title: In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins.
abstract: The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.
authors: GlycoFi, Inc. (a wholly owned subsidiary of Merck & Co., Inc.), Biologics Discovery, Merck Research Laboratories, 16 Cavendish Court, Lebanon, NH, 03766, USA.
authors: Sujatha
authors: S
authors: Gomathinayagam
authors: Stephen R
authors: SR
authors: Hamilton
mesh_terms: D032449:Canavalia / Q000201:enzymology*; D006023:Glycoproteins / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D008361:Mannosidases; D008358:Mannose