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, 20 (4), 6913-24

Accumulation of GD1α Ganglioside in MDA-MB-231 Breast Cancer Cells Expressing ST6GalNAc V

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Accumulation of GD1α Ganglioside in MDA-MB-231 Breast Cancer Cells Expressing ST6GalNAc V

Sandy Vandermeersch et al. Molecules.

Abstract

α-Series gangliosides define a particular sub-class of glycosphingolipids containing sialic acid α2,6-linked to GalNAc residue that was isolated as a minor compound from the brain. The sialyltransferase ST6GalNAc V was cloned from mouse brain and showed α2,6-sialyltransferase activity almost exclusively for GM1b, to form GD1α and is considered as the main enzyme involved in the biosynthesis of α-series gangliosides. Recently, ST6GALNAC5 was identified as one of the genes over-expressed in breast cancer cell populations selected for their ability to produce brain metastasis. However, the capacity of human breast cancer cells to produce α-series gangliosides has never been clearly demonstrated. Here, we show by stable transfection and MS-MS analysis of total glycosphingolipids that ST6GALNAC5 expressing MDA-MB-231 breast cancer cells accumulate GD1α ganglioside (IV3Neu5Ac1, III6Neu5Ac1Gg4-Cer).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
QPCR analysis of ST6GalNAc V expression in control and transfected MDA-MB-231 cells. Quantification of ST6GalNAc V expression was performed by the method described by Pfaffl [21] and normalized to HPRT. The expression of ST6GalNAc V was relative to wild-type (wt), which was regarded as 1. Ctrl, control cells transfected with the empty vector; GFP+, GFP-positive cell population.
Figure 2
Figure 2
Cell sorting for GFP expression of the G418-resistant cell population. The G418-resistant cell population was sorted for GFP expression on an ARIA SORP flow cytometer.
Figure 3
Figure 3
Flow cytometry analysis of α-2,6-sialylation in ST6GalNAc V transfected MDA-MB-231 cells. Detection of α-2,6-sialylation was performed using biotin-labeled SNA and revealed with Dylight-405-conjugated streptavidin. WT, wild-type.
Figure 4
Figure 4
Comparison of MS profiles of permethylated glycosphingolipids purified from MDA-MB-231 wt, clone #13 and GFP+ ST6GalNAc V transfected cells. GSL are present as d18:1/C16:0 (Cer*) and d18:1/C24:0 (Cer**) isomers. ○, Gal; ●, Glc; □, GalNAc; ♦, Neu5Ac.
Figure 5
Figure 5
MS/MS sequencing of permethylated GD1 at m/z 2295 with ceramide moieties d18:1/C24:0 (Cer**). All fragments are observed as [M+Na]+ adducts. Fragment ions were annotated according to nomenclature of Domon and Costello [26]. The nature of monosaccharides was deduced from known biosynthesis of gangliosides. ○, Gal; ●, Glc; □, GalNAc; ♦, Neu5Ac.

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