title: Nitration of tryptophan in ribosomal proteins is a novel post-translational modification of differentiated and naïve PC12 cells.
abstract: Neuron growth factor (NGF) signaling in PC12 cell, which is derived from pheochromocytoma of rat adrenal medulla, induces expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production. Subsequently, NO causes differentiation of PC12 cell to neuronal cell with morphological changes, such as neurite extension. In this study, we showed that 6-nitrotryptophan-containing proteins were produced in PC12 cell (naïve PC12 cell) and/or NGF-induced PC12 cell (differentiated PC12 cell). Western blot analysis of the protein extract of naïve PC12 cell and differentiated PC12 cell using anti 6-nitrotryptophan antibody showed several immunoreactive bands, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. The peptides from five ribosomal proteins, namely, 60S ribosomal protein L7 (Trp154), 60S acidic ribosomal protein P1 (Trp43), 40S ribosomal protein S2 (Trp60), 40S ribosomal protein S6 (Trp45), and 40S ribosomal protein S19 (Trp52), were identified as nitrotryptophan residue-containing proteins with significant ion score levels (p<0.05). Among these, tryptophan nitration was observed only in differentiated PC12 cell for S19 protein, and only in naïve PC12 cell for L7 protein. Tryptophan nitration of the other ribosomal proteins P1, S2, and S6 was observed in both naive and differentiated PC12 cells. The positive signal of nitrotryptophan-containing proteins in the Western blotting around 16 kDa (Band 1), which includes 40S ribosomal protein S19, was suppressed by treatment with NOS inhibitor, L-NAME. The tryptophan nitration of 40S ribosomal protein was not observed by LC-ESI-MS-MS analysis of this sample. This is the first study to identify several specific sites of nitrated tryptophan on proteins not only in viable culture cells but also in a physiological process: cell differentiation.
authors.affiliation: The Institute for Environmental and Gender-Specific Medicine, Urayasu, Chiba 279-0021, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kawasaki
authors.forename: Ayako
authors.initials: A
authors.lastname: Shigenaga
authors.forename: Munehiro
authors.initials: M
authors.lastname: Uda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Baba
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
authors.forename: Kenji
authors.initials: K
authors.lastname: Takamori
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Yamakura
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D020932:Nerve Growth Factor / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D016716:PC12 Cells; D011499:Protein Processing, Post-Translational; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D012269:Ribosomal Proteins; D009569:Nitric Oxide; C055424:6-nitrotryptophan; D014364:Tryptophan; D020932:Nerve Growth Factor; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D019331:NG-Nitroarginine Methyl Ester