Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia

Abstract

Ceramide, a central molecule in the sphingolipid biosynthesis, plays a critical role as second messenger in cellular signaling that regulates antiproliferative processes, including apoptosis, cell differentiation, and cell cycle arrest in different cell types. Levels of ceramide, a highly bioactive molecule, must be tightly controlled by diverse, coordinated mechanisms, including ceramide degradation, phosphorylation, or sphingolipid metabolism. Glucosylceramide synthase (GCS), also defi ned as ceramide glucosyltransferase (CGT), metabolizes ceramide to glucosylceramide (GlcCer), a glycosylated form of ceramide that does not have antiproliferative activity. GCS plays a crucial role in cell survival after apoptotic stimuli, Abstract Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde traffi cking of the encystations-specifi c protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane traffi cking events, and stage differentiation in this parasite. In addition, we identifi ed ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes. -Štefanić,

Glucosylceramide Transferase Activity Is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan, Giardia lamblia

“…Because our current results show that antigGlcT1 morpholino oligonucleotide inhibits gGlcT1 activity and ESV biogenesis (Figs. 3 and 4), and because the activity of this enzyme is low in trophozoites and up-regulated in encysting cells, we thought that the effect of PPMP-inhibiting replication and cytokinesis on nonencysting trophozoites might not occur via GlcCer synthesis, as thought previously (18,26). In fact, there are reports in which PDMP (an analog of PPMP) has been shown to block cell cycle progression in mammalian cells by inhibiting cyclin-dependent kinases, which could be independent of GlcT1 inhibition and ceramide accumulation (42).…”

“…It has been reported that PPMP blocks replication and cytokinesis of giardial trophozoites and is thereby thought to inhibit the encystation and cyst production in culture (18,26). We have shown earlier that PPMP interferes with the formation of cysts when it is added in the culture medium during encystation (17).…”

“…The mixture was incubated for 5 h at 37°C and extracted with chloroform (CHCl 3 )/methanol (MeOH), 1:1 v/v, and the formation of enzymatic product ([ 14 C]GlcCer) was assessed by measuring the radioactivity in a scintillation counter (Beckman, Brea, CA). The assay was conducted in the presence and absence of PPMP (10 M), a common inhibitor of GlcCer synthesis (26).…”

Bioactive Sphingolipids in Response to Chemotherapy: A Scope on Leukemias

“…GluCer is not only important for conferring chemotherapeutic resistance to cancer cells, but it is also important for the pathophysiology of several disorders including Gaucher disease, polycystic kidney disease and asthma [83][84][85]. Membrane trafficking, natural killer lymphocyte polarization and neuronal activation can be given as examples of other cellular processes involving GluCer [86][87][88].…”

The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

“…1A, image a). A similar type of CTXB labeling of Giardia trophozoites was also reported earlier by Stefanić et al (21). Side-by-side with CTXB, parasites were labeled with GM1 antibody to stain raft-like structures.…”