Profiling of O-acetylated Gangliosides Expressed in Neuroectoderm Derived Cells

Abstract

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

O-acetylated Gangliosides as Targets for Cancer Immunotherapy

“…MS/MS fragmentation studies revealed that O-acetylation can occur in a cell type dependent manner on both inner and terminal sialic acid residue of b-and c-series of gangliosides. These data highlight the probable existence of different O-acetylation pathways for gangliosides [51].…”

“…This method avoids the loss of the O-acetyl group and has led to the characterization of OAcGM1, OAcGD3, OAcGD2, OAcGT3 and OAcGT2 in BC, melanoma and neuroblastoma cell lines in a cell type dependent manner, suggesting that O-acetylated ganglioside expression depends mainly on substrate availability and GD3 synthase expression. MS/MS fragmentation of these O-acetylated gangliosides species showed that O-acetylation can occur both on the terminal and on the subterminal sialic acid of the ganglioside carbohydrate moiety [51]. To get further insight into the position of the O-acetyl group on the sialic acid residue, DMB-labeled sialic acid derivatives extracted from gangliosides from BC cells were analyzed by LC-ES-MS. 9-O-acetyl-N-acetyl-neuraminic acid was identified as the main O-acetylated sialic acid species expressed on gangliosides from BC cells [4,51].…”

“…MS/MS fragmentation of these O-acetylated gangliosides species showed that O-acetylation can occur both on the terminal and on the subterminal sialic acid of the ganglioside carbohydrate moiety [51]. To get further insight into the position of the O-acetyl group on the sialic acid residue, DMB-labeled sialic acid derivatives extracted from gangliosides from BC cells were analyzed by LC-ES-MS. 9-O-acetyl-N-acetyl-neuraminic acid was identified as the main O-acetylated sialic acid species expressed on gangliosides from BC cells [4,51].…”

“…O-acetylation is one of the major common modifications of sialic acid bearing gangliosides. A single report has characterized the expression of OAcGM1, OAcGD2, OAcGD3, OAcGT2 and OAcGT3 in neuroectoderm-derived cells [51]. Whereas OAcGD2 and OAcGD3 expression in cancers is very well documented, little is known concerning OAcGM1, OAcGT2 and OAcGT3.…”

“…OAcGT3 expression has been reported as a mixture of OAcGD3/OAcGT3 in the bovine brain, cultured glial cells and BC cells [67,76]. Besides this, OAcGD2 expression has been established in many neuroectoderm-derived tumor types such as neuroblastoma (NB), glioblastoma, SCLC and BC [51,71,72]. The pattern of O-acetylated ganglioside expression is highly tumor type dependent, suggesting a highly regulated biosynthesis mechanism and potential roles in tumor biology.…”