Newsletter Matreya July 2021

Fluorescent BODIPY-Labeled
Ganglioside GM

Understanding ganglioside metabolism and biological function has been made possible in part by using labeled derivatives that can be incorporated into cultured cells and artificial membranes. This includes studies of their distribution in membranes and interactions with neighboring cells as well as investigation of their metabolic fate and intracellular trafficking. As a class of glycosphingolipids that bear sialic acid residues, gangliosides are expressed in all types of cells but are especially abundant in neurons where they were first discovered in ganglion cells. They are anchored to the plasma membrane by fatty acyl chains of the ceramide base, and variations of sialooligosaccharide branches extend from there into the extracellular space, enabling interactions with nearby cells. They also interact within their resident cell’s membrane to regulate responses to signaling proteins.

GM1 is the prototypic ganglioside for all other members of this lipid class. It has roles in neuronal plasticity and repair mechanisms, as well as in the release of neurotrophins in the brain.1 GM1 stimulates neuronal sprouting
and enhances the action of nerve growth factor (NGF) by directly and JULY 2021 tightly associating with Trk, the high-affinity tyrosine kinase-type receptor for NGF. GM1 also acts as the site of binding for both cholera toxin and E. coli
heat-labile enterotoxin.2 Investigation of these functions requires the detection of GM1 in its natural environment within membranes.

Ganglioside derivatives can contain radioactive, paramagnetic, photoreactive, or fluorescent tags for this purpose.3 The type of probe that is chosen is determined by what function it needs to perform in an experiment and which pathway
will be investigated. Fluorescent probes are typically used because they require low concentrations for detection and are well-suited for real-time monitoring at high resolution. The fluorescent tag can be attached to either the
sialooligosaccharide (polar group) or ceramide (nonpolar group) portion and designed with features that resemble those of natural gangliosides so not to alter the integrity of the lipid. Matreya chemists have synthesized N-propanoyl-BODIPY-monosialoganglioside GM1 (C3:0-BODIPY-GM1) wherein a boron-dipyrromethene (BODIPY) tricyclic ring system replaces an acyl group at the amine of the ceramide creating aprobe with excitation/emission maxima of 503/512 nm. GM1 labeled with BODIPY in the ceramide is a valuable tool that can be used to probe the spatial distribution and dynamics in lipid membrane systems and to study their metabolism, trafficking in cells, and specific interactions with proteins at the cell surface.4

This BODIPY derivative offers a high extinction coefficient (>80,000 cm-1M-1), high quantum yield (>0.8), a long excited-state lifetime (4+ ns), and minimal sensitivity to changes in pH.5 However, due to narrow spectral bandwidths and small Stokes shifts, BODIPY probes are usually excited at sub-optimal wavelengths to prevent interferences such as light scattering or cross-over from the wide bandwidth of the excitation source. If used at
high concentrations, these probes are also highly sensitive to dye-dye quenching effects during energy transfer from an excited-state fluorophore to a ground-state fluorophore, making them useful for assays that measure fluorescence resonance energy transfer, fluorescence polarization, or fluorescence intensity. The low polarity of this BODIPY probe also makes it an excellent analog of
natural gangliosides. View article references on page 3 of newsletter.

Staining SH-SY5Y Cells with C3:0-BODIPY-GM1

  • Dissolve C3:0-BODIPY-GM1 (100 μg) in 64 μl DMSO:methanol:water (2:1:0.2) and aliquot. Store aliquots at -20°C until use.
  • Plate cells at density of 10,000/well in a 96-well plate in media with 10% FBS and allow to attach overnight.
  • The following day, remove media and replace with 2.5-10 μM C3:0-BODIPY-GM1 in phenol red-free media. Return cells to incubator for 30 minutes.
  • Remove media and replace with fresh phenol red-free media. Image using a fluorescence microscope with a FITC/GFP laser.

Note that complexing fluorescent lipids with bovine serum albumin (BSA) facilitates cell labeling by eliminating the need for organic solvents to dissolve the lipophilic probe.
A BSA-complexed probe can be directly dissolved in water. However, gangliosides are unique lipids that are soluble in aqueous systems.

Fluorescent Sphingolipids

Matreya has developed fluorescent standards that can be detected in cultures and in biological systems, making them ideal for studies involving the metabolism of sphingolipids. These lipid analogs are useful for determining the localization of various sphingolipids in membranes and organelles. The NBD fluorescent group attached to hexanoic acid has been shown to be readily taken up by cells and used in the biosynthesis of more complex sphingolipids.

Ganglioside Precursors

Lactosylceramides, GM3, GD3, and GT3 can serve as precursors to more complex gangliosides containing 1-3 sialic acid residue(s) linked to the inner galactose residue and/or the inner N-acetylgalactosamine residue. The elaboration of these simple gangliosides is catalyzed by specific glycosyltransferases through various metabolic pathways to create complex gangliosides such as GM1, GD1a, GD1b, GT1b, and GP1c. Matreya’s ganglioside precursors can be used to study these biosynthetic pathways.

Article References
1. Kolter, T. Ganglioside biochemistry. ISRN Biochem. 506160 (2012).
2. Cho, J.A., Chinnapen, D.J.-F., Aamar, E., et al. Insights on the trafficking and retro-translocation of glycosphingolipid-binding bacterial toxins. Front. Cell. Infect. Microbiol. 2, 51 (2012).
3. Schwarzmann, G. Labeled gangliosides: Their synthesis and use in biological studies. FEBS Lett. 592(23), 3992-4006 (2018).
4. Mikhalyov, I., Gretskaya, N., and Johansson, L.B.-A. Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target
interactions. Chem. Phys. Lipids 159(1), 38-44 (2009).
5. Rasmussen, J.-A.M. and Hermetter, A. Chemical synthesis of fluorescent glycero- and sphingolipids. Prog. Lipid Res. 47(6), 436-460 (2008).


Q:What solvents are best to use for sphingolipids?

A: Some lipids, such as sphingolipids, have limited or very poor solubility in many common solvents. To overcome this insolubility problem, various solvent mixtures have been developed. One of the most universal sphingolipid solvents is a mixture of chloroform/methanol/water. However, while this solvent system is very useful for analytical testing, it is very toxic towards cells and cannot be used in live cell cultures or other in vivo applications. Several alternative methods have been developed to provide sphingolipid solutions suitable for live-cell studies. Although these methods were primarily developed for ceramides and glucosylceramides, they may be adapted for use with other lipids. Applicable solubility systems include:

Biological Applications
Method A:
BSA-Lipid Complexes Prepare ~1 mM lipid stock solution in chloroform/methanol (19:1, v/v). Dispense 50 μl of the lipid stock solution into a glass test tube and dry, first under a stream of nitrogen and then in vacuo for at least 1 hour. Redissolve the dried lipid in 200 μl ethanol. Mix 10 ml of buffer (100 mM NaH2PO4/Na2HPO4, pH 7.4) with 3.4 mg (0.34 mg/ml) of fatty acid-free BSA into a 50 ml plastic centrifuge tube.1,2 Agitate on a vortex mixer. Inject the 200 μl lipidsolution into the BSA solution while vortexing. Store the resulting solution (5 μM lipid + 5 μM BSA) in a plastic
tube at -20°C.

Method B:
Solubilization Using Zwitterionic Detergent CHAPS Evaporate a solution containing 15 nmol of lipid under a stream of nitrogen in a 1.5 ml test tube. Add a solution of 1.1 mg of CHAPS (3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate) in 10 μl of phosphate buffer (100 mM NaH2PO4/Na2HPO4, pH 7.4). Thoroughly mix and sonicate for 3 minutes.2

Method C:
Solubilization Using Ethanol/Dodecane (98:2, v/v) for Use with Tissue Homogenates Evaporate a solution containing 15 nmol of lipid under a stream of nitrogen in a 1.5 ml test tube. After dissolving in ethanol/dodecane (98:2, v/v; 1% final concentration), addphosphate buffer (100 mM NaH2PO4/Na2HPO4, pH 7.4) and sonicate the solution for 3 minutes.2

Method D:

Solubilization Using Ethanol/Dodecane (98:2, v/v) for Use in Cell Cultures Dissolve ceramide (from bovine brain sphingomyelin or cerebroside), C2-ceramide, DAG, or sphingosine in ethanol/dodecane (98:2, v/v). Add this solution to DMEM and Ham’s F12 medium in a tube and mix well with themedium using agitation.3 Transfer to culture dishes.

Mass Spectrometry/Organic Chemistry Applications
Method E:

Dissolve the lipid in hexane/2-propanol (3:2, v/v). Can add 5.5% water. Can also add 0.01% BHT to limit oxidation.4 This method is best reserved for gas chromatography applications.

Universal Sphingolipid Solvent (this system will work for most sphingolipids)
Dissolve in chloroform/methanol (2:1, v/v) at a concentration of 10 mg lipid/ml solvent. If the lipid is still not soluble, add water up to 10% of the solvent volume. Sonication or mild heating (up to 40°C) can help to dissolve the lipid faster. A revised version of this solvent using 1:1 butanol/methanol may also be used.

Please visit individual product pages to see specific solvents that we have tested with that particular product.
1. Pagano, R.E. A fluorescent derivative of ceramide: Physical properties and use in studying the Golgi apparatus of animal cells. Methods Cell Biol. 29, 75-85 (1989).
2. Michel, C., van Echten-Deckert, G., Rother, J., et al. Characterization of ceramide synthesis. A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine
at the level of dihydroceramide. J. Biol. Chem. 272(36), 22432-22437 (1997).
3. Ji, L., Zhang, G., Uematsu, S., et al. Induction of apoptotic DNA fragmentation and cell death by natural ceramide. FEBS Lett. 358(2), 211-214 (1995).
4. Norm Radin (unpublished observations)