unlike other members of the galectin family, GRIFIN does not bind to ß-galactoside, and its function is unknown (22). However, the current study found significant amounts of GRIFIN in the insoluble fraction of lens. This suggested that GRIFIN could be a peripheral membrane protein, with a function similar to another recently characterized member of the galectin family, galectin-3. Galectin-3 was postulated to act as a cell adhesion molecule in lens (38). The localization of GRIFIN by immunofluorescence to the interface between lens fiber cells supports this hypothesis (22).
In conclusion, analysis of -crystallin truncation suggested that the recently discovered lens-specific member of the calpain family, Lp82, was more active in young rat lens than m-calpain and was responsible for the majority of crystallin fragmentation during maturation and cataract formation. The regulated activity of Lp82 by calcium may allow an ordered truncation and insolubilization of crystallins in rat lens during normal maturation. Loss of calcium homeostasis in young rats may cause cataract by overactivation of Lp82.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, May 2, 2002, DOI 10.1074/mcp.M200007-MCP200
1 The abbreviation used is: 2-DE, two-dimensional electrophoresis.
* This work was supported in part by National Institutes of Health Grants EY12016 and EY07755 (to L. L. D.) and EY05786 (to T. R. S.) and Core Grant EY10572. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: TRA Urology Research Center, Bayer Yakuhin, Ltd., Kyoto, Japan.
|| To whom correspondence should be addressed: School of Dentistry and Medicine, Oregon Health and Science University, 611 S.W. Campus Dr., Portland, OR 97201. Tel.: 503-494-8625; Fax: 503-494-8772; E-mail: davidl{at}ohsu.edu.
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REFERENCES |
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