Computation of the ratio of fOSGN-P to DLO for different oligosaccharide structures observed in the different cell lines cultivated in the absence or presence of glucosidase and mannosidase inhibitors.
A. Oligosaccharides derived from DLO and fOSG2-P isolated from EBV Ctrl1, EBV Ctrl2, EBV CDG Ia, EBV CDG Ie, EBV CDG Ig, EBV CDG Ih, BW5417.3 and Thy-1 cells were prepared and resolved by HPLC as described in Fig. 4. Peak areas were recorded and used to derive the ratio fOSGN2-P/DLO for each oligosaccharide structure. These values were multiplied by 1000 and imposed on a logarithmic scale. Abbreviations: M1-9; Man1-9GlcNAc2, G1-3M1-9; Glc1-3Man1-9GlcNAc2, G1-3M1-7; Glc1-3Man1-7GlcNAc2, G1-3M1-5; Glc1-3Man1-5GlcNAc2. In a separate experiment EBV Ctrl2 (B) and EBV CDG Ig (C) cells were preincubated and then radiolabeled as described above, in the presence of the mannosidase inhibitors swainsonine (SW) and kifunensin (KIF) or the glucosidase inhibitor castanospermine (CST). Oligosaccharides derived from DLO and fOSGN2-P were resolved by TLC and, after elution of radioactive components from the chromatography plates followed by scintillation counting, the ratio fOSGN2-P/DLO for the oligosaccharide structures M7–G3M9 were generated and presented as described above. D. Using data from the experiment described in B and C the percentage of total DLO species occurring as triglucosylated species was computed for EBV Ctrl2 and EBV CDG Ig cells radiolabeled in either the absence or presence of CST. E. Using data from the experiment described in B and C, DLO-, and fOSGN2-P-derived oligosaccharides possessing 7 residues of mannose were quantitated. The amounts of these components that were generated in cells treated with CST have been expressed as a percentage of those generated in cells radiolabeled in the absence of this reagent.
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