Fig. 2: Glycosylation of non-canonical sequons by Desulfovibrio PglB homologs. | Nature Communications

Fig. 2: Glycosylation of non-canonical sequons by Desulfovibrio PglB homologs.

From: Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in bacteria

Fig. 2

a Immunoblot analysis of periplasmic fractions from CLM24 cells transformed with the following plasmids: pMW07-pglΔBCDEF for making GalNAc5(Glc)GlcNAc; a derivative of pMLBAD encoding one of the PglB homologs as indicated; and pBS-scFv13-R4AQNAT encoding the scFv13-R4(N34L/N77L) acceptor protein with AQNAT sequon. Blots were probed with anti-His antibody (top panel) and hR6 serum (bottom panel). Molecular weight (MW) markers are indicated on the left. The g0 and g1 arrows indicate un- and monoglycosylated acceptor proteins, respectively. Blots are representative of biological replicates (n = 3). b Glycosylation efficiency was determined by densitometric analysis, with data reported as mean ± SD. Red bars correspond to positive and negative controls generated by CjPglB with scFv13-R4DQNAT or scFv13-R4(N34L/N77L)AQNAT as acceptors, respectively; blue bars correspond to samples generated with Desulfovibrio PglBs. (c, d) Same as (a, b) but with plasmid pBS-scFv13-R4QYNST encoding scFv13-R4(N34L/N77L) with QYNST sequon. Red bars correspond to positive and negative controls generated by CjPglB with scFv13-R4DQNAT or scFv13-R4(N34L/N77L)QYNST as acceptors, respectively; blue bars correspond to samples generated with Desulfovibrio PglBs. The first two lanes in left and right panels of (a) and (c) were loaded with the same positive and negative control samples (marked with asterisk). Schematics created with BioRender. DeLisa, M. (2025) https://BioRender.com/wesqljn. Statistical significance was determined by unpaired two-tailed Student’s t-test with calculated p-values represented as: *, p < 0.05; ***, p < 0.001; ns, not significant. Actual p-values relative to CjPglB (DQNAT) in panel (b) are (from left-to-right): p = 0.1072, p = 0.3838, p = 0.0008, p = 0.2436, and p = 0.2462. Actual p-values relative to CjPglB (DQNAT) in panel (d) are (from left-to-right): p = 0.6619, p = 0.0144, p = 0.0363 (DgPglB vs. DmPglB), p = 0.0017, and p = 0.0027. Source data are provided as a Source Data file.

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