FIGURE 5.

Analysis of PC processing of FGF23 and ANGPTL3 reporter constructs in CHO ldlD cells. A, schematic depiction of design of reporter constructs. Vertical arrows indicate sites of furin cleavage, and filled circles indicate sites of O-glycosylation. All potential O-glycosylation sites are shown in gray. B, SDS-PAGE Western blot analysis of a representative clone expressing ANGPTL3 (6F7) or FGF23 (7B2) reporter constructs. Due to extensive O-glycosylation in the MUC1 tandem repeat region, the reporter construct migrates at 48 kDa compared with 44 kDa for the construct without O-glycosylation. The PC-processed N-terminal fragments with EYFP migrate at 38 kDa (ANGPTL3 reporter) and 39 kDa (FGF23 reporter). The upper panels are labeled with anti-EYFP, whereas the lower panels are labeled with mAb 5E5 specifically recognizing the GalNAc O-glycosylated MUC1 tandem repeat sequence in the C-terminal fragment (22). Migration of O-glycosylated, non-glycosylated, and processed proteins are indicated by arrows, and the additions of Gal and GalNAc to growth medium are indicated as described in the legend to Fig. 4.