1 - The Overlooked Ditryptophan Cross-Link Is Present in Bovine Beta Crystalline Submitted to UVC or Solar Simulator Irradiation

Abstract

During aging and pathological conditions associated with oxidative stress, the levels of over-oxidized and cross-linked proteins build-up because these species are poor substrates for the intracellular proteolytic systems, leading to protein aggregation. The chemical nature of protein cross-links remains largely uncharacterized despite their potential importance in protein aggregation. Even for characterized cross-links, such as dityrosine, information about their occurrence on specific proteins and on specific residues remains limited because bioinformatic tools to analyze LC-MS/MS proteomic data are under development. Not surprisingly, the ditryptophan cross-links (Trp-Trp), which are produced by recombination of protein-tryptophanyl radicals (protein-Trp•), have been largely disregarded in the literature. Previously, we showed that Trp-Trp cross-links are formed on human SOD1 and lysozyme submitted to the carbonate radical and on lysozyme irradiated with UVC light. Here, we report that Trp-Trp cross-links are also produced upon bovine beta crystalline irradiation. Beta crystalline (40 μM) and DTPA (0.1 mM) in phosphate buffer, pH 7.0 was irradiated for different times with UVC (254 nm, 6.3 mW/cm2) (0.5-10 min) or with a solar simulator (SOL-UV-2 (Newport); 5.0 mW/cm2 at 315 nm) (1-120 min). After irradiation, the samples analyzed by SDS-PAGE showed formation of dimers (~50 kDa) that attained a maximum at 2 min and decreased to produce higher oligomers (> 100 kDa); in the case of the simulator, dimers were clearly noted after 60 min irradiation. The bands corresponding to the irradiated samples at the required time were excised and pooled from the gels, reduced, alkylated, digested with trypsin and submitted to nano-ESI-Q-TOF MS/MS analysis. In the case of the samples irradiated with UVC, the results characterized the residues oxidized in the monomer at chain B3 (Trp82, Trp151, Trp158andTyr161) and chain A3 (Trp198) and in the dimer, a Trp151-Trp151crosslink was characterized in chain B2. In the case of samples irradiated with the simulator, the results characterized the residues oxidized in the monomer at chain B2 (Trp82) and chain A3 (Trp198). In the dimer, a Trp150-Trp150cross-link was characterized in chain A2. Although different dimers were produced under the 2 irradiation conditions, the results indicate that the formation of Trp-Trp cross-links is more common than previously considered.