De Novo Ceramide Synthesis Is Required for N-Linked Glycosylation in Plasma Cells

Abstract

Plasma cells (PCs) are terminally differentiated B lymphocytes responsible for the synthesis and secretion of Igs. The differentiation of B cells into PCs involves a remarkable expansion of both lipid and protein components of the endoplasmic reticulum. Despite their importance in many signal transduction pathways, the role of ceramides, and of complex sphingolipids that are derived from ceramide, in PC differentiation has never been directly studied. To assess their putative role in PC differentiation, we blocked ceramide synthesis with fumonisin B1, a specific inhibitor of ceramide synthase. Under fumonisin B1 treatment, N-linked glycosylation was severely impaired in LPS-activated, but not in naive, B cells. We also show that ceramide synthesis is strongly induced by XBP-1 (X box-binding protein-1). In the absence of ceramide synthesis, ER expansion was dramatically diminished. Our results underscore ceramide biosynthesis as a key metabolic pathway in the process of PC differentiation and reveal a previously unknown functional link between sphingolipids and N-linked glycosylation in PCs. The Journal of Immunology, 2009, 182: 7038 -7047. P lasma cells (PCs)3 are terminally differentiated B lymphocytes responsible for the synthesis and secretion of Igs. The differentiation of B cells into PCs involves a series of remarkable phenotypic changes, most prominent of which are a cessation of cell division and remodeling of the secretory pathway (1). IRF4 (IFN regulatory factor 4), Blimp-1, and XBP-1 (X boxbinding protein-1) are transcription factors essential for this transition (2-4). While IRF4 and Blimp-1 are required to extinguish the mature B cell program and terminate the germinal center reaction, XBP-1 is required to initiate the PC program, which involves a steep up-regulation of the synthesis of Ig molecules in their secreted form and the expansion of the endoplasmic reticulum (ER) (4 -6).The mechanism behind the expansion of the ER entails a marked increase of both its lipid and protein components (7,8). These changes are largely regulated by XBP-1 in a process that is sensitive to conditions of ER stress, which ensue when the amount of client proteins that emerge into the ER exceeds its overall folding capacity. XBP-1 is a key element of an intricate cytoprotective ER-to-nucleus signaling pathway, which responds to conditions of ER stress. This pathway is collectively referred to as the unfolded protein response (UPR). The mammalian UPR is composed of at least three transducers: PERK (PKR-like ER kinase), ATF6 (activating transcription factor 6), and IRE1. While PERK reduces protein translation by phosphorylation of eIF2␣ (eukaryotic initiation factor-2␣) on serine 51, ATF6 and IRE1 largely control the transcription of UPR targets. When activated, IRE1 oligomerizes and undergoes autophosphorylation. Phosphorylated IRE1 activates a nuclease activity in its cytosolic tail, which then removes a small intron in the mRNA of XBP-1. The conversion of unspliced XBP-1 (XBP-1u) into the spliced form (XBP-1s) in the ...

Glucose-dependent de Novo Lipogenesis in B Lymphocytes

“…A doubling of membrane phospholipid content precedes proliferation, whereas differentiation involves a major morphological change of the cell interior, including a substantial expansion of the rough ER and Golgi network (17)(18)(19). Distinct changes in the ER network are evident as early as 24 h after exposure of naive B lymphocytes to LPS (14,15).…”

“…CM synthesis is required for ER expansion and protein glycosylation in plasma cells (19). CM synthesis is up-regulated in an X-box binding protein 1 (Xbp1)-dependent manner and in turn results in increased levels of sphingolipids (19,20). XBP-1 is a key transcription factor that drives the unfolded protein response (20).…”

“…6A); both of these lipids are required for ER expansion in B lymphocytes (17)(18)(19). It therefore seemed likely that ACLY activity might contribute to expansion of the endomembrane secretory network in response to LPS stimulation.…”

“…The molecular mechanisms underlying how membrane phospholipid and cholesterol content increases during B lymphocyte differentiation have received little attention (18,19,21,22). Overall rates of fatty acid synthesis in most mammalian tissues are relatively low, in part, because lipids are acquired from the circulation.…”

Regulated IRE1‐dependent decay participates in curtailing immunoglobulin secretion from plasma cells

“…CD19-Cre-driven conditional XBP-1 KO was previously described [35]. Floxed IRE1 bearing mice were provided by Dr. Takao Iwawaki and crossed to the CD19-Cre strain for five generations.…”