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Human and Monkey Lenses Cultured with Calcium Ionophore Form B

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Human and Monkey Lenses Cultured with Calcium Ionophore Form B Human and Monkey Lenses Cultured with Calcium Ionophore Form ␣B-Crystallin Lacking the C-Terminal Lysine, a Prominent Feature of Some Human Cataracts Emi Nakajima,1,2 Larry L. David,3 Michael A. Riviere,2 Mitsuyoshi Azuma,1,2 and Thomas R. Shearer2 PURPOSE. Elevation of lens calcium occurs in both human and ataracts are opacities in the lens of the eye. This disease is experimental animal cataracts, and opacification may result Cthe leading cause of preventable blindness in the world,1 from calcium-activated proteolysis. The purpose of the present yet an effective therapeutic strategy to slow the rate of cataract study was to determine whether calcium accumulation in cul- progression is unavailable. The human lens faces several chal- tured human and Macaca mulatta lenses results in proteolysis lenges in attempting to remain transparent during its long of crystallins, the major lens proteins. lifespan. It is exposed to several possible oxidants,2,3 under- 4 METHODS. Two-dimensional electrophoresis and mass spec- goes a large number of posttranslational modifications, and is trometry were used to construct detailed maps of human and unable to replace the damaged proteins contained in its oldest 5 monkey lens crystallins so that proteolysis after calcium accu- central region because of a programmed loss of organelles. mulation could be monitored and the altered crystallins iden- Opacification results from a loss of the short-range order of tified. Human and macaque lenses cultured in A23187 showed crystallins, the major proteins of the lens, due to their aggre- elevated lenticular calcium and superficial cortical opacities. gation on unfolding or separation into protein-rich and -poor 6 The carboxypeptidase E (CPE) gene is expressed in human phases. Three factors that may normally prevent opacification lens, and its presence in lens fibers was demonstrated by with increasing age are a healthy epithelium on the anterior Western blot. To investigate whether CPE could cause similar surface of the lens where the bulk of metabolic activity re- 7 truncation, purified ␣B-crystallin and CPE were incubated in sides, a complex network of pumps and channels allowing vitro. movement of ions and small molecules to and from the lens interior,8 and the maintenance of a reducing environment, RESULTS. The major change observed in the crystallins of these especially in the center of the lens.9 Although oxidation of cultured lenses was the accumulation of ␣B -crystallin re- 1-174 proteins in the lens interior may directly precede opacification, sulting from the loss of a C-terminal lysine. This result was alterations in lens epithelium may initiate the process. significant, because similar appearance of ␣B is a promi- 1-174 Global genomic10 and proteomic11 analyses have been used nent change in some human cataracts. ␣B-crystallin and CPE to examine the differences between gene expression and pro- incubation result in the formation of ␣B -crystallin. This 1-174 tein composition in normal and cataractous human lenses. A truncation was specific to ␣B -crystallin, since other crys- 1-174 major finding in the proteomic analysis, performed by two- tallins were not proteolyzed. Although a weaker activator than zinc, calcium activated CPE in vitro. dimensional gel electrophoresis (2-DE), was a 10% to 90% loss of the C-terminal Lys175 from a major lens protein known as CONCLUSIONS. Since zinc concentrations did not increase during ␣B-crystallin.11 Additional changes in this normally 20-kDa pro- culture in A23187, calcium uptake in the lens may be respon- tein also included an N-terminal cleavage yielding a 16.4-kDa ␣ sible for CPE activation and B1-174 formation during cataract. fragment and an overall elevation in the concentration of ␣B- (Invest Ophthalmol Vis Sci. 2009;50:5828–5836) DOI:10.1167/ crystallin. iovs.09-4015 ␣B-crystallin and its binding partner ␣A-crystallin belong to the small heat shock protein family and form a polydisperse aggregate of 300 to 1200 kDa. These proteins are capable of ␤ ␥ From the 1Laboratory of Ocular Sciences, Senju Pharmaceutical binding to partially unfolded intermediates of the / -cystallin Corporation Limited, Beaverton, Oregon; and the Departments of 2In- families and prevent their irreversible aggregation and precip- 6 tegrative Biosciences and 3Molecular Biology, Oregon Health and Sci- itation during aging. Thus, ␣-crystallins may function in the ence University, Portland, Oregon. human lens, where they contribute 28% of the total protein,12 Supported by NIH Grants EY07755 (LLD), EY05786 (TRS), and by preventing opacity, until they are “titrated out” by other Core Grant EY10572. TRS is a paid consultant for Senju Pharmaceutical unfolded lens proteins.6 The study of lens epithelial cells from Co., Ltd., a company that may have a commercial interest in the results knockout mice missing ␣-crystallins suggests these proteins of this research and technology. MA is and EN was (during the study) may also influence cell proliferation. Lens epithelial cells from an employee of Senju Pharmaceutical Co., Ltd. This potential conflict ␣ Ϫ/Ϫ 13 of interest was reviewed, and a management plan approved by the A mice exhibited reduced survival and growth, whereas ␣ Ϫ/Ϫ OHSU Conflict of Interest in Research Committee was implemented. epithelial cells from B mice exhibited genomic instability 14 Submitted for publication May 20, 2009; revised June 16, 2009; and hyperproliferation. The reported cataract-specific accepted August 25, 2009. changes in ␣B may alter one or more ␣B functions and con- Disclosure: E. Nakajima, Senju Pharmaceutical Co., Ltd. (E, F); tribute to cataract formation. L.L. David, Senju Pharmaceutical Co., Ltd. (F); M.A. Riviere, Senju ␣ The protease producing truncated B-Lys1-174 is unknown, Pharmaceutical Co., Ltd. (F); M. Azuma, Senju Pharmaceutical Co., but this cleavage was most likely due to the activation of a Ltd. (F); T.R. Shearer, Senju Pharmaceutical Co., Ltd. (C, F) carboxypeptidase. Although aminopeptidases have been thor- The publication costs of this article were defrayed in part by page oughly examined in the lens,15 carboxypeptidases have not charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. been characterized. However, several metallocarboxypepti- Corresponding author: Emi Nakajima, Senju Laboratory of Ocular dases have been described in other tissues and are capable of Sciences, OHSU West Campus, 20000 NW Walker Rd., Suite JM508, removing basic residues from both peptides and proteins. Beaverton, OR 97006; [email protected]. Transcripts for carboxypeptidases E/H (NbLi0012; NEI Bank, Investigative Ophthalmology & Visual Science, December 2009, Vol. 50, No. 12 5828 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 10/03/2021 IOVS, December 2009, Vol. 50, No. 12 Truncated ␣B-crystallin in Primate Lenses 5829 available at http://www.neibank.nei.gov/, provided in the pub- with 50 ␮g/mL gentamicin and 10% fetal bovine serum and cultured at lic domain by the National Eye Institute, Bethesda, MD), D, and 37°C under 5% CO2 for up to 7 days. Ten micromoles of A23187 (EMD X16 have been detected in the human lens. Many human Biosciences, Inc., San Diego, CA) were added on day 1 only (A23187 cataracts, especially those in the cortex contain increased lev- group). Paired lenses were used for control and experimental proto- els of calcium,17 and calcium is an activator of some proteases. cols. Therefore, the purpose of the present study was to determine whether elevation of lens calcium in cultured primate lenses is Two-Dimensional Gel Electrophoresis capable of reproducing the previously reported alterations in ␣ First-dimension isoelectric focusing was performed using custom, 18- lens B-crystallin. In the present study, calcium loading in both cm, linear pH 5 to 10 immobilized pH gradient (IPG) strips produced human and monkey lenses caused the loss of the C-terminal 18 ␮ ␣ as previously described, and reswelled in 340 L of solution contain- Lys175 from B-crystallin, but did not produce the 16.4-kDa ing 400 ␮g protein, 8 M deionized urea, 2% CHAPS, 50 mM DTT, 2% form of the protein. These results were unlike those in similar glycerol, 2% pH 6 to 11 IPG buffer (GE Health Care, Piscataway, NJ), experiments with rodent lenses, where calcium loading caused and a trace of bromophenol blue. Isoelectric focusing was performed activation of the calcium-dependent endopeptidase calpain. (Protean IEF Cell; Bio-Rad, Hercules, CA) using a program with the voltage ramp set at rapid, a final 3,500-V setting, 50,000 total volt hour, ␮ MATERIALS AND METHODS 50 A limit per gel, and 20°C temperature. The gel strips were then reduced/alkylated, and the second-dimension separation was per- Source of Lenses formed on 24 ϫ 18.5-cm 12% SDS PAGE gels, as previously de- scribed.18 The gels were then either stained with Coomassie blue Human eyes from 3-, 71-, 76-, and 81-year-old donors were obtained G-25019 in preparation for in-gel digestion and identification of pro- from the Lions Eye Bank (Portland, OR). The research adhered to the teins or negative stained with zinc-imidazole20 in preparation for pro- tenets of the Declaration of Helsinki and was approved by the institu- tein elution and mass measurement. tional human experimentation committee or institutional review board (IRB). Eyes from monkeys (Macaca mulatta) ranging in age from 1 to Protein Analysis by Mass Spectrometry 12 years were obtained from procedures unrelated to the present 21 studies. Experimental animals were handled in accordance with the In-gel trypsinization was performed as previously described, and 21 ARVO Statement for the Use of Animals in Ophthalmic and Vision digests were analyzed by either LC-MS or atmospheric pressure 22 Research and the NIH Guiding Principles in the Care and Use of MALDI-MS to collect MS/MS spectra using an ion trap mass spectrom- Animals.
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