Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled l-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of l-cysteine in E. histolytica. [U-(13)C3, (15)N]l-cysteine was rapidly metabolized into three unknown metabolites, besides l-cystine and l-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of l-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of l-cysteine. Liberation of l-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these l-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.