Quantifying Changes in the Cellular Thiol-Disulfide Status during Differentiation of B Cells into Antibody-Secreting Plasma Cells

Plasma cells produce and secrete massive amounts of disulfide-containing antibodies. To accommodate this load on the secretory machinery, the differentiation of resting B cells into antibody-secreting plasma cells is accompanied by a preferential expansion of the secretory compartments of the cells and by an up-regulation of enzymes involved in redox regulation and protein folding. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated proteins in whole cells. The results show that while the global thiol-disulfide state is affected to some extent by the differentiation, steady-state levels of glutathionylated protein thiols are less than 0.3% of the total protein cysteines, even in fully differentiated cells, and the overall protein redox state is not affected until late in differentiation, when large-scale IgM production is ongoing. A general expansion of the ER does not affect global protein redox status until an extensive production of cargo proteins has started. 1. Introduction The cellular thiol-disulfide redox environment is defined by protein thiols (PSH) and disulfides as well as low molecular weight thiols and disulfides. In mammalian cells, by far the most abundant low molecular weight sulfhydryl molecule is glutathione (GSH). Together with its disulfide (GSSG), this pair is often referred to as the cellular thiol-disulfide redox buffer. In the cytosol of eukaryotic cells, glutathione is highly reducing with a ratio of GSH to GSSG of at least 3,000 [1, 2], and consequently the majority of protein cysteines are found as PSH. The high concentrations of PSH and GSH in this compartment are important in the cellular defense against thiol oxidants [3], during thiol-disulfide stress, formation of mixed disulfides between protein and glutathione (PSSG) serves as a mechanism for protecting PSH and GSH from irreversible oxidation. In contrast to cytosolic proteins, secretory proteins often contain disulfide bonds, and the glutathione redox pool in the secretory compartments of the cell is found to be considerably more oxidizing than the cytosolic pool [4]. Disulfide bond formation is an essential step for the correct folding of many secretory proteins [5], and in eukaryotic cells their folding and assembly takes place in the endoplasmic reticulum (ER). In this compartment, molecular chaperones and enzymes for disulfide bond formation and glycosylation support protein folding. The maintenance of a proper ER redox environment is crucial for the folding of secretory proteins. If the redox environment becomes too