Interactions of Gliotoxin with Protein Cysteines

Abstract

Gliotoxin is a secondary metabolite that is produced by several species of fungi, and is toxic to mammalian cells. It is immunosuppressive, affects antigen presentation by macrophages, and causes apoptosis of some cells. Gliotoxin is an epipolythiodioxopiperazine molecule and contains an internal disulfide bridge that is highly reactive and essential for its toxicity. Suggested mechanisms of action include modification of thiol groups of cysteine residues in target proteins by generating oxidative stress or through covalent modification. The goal of this project was to develop mass spectrometry methods to detect protein modification by gliotoxin. Creatine kinase was used as a model protein. The measured mass of creatine kinase from 45 spectra gave a mean of 42,944 ± 24 which was consistent with the predicted mass of creatine kinase. A tryptic digest of creatine kinase indicated ions consistent with the predicted masses of the four cys-containing peptides including abundant ions at m/z 794, 1130 and 2870 and an ion at low intensity at 4373. The reaction of creatine kinase with gliotoxin showed a time dependent reaction that after 14 h was consistent with formation of a gliotoxin adduct. Reduction of the product with dithiothreitol released creatine kinase. Analysis of the tryptic peptides using MALDI mass spectrometry indicated complex modification of cysteines possibly including formation of a mixed disulfide adduct, intramolecular disulfides of CK, and sulfur oxidation products. Further analysis using the ICAT (isotope-coded affinity tag) method suggested modification of Cys-254 and Cys-283 by gliotoxin. Preliminary experiments examined the effects of gliotoxin on HL-60 cells using ICAT. Proteins of gliotoxin-treated and untreated cells were labeled with Heavy and Light ICAT reagents. Potential ICAT pairs were detected in the mass spectrum as a preliminary search for proteins affected by gliotoxin. The results indicate that ICAT labeling should be an effective strategy for characterization of the protein targets of gliotoxin.