Identification of Photooxidation Sites in Bovine α‐Crystallin

Abstract

Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine alpha-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine alpha-crystallin was irradiated with UV light (> 293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of alpha A-crystallin and 1-11, 12-22 and 57-69 of alpha B-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of alpha B 12-22. The specific sites of photooxidation indicate that the N-terminal regions of alpha A- and alpha B-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.

Modulating the Structure of EGFR with UV Light: New Possibilities in Cancer Therapy

“…1). These reactions are likely to lead to changes or to complete loss of protein structure and function [22], [43]. UVB excitation also leads to electron ejection from the side chains of aromatic residues [20], [24], [44]–[46].…”

The Major in vivo Modifications of the Human Water-insoluble Lens Crystallins are Disulfide Bonds, Deamidation, Methionine Oxidation and Backbone Cleavage

“…Among the many modi®cations proposed to affect crystallin solubility, and consequently lens opacity and cataract, are disul®de bonding (Spector and Roy, 1978;Kodama and Takemoto, 1988), modi®cation of lysines by carbamylation (Beswick and Harding, 1987) and glycation (Garlick et al, 1984;Liang and Rossi, 1990;van Boekel and Hoenders, 1992;Swamy et al, 1993;Lee, Mossine and Ortwerth, 1998), formation of cross-links involving lysines and arginines (Hayase et al, 1989;Sell and Monnier, 1989;Nagaraj et al, 1991;Nagaraj, Shipanova and Faust, 1996;Frye et al, 1998), oxidation by UV light (Lerman, Megaw and Morgan, 1985;Ohmori and Nose, 1985;Jose, 1986;Andley and Clark, 1989;Dillon et al, 1989;Li et al, 1990;Finley et al, 1997) and free radicals (Bessems et al, 1987;Taylor and Davies, 1987;Zigler, Huang and Du, 1989;, formation of transglutaminase mediated cross-links (Lorand et al, 1981;Pucci et al, 1988), formation of mixed disul®des (Lou, Dickerson and Garadi, 1990) and a variety of degradation products (Takemoto et al, 1987;Srivastava, 1988;Takemoto and Emmons, 1991;Emmons and Takemoto, 1992). Many of these reactions have been studied using in vitro incubations and the products analysed by techniques that did not permit the unambiguous identi®cations now possible.…”

Quantification of anti-aggregation activity of UV-irradiated α-crystallin

“…UV irradiation results in oxidative damage of lens proteins, especially γ-and β-crystallins, and the formation of insoluble aggregates of high molecular weight, whereas UV irradiation of α-crystallin does not cause significant rearrangement of protein quaternary structure, aggregation of oligomers or loss of solubility [9][10][11][12][13]. However, UV irradiation brings about oxidative modification of α-crystallin [14][15][16]. α-Crystallin, a member of the small heat shock protein (sHSP) superfamily, exhibits chaperone-like activity and protects β-and γ-crystallins in mammal eye lens against non-specific aggregation [17,18].…”