FIGURE 3.

Glycan occupancy of Asn³⁴⁷ abolishes the PAE-induced RCL cleavage. A, unlike NE-digested (light blue) CBG (two penultimate left lanes), crudely isolated and pure PAE (green) generated two relatively homogeneous complementary CBG fragments (i.e. a single CBG-Ct (band 3, 5 kDa) and a CBG-Nt (50–55 kDa) fragment under extended incubation (4 and 16 h, right lanes) as evaluated by SDS-PAGE. Undigested native CBG is shown in the far left lane. Broken line, non-neighboring gel lanes from the same gel. Solid line, gel lanes from different gels. B, amino acid residues of the exposed RCL of CBG in the vicinity of the observed PAE cleavage sites (green arrows). The CBG-Nt (gold) and CBG-Ct (brown) peptides covering the Asn³⁴⁷ glycosylation site are shown. C, the presence of ³⁴⁶LNLTSKPIILR³⁵⁶ and ³⁴⁸LTSKPIILR³⁵⁶ (data for the latter not shown) in an Asn³⁴⁷-non-glycosylated form (top EIC, left) and the absence of their corresponding Asn³⁴⁷-glycosylated forms (bottom EIC, left; as exemplified by the absence of an EIC signal for the TS3 Asn³⁴⁷ glycopeptide) in the CBG-Ct fragment was confirmed using CID-MS/MS (right). D, peptides of the complementary CBG-Nt fragments (i.e. ³²⁷AVLQLNEEGVDTAGSTGVTLN³⁴⁷ and ³²⁷AVLQLNEEGVDTAGSTGVT³⁴⁵ (data for the latter not shown) were also exclusively identified in their non-glycosylated state by LC-MS/MS. No EIC signals were detected in any charge states for any glycoforms of either the CBG-Nt or the CBG-Ct peptides. *, CBG-Nt fragments occurring after extended digestion of CBG with NE that were not further investigated in this study. ‡, crude PAE migrated around 33 kDa as validated by LC-MS/MS analysis, whereas the commercial form of PAE (“pure PAE”) was observed in separate experiments to co-migrate with human CBG around 55–60 kDa (data not shown).