American Society of Hematology

Figure 5.

$Figure 5. The S158C mutation in C2GnT-I reduces enzyme activity. BW5147 murine T cells lack C2GnT-I. BW5147 cells were transfected with wild-type C2GnT-I (WT), mutant C2GnT-I with the S158C mutation (mut), or vector alone. (A) Relative expression of WT or mut C2GnT-I mRNA in transfected cells was analyzed by real-time RT-PCR. Clones with similar C2GnT-I mRNA expression, calculated as C2GnT-actin ratios, were selected for further analysis; these were designated WT clone 6 and mut clone 2. (B) Total membrane extracts of selected clones were analyzed for C2GnT activity. Cells expressing mutant C2GnT-I or vector alone had comparable activity, whereas cells expressing WT C2GnT-I had increased enzyme activity (*P < .001 for WT compared with vector or mut). Values are mean ± SD for duplicates and are from 1 of 6 independent experiments. (C) The S158C mutation does not affect enzyme dimerization. For analysis of C2GnT, total membrane extracts of the clones in panel A were separated by 10% SDS-PAGE under nonreducing conditions, blotted to nitrocellulose, and probed with rabbit polyclonal antiserum to C2GnT. C2GnT dimers (∼ 115 kDa) were observed in cells expressing WT or mutant C2GnT-I. A minor fraction of monomeric enzyme (∼ 64 kDa) was detected in both samples. (D) Glycosylation of the primary C2GnT acceptor substrates, CD43 and CD45. (Top) CD45 was precipitated from BW5147 cells transfected with vector alone, WT C2GnT-I, or mutant C2GnT-I, or PhaR2.1 cells that express endogenous C2GnT. BW5147 cells expressing WT C2GnT-I demonstrated increased CD45 heterogeneity comparable to that seen for CD45 from PhaR2.1 cells, indicating that WT C2GnT-I expression increased CD45 glycosylation. BW5147 cells expressing mutant C2GnT-I demonstrated minimal CD45 heterogeneity, comparable to that seen from cells transfected with vector alone. (Bottom) CD43 was precipitated with the 1B11 mAb that recognizes murine CD43 modified with core 2 O-glycans or the S7 mAb that recognizes murine CD43 modified with core 1 O-glycans, and immunoblotted with the same mAb used for precipitation. 1B11-reactive CD43 was detected in cells transfected with C2GnT-I, whereas S7-reactive CD43 was detected in control cells and was the primary band detected in cells transfected with mutant C2GnT-I.$

The S158C mutation in C2GnT-I reduces enzyme activity. BW5147 murine T cells lack C2GnT-I. BW5147 cells were transfected with wild-type C2GnT-I (WT), mutant C2GnT-I with the S158C mutation (mut), or vector alone. (A) Relative expression of WT or mut C2GnT-I mRNA in transfected cells was analyzed by real-time RT-PCR. Clones with similar C2GnT-I mRNA expression, calculated as C2GnT-actin ratios, were selected for further analysis; these were designated WT clone 6 and mut clone 2. (B) Total membrane extracts of selected clones were analyzed for C2GnT activity. Cells expressing mutant C2GnT-I or vector alone had comparable activity, whereas cells expressing WT C2GnT-I had increased enzyme activity (*P < .001 for WT compared with vector or mut). Values are mean ± SD for duplicates and are from 1 of 6 independent experiments. (C) The S158C mutation does not affect enzyme dimerization. For analysis of C2GnT, total membrane extracts of the clones in panel A were separated by 10% SDS-PAGE under nonreducing conditions, blotted to nitrocellulose, and probed with rabbit polyclonal antiserum to C2GnT. C2GnT dimers (∼ 115 kDa) were observed in cells expressing WT or mutant C2GnT-I. A minor fraction of monomeric enzyme (∼ 64 kDa) was detected in both samples. (D) Glycosylation of the primary C2GnT acceptor substrates, CD43 and CD45. (Top) CD45 was precipitated from BW5147 cells transfected with vector alone, WT C2GnT-I, or mutant C2GnT-I, or PhaR2.1 cells that express endogenous C2GnT. BW5147 cells expressing WT C2GnT-I demonstrated increased CD45 heterogeneity comparable to that seen for CD45 from PhaR2.1 cells, indicating that WT C2GnT-I expression increased CD45 glycosylation. BW5147 cells expressing mutant C2GnT-I demonstrated minimal CD45 heterogeneity, comparable to that seen from cells transfected with vector alone. (Bottom) CD43 was precipitated with the 1B11 mAb that recognizes murine CD43 modified with core 2 O-glycans or the S7 mAb that recognizes murine CD43 modified with core 1 O-glycans, and immunoblotted with the same mAb used for precipitation. 1B11-reactive CD43 was detected in cells transfected with C2GnT-I, whereas S7-reactive CD43 was detected in control cells and was the primary band detected in cells transfected with mutant C2GnT-I.

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