Fig. 5

ADAMTS16 is C-mannosylated by DPY19L1 and DPY19L3.A, scheme of the ADAMTS16 fragment expressed in WT, DPY19L1 KO, and DPY19L3 KO CHO-K1 cells. The fragment contains TSR1 of ADAMTS16 resulting in the indicated peptides upon tryptic digestion. B, Western blot of the TSR1-containing ADAMTS16 fragment secreted by WT and KO cells. Coexpression of a myc-tagged fragment of mouse Notch1 comprising EGF repeats 9–14 served as transfection and secretion control. The amount of secreted ADAMTS16 relative to secreted EGF is depicted in the bar graph; results of individual experiments are represented by dots. C, MS analysis of a tryptic digest of the purified ADAMTS16 fragment secreted from WT, DPY19L1 KO, or DPY19L3 KO CHO-K1 cells. Extracted ion chromatograms (EICs) of the TSR1-derived peptides ([M + H⁺]⁴⁺) with different numbers of hexoses (Hex) are displayed. Corresponding spectra of each sample were adjusted to the intensity of the most intense glycoform (maximum intensity, as indicated at the bottom). Annotation of peaks was based on the parental ion mass, RT, and fragmentation spectra. Numbers 1 to 4 refer to the fragmentation spectra of the respective peaks provided in supplemental Figure S5. For each peptide, only tryptophan (W) residues are depicted, with respective C-mannoses (green circles). ADAMTS, A Disintegrin And Metalloproteinase with ThromboSpondin motifs; EGF, epidermal growth factor; RT, retention time; TSR, thrombospondin type 1 repeat.