FIGURE 4.

PC processing of ANGPTL3 is blocked by O-glycosylation in CHO ldlD cells. A, schematic depiction of the full coding sequence of ANGPTL3 and design of the expression constructs used. Heparin binding site (VHKTKG), furin cleavage site (RAPR), and N-glycans are indicated. B, transient expression of full coding ANGPTL3 in CHO ldlD cells grown with Gal and GalNAc (G/Gn), with GalNAc (Gn) (allowing GalNAc O-glycosylation only), and with Gal (G) (allowing complete N-glycosylation but no O-glycosylation). Medium from transfected 6-well plates were harvested after 72 h, and His-tagged ANGPTL3 was affinity-purified on Talon beads (Co²⁺) and analyzed by Western blotting with anti-V5 antibody. The protein load was normalized to expression of ANGPTL3 evaluated by capture ELISA (not shown). The ANGPTL3 proprotein migrating at 62 kDa and the mature protein migrating at 39 kDa are indicated by arrows. C, Western blot analysis of 50 ng of commercial recombinant human ANGPTL3 with mAb 1D10 to ANGPTL3.