Article pubs.acs.org/JAFC
Carnosine Ameliorates Lens Protein Turbidity Formations by Inhibiting Calpain Proteolysis and Ultraviolet C‑Induced Degradation Jiahn-Haur Liao,† I-Lin Lin,§ Kai-Fa Huang,† Pei-Ting Kuo,§ Shih-Hsiung Wu,† and Tzu-Hua Wu*,§ †
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Department of Clinical Pharmacy, School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
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ABSTRACT: Carnosine (CAR) is an endogenous peptide and present in lens, but there is little evidence for its effectiveness in calpain-induced proteolysis inhibition and its differential effects toward different wavelengths of ultraviolet (UV) irradiation. This study aimed to develop three in vitro cataract models to compare the mechanisms underlying the protective activities of CAR. Crude crystallins extracted from porcine lenses were used for antiproteolysis assays, and purified γ-crystallins were used for antiUV assays. The turbidity in those in vitro models mimics cataract formation and was assayed by measuring optical density (OD) at 405 nm. The effectiveness of CAR on calpain-induced proteolysis was studied at 37 and 58 °C. Patterns of proteins were then analyzed by SDS-PAGE. The turbidity was reduced significantly (p < 0.05) at 60 min measurements with the increased concentration of CAR (10−300 mM). SDS-PAGE showed that the decreased intensities at both ∼28 and ∼30 kDa protein bands in heat-enhanced assays were ameliorated by CAR at ≥10 mM concentrations. In UV-B studies, CAR (200, 300 mM) reduced the turbidity of γ-crystallin significantly (p < 0.05) at 6 h observations. The turbidity of samples containing γ-crystallins was ameliorated while incubated with CAR (100, 300 mM) significantly (p < 0.05) following 4 h of exposure to UV-C. SDS-PAGE showed that the presence of CAR reduced UV-B-induced aggregation of γ-crystallins at ∼44 kDa and resulted in less loss of γcrystallin following UV-C exposure. The result of modeling also suggests that CAR acts as an inhibitor of calpain. In conclusion, CAR protects lens proteins more readily by inhibiting proteolysis and UV-C-induced degradation than aggregation induced by UV-B irradiation. KEYWORDS: carnosine, calpain proteolysis, UV insults, lens turbidity, molecular modeling
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INTRODUCTION
In addition, increased exposure to ultraviolet light results in increased photo-oxidative stress and will increase the risk of cataractogenesis.14 The human lens can absorb visible light, UV-A and UV-B, but ocular protection from UV-B (290−320 nm) is recommended.15 Moreover, accidental overexposure to UV-C (200−290 nm) during germicidal therapy can cause corneal burns and severe sunburn to the face and eyes. The consequences of overexposure to UV-C include increased production of free radicals, increased sensitivity of proteolysis, and decreased chaperone activity of α-crystallin16,17 and further cause lens protein conformation destruction by cross-linking lens protein and further decreasing lens transparency. Cataract is the leading cause of blindness, accounting for approximately 50% of blindness worldwide. Previously, we demonstrated that compounds that protect γ-crystallins from UV-C damage18,19 or interact with calcium20−22 may potentially be used as anticataract agents. In certain countries, CAR is available as a dietary supplement or eye drops in the health promotion market. Clinical eye drops containing its prodrug, N-acetylcarnosine, were also demonstrated to be absorbed intraocularly to prevent and reverse cataract.23,24 However, claims that CAR possesses ophthalmological benefits are, as yet, insufficiently supported for the medical community to recommend CAR as a topical treatment for cataracts. For
Carnosine (β-alanyl-L-histidine; CAR), discovered by Russian scientists in 1990, is a dipeptide present in meat,1 lens,2 and other tissues. The endogenous CAR acts as a natural antioxidant to scavenge free radicals3 and lipid peroxides.4 Its L-histidine, more specifically its imidazole moiety, appears to be the prime bioactive component and may react nonenzymically with protein carbonyl groups, a process termed “carnosinylation” to show its antiaging actions.5 In eye lenses, crystallins are the proteins essential for their transparency. Among lens crystallin proteins, α-crystallin is able to prevent aggregation of the other destabilized proteins (especially of γ- and βcrystallins). The reported abilities of CAR in disaggregating crystallins, inhibiting fibril formations,6,7 and protecting αcrystallins against glycation8 provide an explanation for the anticataract activity exerted by CAR eye drops. The aggregations or degradations of crystallins in cataractous eye lenses may result from intracellular or environmental insults. Recent studies have suggested that a major cause of the insolubilization of crystallins may be the unregulated proteolysis by calpains.9 Calpain is one of the widely distributed calcium-dependent cysteine proteases. Among the subtypes of calpains, calpain II is the major type in human and rodent lenses10,11 and is present in the superior cortical region.12 Calpastatin is an endogenous inhibitor of calpain II in certain tissues. Inappropriate regulation of the calpain−calpastatin proteolytic system is associated with several important human pathological disorders including cataract formation.13 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5932
April 15, 2014 May 29, 2014 June 5, 2014 June 16, 2014 dx.doi.org/10.1021/jf5017708 | J. Agric. Food Chem. 2014, 62, 5932−5938
Journal of Agricultural and Food Chemistry
Article
of the structures of rat calpain II bound to calpastatin, the model of CAR was manually docked into the active site of S. scrofa calpain II model with the program Coot to generate an initial binding pose of carnosine in S. scrofa calpain II. This model of the calpain II−CAR complex was optimized by energy minimization with the program Discovery Studio, and the resulting model with the lowest value of potential energy was selected. The stereochemical quality of the model was further checked with the programs 3D-profile and PROCHECK. The structure figures were generated with the program PyMOL (Schrödinger, New York, NY, USA). Statistical Analysis. Average turbidity formation (in absolute OD 405 nm) changes from the baseline or percentage changes versus the controls were calculated. The changes of turbidity after various treatments are expressed as the mean ± standard error of the mean (n ≥ 3). The data were analyzed using an unpaired t test between two groups or by Kruskal−Wallis one-way analysis of variance (ANOVA) on ranks with a post hoc Dunn’s test, using SigmaStat 2.03 (Systat Software, San Jose, CA, USA). Differences were considered significant when the p value was
