STT3B-independent glycosylation of closely spaced sites in hemopexin. (A) Diagram of hemopexin (Hpx) showing the signal sequence (black), glycosylation sites, disulfide bonds (red lines) and DDK-His tag. The five sequons are numbered 1–5; Hpx mutants lacking one or more sequons are designated as Hpx-ΔXYZ, where XYZ is the list of mutated sequon(s). HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B as indicated (B–E) for 48 hours prior to transfection with Hpx expression vectors. Cells were pulse labeled for 4 minutes and chased for 20 minutes. EH designates digestion with endoglycosidase H. Hpx glycoforms (0–5 glycans) were immunoprecipitated with anti-DDK sera and resolved by SDS-PAGE. Quantified values below gel lanes are for the displayed image that is representative of two or more experiments.

Sequon skipping of adjacent NxS sites. (A) Diagram of ZAG showing the signal sequence (black), glycosylation sites, disulfide bonds (red lines) and Myc-DDK tag. The four sequons are numbered 1–4; ZAG mutants lacking one or more sequons are designated as ZAG-ΔXYZ, where XYZ is the list of mutated sequon(s). (B) HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B for 48 hours prior to transfection with wild-type ZAG. Cells expressing wild-type ZAG (B), or various ZAG glycosylation site mutants (C) were pulse labeled for 4 minutes and chased for 20 minutes. ZAG glycoforms (0–3 glycans) were immunoprecipitated using anti-DDK antibody and resolved by SDS-PAGE. Arrowheads designate a transient form of ZAG (see supplementary material Fig. S1). EH designates digestion with endoglycosidase H. Quantified values below gel lanes are for the displayed image that is representative of two or more experiments.

Glycosylation of haptoglobin. (A) Diagram of the haptoglobin precursor (Hp) showing the signal sequence (black), protease processing site at the α-β junction (arrow), glycosylation sites, disulfide bonds (red lines), cysteine residues that form interchain disulfides to link two α-subunits (elongated diamonds) and a DDK-His tag. The four sequons are numbered 1–4; Hp mutants lacking one or more sequons are designated as HpΔXYZ, where XYZ is the list of mutated sequon(s). (B,D) HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B as indicated for 48 hours prior to transfection with Hp expression constructs. Cells were then pulsed for 4 minutes and chased for 20 minutes. (C) Cells expressing Hp-wt were pulse labeled for 4 minutes and chased as indicated. Hp glycoforms were precipitated with anti-DDK sera and resolved by SDS-PAGE. EH designates digestion with endoglycosidase H. Quantified values below gel lanes are for the displayed image that is representative of two or more experiments.

Sequence and gap-length dependence of sequon skipping. HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B as indicated for 48 hours prior to transfection with Hp expression constructs. The HpΔ14 derivatives had closely spaced NxT sites (A) or NxS sites (B) separated by 0–2 alanine residues. HeLa cells expressing Hp glycosylation site mutants were pulsed for 4 minutes and chased for 20 minutes. EH designates digestion with endoglycosidase H. Quantified values below gel lanes are for the displayed image that is representative of two or more experiments.

Glycosylation of consecutive gap-1 sequons. (A) The sequences of HpΔ14 derivatives that have two to five closely spaced sites. (B–D) HeLa cells expressing Hp glycosylation site mutants were pulsed for 4 minutes and chased for 20 minutes. (C) Limited digestion of anti-DDK immunoprecipitates with endoglycosidase H (EH, 0–15 minutes). (D) HeLa cells were treated with negative control (NC) siRNA or siRNAs specific for STT3A or STT3B as indicated for 48 hours prior to transfection with Hp expression constructs. Quantified values below gel lanes are for the displayed image that is representative of two or more experiments.