Fig. 5: Glycosylation of native acceptor site in IgG Fc domain by DmPglB.
a Non-reducing immunoblot analysis of protein A-purified proteins from whole-cell lysate of CLM24 cells transformed with the following plasmids: pMW07-pglΔBCDEF for making GalNAc5(Glc)GlcNAc (left) or pMW07-pglΔBICDEF for making GalNAc5GlcNAc (right); pMLBAD encoding CjPglB, DgPglB, DmPglB, or DmPglBmut; and pTrc99S-spDsbA-hinge-Fc encoding hinge-Fc derived from human IgG1. Blots were probed with anti-human IgG (anti-IgG) to detect human Fc (top panel) and hR6 serum (bottom panel). Molecular weight (MW) markers are indicated on the left. The g0, g1, and g2 arrows indicate un-, mono-, and diglycosylated Fc proteins, respectively. Blots are representative of biological replicates (n = 3). b Glycosylation efficiency was determined as above with data reported as the mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test with calculated p-values represented as: **p < 0.01; ***p < 001; ****p < 0.0001. Actual p-values in top panel are (from left-to-right): p < 0.0001 and p = 0.0002. Actual p-values in bottom panel are (from left-to-right): p = 0.0017 and p = 0.0017. c Same as in (a) but with JUDE-1 cells transformed with plasmid pMAZ360-YMF10-IgG encoding a full-length chimeric IgG1 specific for PA along with plasmids for glycan biosynthesis and ssOST as indicated. Asterisks denote band shifts due to glycosylation of HC-LC dimer. Schematics created with BioRender. DeLisa, M. (2025) https://BioRender.com/wesqljn. Source data are provided as a Source Data file.
