3-nitrotyrosine and 6-nitrotryptophan

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has been indicated in the pathogenesis of various ocular disorders. The PON induces mobilization of intra cellular calcium which plays an important function in structure and activity of lens proteins. Moreover, the amount of calcium increases to the pathogenic level in the cataractous lenses. The aim of this study was to assess the impact of calcium ion on structure and aggregation of PON-modified lens crystallins, using spectroscopic techniques and gel mobility shift assay. The PON modification of lens proteins was confirmed with detection of the significantly increased quantity of carbonyl group, dityrosine, nitrotyrosine and nitrotryptophan. Moreover, the modified proteins indicated high levels of solvent exposed hydrophobic surfaces and markedly elevated proteolytic instability which can be explained with their structural alteration upon this type of modification. The results of UV-vis absorption studies suggest that PON-modified lens crystallins are highly sensitive to aggregation in the presence of both physiological and pathological ranges of calcium ion. Also, the results of thioflavin T fluorescence study indicated absence of any ordered aggregate entity in the calcium-induced aggregate samples. The results of gel mobility shift assay demonstrated the importance of calcium ion in the induction of disulfide and dityrosine covalent cross-linking and formation of the oligomeric structure with relatively larger sizes in the PON-modified crystallins compared to the non-modified protein counterparts. Overall, this study may suggest that a simultaneous raise of calcium ion and PON in the eye ball is an important risk factor for development of cataract diseases.

Topics: Animals; Benzothiazoles; Calcium; Cattle; Crystallins; Hydrophobic and Hydrophilic Interactions; Lens, Crystalline; Oxidants; Peroxynitrous Acid; Protein Aggregates; Protein Carbonylation; Protein Stability; Proteolysis; Spectrometry, Fluorescence; Thiazoles; Tryptophan; Tyrosine

One of the important sites of peroxynitrite action that affects cellular function is known to be nitration of tyrosine residues. However, tryptophan residues could be another target of peroxynitrite-dependent modification of protein function, as we have shown previously using a model protein (F. Yamakura et al., J. Biochem. 138:57-69; 2005). Here, we report the identification of several proteins that allowed us to determine the position of nitrotryptophan in their amino acid sequences in a more complex system. We modified lysates from PC12 cells with and without nerve growth factor (NGF) by treatment with peroxynitrite (0.98 or 4.9 mM). Western blot analyses using anti-6-nitrotryptophan antibody showed several immunoreactive bands and spots, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. We identified several tryptic peptides including nitrotryptophan residues, which were derived from L-lactate dehydrogenase A, malate dehydrogenase 1, M2 pyruvate kinase, and heat-shock protein 90 α, in peroxynitrite-treated lysates from PC12 cells, and l-lactate dehydrogenase A, malate dehydrogenase 1, transaldorase, and lactoylglutathione lyase, in peroxynitrite-treated lysates from NGF/PC12 cells. The molar ratio of 3-nitrotyrosine to 6-nitrotryptophan in protease-digested PC12 cell lysates treated with peroxynitrite was determined to be 5.8 to 1 by using an HPLC-CoulArray system. This is the first report to identify several specific sites of nitrated tryptophan on proteins in a complex system treated with peroxynitrite and to compare the susceptibility of nitration between tryptophan and tyrosine residues of the proteins.

Topics: Amino Acid Sequence; Animals; Cell Extracts; Chromatography, Liquid; Enzymes; HSP90 Heat-Shock Proteins; Models, Molecular; Molecular Sequence Data; PC12 Cells; Peroxynitrous Acid; Protein Structure, Tertiary; Rats; Tandem Mass Spectrometry; Tryptophan; Tyrosine