Glucosylceramide Analysis Service

What Is Glucosylceramides

Glycosphingolipids (GSLs) are ubiquitous components of eukaryotic cell membranes. GSLs are synthesized by sequential addition of saccharides to the hydroxyl group at the C-1 position of the ceramide (Cer) backbone. GSLs are mainly located in the extracellular leaflet of the plasma membrane, where they are involved in several functions such as cell-to-cell interaction and recognition. Glucosylceramide (GlcCer), one of the simplest GSLs, is widely distributed in mammalian tissues. GlcCer is formed at the cytosolic leaflet of Golgi apparatus by glycosylation of Cer by GlcCer synthase (GCS). After its synthesis, GlcCer is transported to the luminal side of the Golgi apparatus where is converted into more complex GSLs, or transported by FAPP2 (4-phosphate adaptor protein-2) to the cytoplasmic leaflet of the plasma membrane (PM) or of the endoplasmic reticulum (ER). In the PM, GlcCer may remain on the cytoplasmic leaflet or translocate to the cell surface.

Figure 1. The synthetic pathways of major glycosphingolipids (GSLs). Note that ceramide is metabolized to glucosylceramide (GlcCer), galactosylceramide (GalCer) or sphingomyelin (SM).

Fuction Of Glucosylceramides

GlcCer is an important intracellular messenger that plays key roles in cell maintenance and regulation. However, the molecular mechanisms by which GlcCer regulates cellular processes are unknown. GSLs are thought to be involved in the formation of lipid rafts, which, because of their specific biophysical properties, can act as signaling platforms. In the last decade, much effort has been made to understand the biophysical properties of lipid rafts; however, little attention has been given to GlcCer. GlcCer has a high main transition temperature (T_m) and a complex thermotropic phase behavior, with multiple transitions between different stable and metastable phases. Due to their high T_m and extensive hydrogen-bond network, GSLs with small uncharged headgroups are expected to segregate from low T_m phospholipids and form tightly packed domains, with a higher packing density than sphingomyelin with a similar backbone. These observations suggest that GlcCer might promote similar changes to those induced by Cer in the biophysical properties of fluid membranes. Previous studies from our group have showed that Cer has a complex phase behavior promoting extensive alterations in the biophysical properties of membranes that are dependent both on Cer content and membrane lipid composition, particularly on cholesterol (Chol) content.

GlcCer is synthesized on the cytoplasmic leaflet of the Golgi apparatus, and can translocate across the Golgi membrane to the lumen, where it is a substrate for glycosyltransferases involved in the biosynthesis of higher glycolipids. Transport vesicles can be formed at the Golgi and reach the plasma membrane. GlcCer can be present in the lumenal leaflet of the transport vesicles, and after fusion with the plasma membrane, ends up at the cell surface. GlcCer can also reach the cytosolic leaflet of the plasma membrane, either by monomeric transportation, or via the cytosolic leaflet of the transport vesicles. GlcCer could then be translocated at the cell surface by the action of MDR1 PgP. There are bi-directional vesicular pathways between the plasma membrane, the Golgi apparatus and the endosomes. GlcCer is finally degraded either in lysosomes by the lysosomal glucocerebrosidase, or by the non-lysosomal glucocerebrosidase.

Figure 2. Synthesis, transportation and degradation of GlcCer.

GlcCer is required for the vesicular pathway from the Golgi complex to the melanosome. As this pathway involves the budding of carrier vesicles from the trans-Golgi network or from an organelle intermediate between the trans-Golgi network and the melanosome, the simplest mechanism would be that GlcCer is required for the formation of a functional protein coat on the cytosolic surface of these membranes. After budding and transportation to the melanosome the active lipid GlcCer might be removed from the cytosolic surface by a translocator, which would make the process unidirectional. A defect in this machinery may lie at the basis of some forms of the Hermansky–Pudlak syndrome, a group of related recessively inherited human pigmentation disorders, as has already been demonstrated for the AP-3 complex. Finally, melanosomes are part of the endosomal membrane system. It will be interesting to see the consequences of the absence of glycosphingolipids on the structure and function of endosomes in regular cells that do not make pigment.

To provide bioanalytical support to studies of neuronopathic Gaucher disease, a liquid chromatography-mass spectrometry (LC-MS/MS) method was developed to separate GlcCer from the predominant isomer found in brain tissue (galactosylceramide, GalCer).

Summary

• Identification and quantification of glucosylceramide.

Report

• A detailed technical report will be provided at the end of the whole project, including the experiment procedure, instrument parameters.
• Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
• The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective glucosylceramide targeted metabolomics services.

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