Journal: The Journal of Cell Biology

Article Title: Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins

doi: 10.1083/jcb.201404083

Figure Lengend Snippet: Disulfide bonds in MagT1-dependent substrates. (A) HeLa cells treated with NC or MagT1 siRNA were treated with 3 mM DTT for 5 min before a 5-min pulse, 10-min chase labeling period. Endogenous cathepsin C was immunoprecipitated using anti-CatC sera and resolved by SDS-PAGE. Diagrams of pCatCΔ234-HA (B) and FVII N183Q (D) showing the signal sequence (black), glycosylation sites, disulfide bonds (red lines), free cysteine residues (diamonds), mature protein domains (green, cyan, magenta, and yellow segments), and the C-terminal HA tag on pCatCΔ234-HA. Disulfides that link (pCatCΔ234) or bracket (FVII N183Q) a STT3B-dependent glycosylation site are indicated. (C and E) HeLa cells were treated with NC, or siRNAs specific for STT3A, STT3B, or MagT1 for 48 h as indicated, then transfected with pCatCΔ234-HA (C) or FVII N183Q (E and F) expression vectors and cultured for an additional 24 h before pulse labeling. Cells were pulse labeled for 4 min (C), pulse labeled for 2 min, and chased for 30 min (E), or pulsed for 2 min and chased as indicated (F). Glycoprotein substrates were precipitated with anti-HA sera (C) or anti-factor VII sera (E and F). Quantified values below gel lanes (A, C, and E) are for the displayed image that is representative of two or more experiments. Data points in F are the mean of two determinations, with individual data points indicated by error bars.

Article Snippet: Goat anti–human cathepsin C (AF1071), goat anti–human factor VII (AF2338), goat anti–human progranulin (AF2420), and goat anti–human SHBG (AF2656) were purchased from R&D Systems.

Techniques: Labeling, Immunoprecipitation, SDS Page, Sequencing, Glycoproteomics, Transfection, Expressing, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins

doi: 10.1083/jcb.201404083

Figure Lengend Snippet: Formation of mixed disulfides between MagT1 and glycoprotein substrates. (A) Diagram of the pCatC-Insert-Δ234 construct. (B–D) HeLa cells were treated with the NC or MagT1 siRNA for 48 h before cotransfection with wild-type or mutant versions of the pCatCΔ234-HA (B and C), FVII N183Q (D), and MagT1-V5 expression vectors (B–D). The m1, m2, and m3 mutants of MagT1 are defined in . Cells were pulse labeled for 4 min (A–D) and chased for 10 min (B and C) or 40 min (D). Glycoproteins were immunoprecipitated with anti-HA sera (B and C) or anti-FVII sera (D) and quantified after SDS-PAGE. (E) Cells expressing wild-type or mutant versions of MagT1-V5 were treated with NEM to prevent disulfide exchange during cell lysis and sample preparation. Total cell extracts were resolved by nonreducing (−DTT) or reducing (+DTT) SDS-PAGE as indicated, and analyzed by protein immunoblotting using anti-V5 sera. (F) In vivo redox status of MagT1 and PDI in HeLa cells was assayed using a maleimide-shift protocol. The arrows designate oxidized and reduced forms of MagT1 and PDI. A minor MagT1 reactive band in the DPS-oxidized lane (asterisk) is probably due to inefficient formation of a disulfide between cysteine residues located on the cytoplasmic face of TM3 and TM4 (see for a map of MagT1 cysteine residues). Quantified values below gel lanes (B–D) are for the displayed image, which is representative of two experiments.

Article Snippet: Goat anti–human cathepsin C (AF1071), goat anti–human factor VII (AF2338), goat anti–human progranulin (AF2420), and goat anti–human SHBG (AF2656) were purchased from R&D Systems.

Techniques: Construct, Cotransfection, Mutagenesis, Expressing, Labeling, Immunoprecipitation, SDS Page, Lysis, Sample Prep, Western Blot, In Vivo