Extended Data Fig. 6: Analysis of GSLs from B3GNT5-KO + OE cells.
From: Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication
(A) Glycolipids were isolated from B3GALT5-KO m1 or B3GNT5-KO + OE cells. Glycans were released with endoglycoceramidase, labeled with procainamide, and analyzed by mass spectrometry with HILIC-FL separation. (B, C) LC-ESI/MS analysis of oligosaccharides obtained by digestion of neutral GSLs from B3GNT5-KO + OE cells with endoglycoceramidase I. Diagnostic ions indicate carbohydrate linkage positions. (C) An MS spectrum displaying a series of C type fragment ions (C2α at m/z 528, C3α at m/z 690, C4α at m/z 1039, C5α at m/z 1201, C6 at m/z 1550, and C7 at m/z 1712), which identified an oligosaccharide with Hex-(Fuc-)HexNAc-Hex-(Fuc-)HexNAc-Hex-(Fuc-)HexNAc-Hex-Hex sequence. The ion at m/z 364 is obtained by double glycosidic cleavage of the 3-linked branch (C2/Z3β), and characteristic for an internal 4-linked GlcNAc substituted with a Fuc at 3-position that is a terminal Lex (refs. 90,94). Taken together this indicated an undecasaccharide with a terminal Lex determinant. (D, E) B3GALT5 + B3GNT5-dKO cells were incubated with GSLs extracted from B3GNT5-KO + OE cells, a commercial mixture of neutral GSLs, or purified GM1. Cells were then treated with BFA or a vehicle control for 0.5 h prior to incubation with CT (1 nM) for analysis of cAMP accumulation (D). Alternately, cells were treated with forskolin (10 µM) for 0.5 h (n = 2) (E). Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate (n = 3 for panel D, n = 2 for panel E) consisting of 2 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates (panel D). Statistical analyses were performed by two-way ANOVA with Tukey correction. No significant differences were observed.
