Journal: Frontiers in Immunology
Article Title: Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
Figure Lengend Snippet: Schematic representation of transglycosylation of palivizumab to generate homogeneous glycoforms. (A) Deglycosylation of commercially-available palivizumab using EndoS D233Q generates the truncated GlcNAcFuc disaccharide (blue square = GlcNAc; red triangle = fucose) remaining on palivizumab. Defucosylation is achieved using fucosidase GH29, and subsequent glycan ligation of purified glycan oxazoline is achieved using EndoS 233Q. (B) Enzymatic reaction and purification of truncated glycans (mono-galactosylated at the α3 antenna (G1 (3)T) and digalactosylated (G2T)) using EndoS and α2-3,6,8 neuraminidase to cleave truncated glycans between the reducing end chitobiose core, followed by purification by porous graphitic carbon (PGC)-HPLC. (C) Synthesis of truncated agalactosylated (G0T) and monogalactosylated at the α6 antenna (G1 (6)T) glycans using LacZ β1-4 galactosidase (* indicates reaction byproduct peaks), followed by purification by PGC-HPLC. HPAEC-PAD trace of overlaid spectra of purified glycans are shown on the right in each of (B, C) .
Article Snippet: 5 µL α2-3,6,8 neuraminidase (NEB, Cat #P0720) was then added to desialylate glycans.
Techniques: Ligation, Purification, Pyrolysis Gas Chromatography, High Performance Liquid Chromatography