Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore.

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.

Activated neutrophils oxidize extracellular proteins of endothelial cells in culture: effect of nitric oxide donors.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.