B GalCer synthesis occurs at luminal site of ER, while galactosylation of Chol occurs at the cytosolic site of ER C Glycosylated Chol is secreted into cultured medium as EVs (exosomes). Plant type glucosylated sitosterol is GSL, and determined its structure and biosynthetic pathway. Interestingly, the glycosylation of Chol is carried out by retained trans-glycosylation by glucosylceramide (GlcCer)-degrading enzymes, GBA1 and 2 3,4) (Figure 2A,2B). We have demonstrated that GBAs act not only on GlcCer but also on GalCer to biosynthesize galactosylcholesterol (GalChol). Unexpectedly, GBAs, mainly GBA2 act on GalCer as well as on GlcCer to synthesize GalChol 5). These glycolipids are secreted as extracellular vesicle (EVs) suggesting that UDP-sugar independent glycosylation of the lipids is involved in membrane GSL/sterol homeostatic mechanisms.Figure 2. Membrane topology of lipid glucosylation and destination of glycosylated lipidsA Topology of GlcChol synthesis. GlcCer cytosolically produced by UGCG (GCS) is mostly translocated into the luminal compartment of the Golgi apparatus serving as precursor for the synthesis of LacCer and the complex type of GSLs. A part of cytosolic GlcCer is hydrolyzed by GBA2 and generates GlcChol, or GlcCer is taken by glycolipid transporter protein (GLTP) and possibly transported to other membrane compartment. Modification of GlcCer by galactosyltransferase (B4galt5 and 6) is capping reaction to prevent GlcCer back to cytosolic side.Characterization of unusual glycolipids in rodent brainWe have developed HILIC-LC-MS system to separate isobaric glycolipids and found that vertebrate brain tissues contained GalChol in addition to GlcChol (Figure 1A). To confirm the chemical structure, we isolated GalChol to pure form from adult rat brain and determined its structure by NMR and RPLC–ESI–MS/MS. To identify the responsible enzyme to form GalChol, we established in vitro assay system of glycosylation using NBD-Chol as substrate. We found GalChol formation is carried out by mainly GBA2 6). Galactosylceramide synthase, UGT8 has no activity to synthesize GalChol. Since UGT8 generates GalCer in the luminal ER, GalCer is flip-flopped to the cytosolic site, which is then cleaved by GBA2 forming GalChol and ceramide (Figure 1B, 2B). membrane. It remains elusive how GlcCer and GalCer are flip-flopped in the lipid bilayer.detectable in primary cultured neurons, which may be derived culture medium (serum). GSX2, unidentified glucosylated sterol.81
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