Fig. 6.

RNAi of VCP affects the glycosylation pattern of multiple glycoproteins, including αTCR. (A) RNAi of VCP using two different siRNAs (vcp2 and vcp6) induces a 20-30% increase of the levels of glycosylated αTCR (arrowhead, A); when the samples are run on a modified 15% SDS-PAGE (pH 9.4) the corresponding band migrates as a doublet and a shift of the ratio between the two adjacent forms of glycosylated αTCR (arrows, A) is apparent as quantified by an increase in the ratio of the lower to the upper band (B). (C) Treatment with 1-deoxymannojirymycin induces the disappearance of the lower band suggesting that it reflects a form of αTCR resulting from the cleavage of a mannose residue. (D) This is confirmed by digestion of the immunoprecipitated αTCR with jack bean mannosidase, which collapses the upper band into a faster migrating form (arrowhead) while the lower band of the doublet is resistant to endoglycosidase H treatment.(E) MALDI of N-linked glycans isolated from cellular glycoproteins reveals that RNAi of VCP with either vcp2 or vcp6 induces a decrease in polymannosylated oligosaccharides and an increase in oligomannosylated oligosaccharides as well as changes in the levels of complex glycans. Symbols used to denote different sugar moieties: open circle – mannose, yellow square – N-acetylglucosamine: red circle – fucose; red triangle – sialic acid; open square – galactose.