TABLE 2.
Glycosaminoglycan synthesis and disaccharide composition of labeled HS isolated after mock or siRNA mediated knockdown of EXTL2 in HEK 293 cells
Labeled glycosaminoglycans were extracted from the cell surface and quantified. The percentage values of HS and CS are given from a single experiment (values within parentheses refer to a [3H[GlcN-labeled sample) or as means from three independent 35S labeling experiments ± mean deviation.35S-labeled samples were degraded by deamination at pH 1.5, followed by reduction of products with NaBH4. The resulting labeled disaccharides were analyzed by anion-exchange HPLC (see “Experimental Procedures”). The values for mock-treated and cells treated with siRNA against EXTL2 are given as means from two independent labelling experiments ± mean deviation. Mock, mock transfected cells; siNC, cells transfected with non-target control siRNA; siL2, cells transfected with siRNA against EXTL2.
| Cell type | Labeled glycosaminoglycans |
35S-labeled deamination productsa |
||||
|---|---|---|---|---|---|---|
| HSb | CSc | GlcA-aManR6Sd | IdoA-aManR6S | IdoA2S-aManR | IdoA2S-aManR6S | |
| % | % | |||||
| Mock | 51 ± 10 (42) | 49 ± 10 (52) | 18 ± 1.4 | 7 ± 1.4 | 56.5 ± 5.5 | 19 ± 4 |
| siNC | NDe | ND | 14 | 6 | 55 | 24 |
| siL2 | 57 ± 9 (57) | 44 ± 9 (40) | 17 ± 1 | 6.5 ± 0.5 | 58.5 ± 2.5 | 19 ± 4 |
a % of total O-sulfated disaccharides.
b Material resistant to chondroitinase ABC.
c Material susceptible to chondroitinase ABC.
d aManR, the 2,5-anhydromannitol deamination product of GlcNS residues.
e ND, not determined.