The Journal of Immunology, Vol 156, Issue 5 1973-1980, Copyright © 1996 by American Association of Immunologists

ARTICLES

Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress

T Seres, V Ravichandran, T Moriguchi, K Rokutan, JA Thomas and RB Johnston Jr
Department of Pediatrics (Immunology), Yale University School of Medicine, New Haven, CT 06520, USA.

Stimulation of the respiratory burst in mouse macrophages or human neutrophils results in the formation of disulfide bonds between low m.w. thiols and sulfhydryl groups on specific cytosolic proteins (S- thiolation). S-thiolation is reversible in certain chemical systems. The aim of the present study was to analyze the dynamic nature of this process in human monocytes under physiologic conditions. We report here that the extent of S-thiolation and the rate of respiratory burst stimulated by opsonized zymosan or phorbol diester increased for 10 to 20 min and then declined (dethiolation) in close association. Individual proteins underwent S-thiolation and dethiolation at different rates. H2O--appeared particularly effective in mediating S- thiolation, based on inhibition of S-thiolation by added catalase and accentuation by azide, which inhibits cellular catalase. S-thiolation did not occur in stimulated monocytes from patients with chronic granulomatous disease. The addition of H2O2 to monocytes or lymphocytes induced rapid S-thiolation (1 to 3 min); a subsequent dethiolation returned most of the proteins to baseline by 15 to 30 min. At 0 degrees C and after addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea, there was effective S-thiolation on exposure to H2O2, but dethiolation was inhibited, suggesting a possible role for glutathione (GSH)/thioredoxin reductase systems in this process. GSH was determined to be the most abundant low m.w. thiol bound to S-thiolated proteins, but gamma- glutamylcysteine and cysteine were also bound. The time of maximal reduction in cytosolic GSH during the respiratory burst (10 min) coincided with the time at which protein-bound GSH was highest. S- thiolation-dethiolation represents a reversible post-translational modification that could protect cellular proteins from irreversible oxidative damage.

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