Role of Intracellular Lipid Logistics in the Preferential Usage of Very Long Chain-Ceramides in Glucosylceramide

Abstract

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20–26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It is known that the proportion of VLC species in GSLs is higher than that in SM. To address the mechanism of the VLC-preference of GSLs, we used genome editing to establish three HeLa cell mutants that expressed different amounts of CERS2 and compared the acyl chain lengths of SM and GSLs by metabolic labeling experiments. VLC-sphingolipid expression was increased along with that of CERS2, and the proportion of VLC species in glucosylceramide (GlcCer) was higher than that in SM for all expression levels of CERS2. This higher proportion was still maintained even when the proportion of C16-Cer to the total ceramides was increased by disrupting the ceramide transport protein (CERT)-dependent C16-Cer delivery pathway for SM synthesis. On the other hand, merging the Golgi apparatus and the endoplasmic reticulum (ER) by Brefeldin A decreased the proportion of VLC species in GlcCer probably due to higher accessibility of UDP-glucose ceramide glucosyltransferase (UGCG) to C16-rich ceramides. These results suggest the existence of a yet-to-be-identified mechanism rendering VLC-Cer more accessible than C16-Cer to UGCG, which is independent of CERT.

Blood group P1 antigen–bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors

“…Metabolic labeling experiments using D-[1-14 C]galactose, including mild alkaline methanolysis, were performed as described previously (59). Cells (3 3 10 5 /well in a 6-well plate) were cultured overnight at 37°C and then incubated for 16 h with 7.4 kBq of D-[1-14 C]galactose in Opti-MEM with 1% Neutridoma-SP (Roche).…”

Defining lipid mediators of insulin resistance: controversies and challenges

“…Ceramides are primarily generated de novo in the ER, but also from the hydrolysis of sphingomyelin at the plasma membrane and via sphingosine salvage in the mitochondria. Subcellular transport mechanisms, particularly to the Golgi where ceramides are further metabolised, show preferential targeting for specific ceramide species (Kumagai et al 2005, Konstantynowicz-Nowicka et al 2015, Kakazu et al 2016, Yamaji et al 2016, indicating the likelihood of differential function according to spatial distribution. Temporal studies have further found that ceramide species in the nucleus are differentially composed and regulated to those found in mitochondria (Aviram et al 2016).…”

Structure, functions and regulation of CERT, a lipid‐transfer protein for the delivery of ceramide at the ER–Golgi membrane contact sites

“…This paradox might be explained by recent findings. First, CERT may preferentially transport ceramide subspecies that are preferential substrates for SMSs . Second, when GlcCer synthase is partitioned into the same Golgi cisterna as SMS1, the activity of the former enzyme is inhibited .…”