Figure 3. ADAMTS17 is N-glycosylated and O-fucosylated and requires O-fucosylation for secretion.

(a) Predicted N-glycosylation (branched stems) and O-fucosylation sites (asterisks) in ADAMTS17 and constructs used for LC-MS/MS and the ADAMTS17 secretion analysis. (b) Western blot analysis of ADAMTS17^EA-containing medium (Med) with or without treatment with peptide-N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH). M = mature enzyme; Z = zymogen. For the full-length western blot see Supplemental Fig. 5a. (c) Schematic of TSR O-fucosylation depicting the enzymes and substrates involved. (d) Extracted ion chromatograms of the ions corresponding to unmodified (black), O-fucose (red), and O-fucose-glucose (blue) glycoforms of peptides from ADAMTS17 TSRs as identified by nano-LC-MS/MS. The O-fucosylation consensus sequence of the respective TSR and the modified residue (blue) are indicated above each chromatogram. (e) Western blot analysis of medium (Med) and lysate (Lys) from HEK293T cells with inactivated B3GLCT or POFUT2 (null) and transiently transfected with ADAMTS17-1C (1C), ADAMTS17-25P (25P) and ADAMTS17^EA (red). Co-transfected IgG (green) was used as the secretion control for quantification since it does not contain TSRs. For the full-length western blots see Supplemental Fig. 5b,c. (f) Quantification of the integrated densities of the respective bands normalized to IgG (n = 3). Statistical significance was calculated using a two-sided Student t-test and compared to the band intensity measured after secretion from wild-type cells.