title: Production of sialylated O-linked glycans in Pichia pastoris.
abstract: The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.
authors.affiliation: Merck Research Laboratories, GlycoFi, Inc., Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH 03766, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
authors.forename: W James
authors.initials: WJ
authors.lastname: Cook
authors.forename: Sujatha
authors.initials: S
authors.lastname: Gomathinayagam
authors.forename: Irina
authors.initials: I
authors.lastname: Burnina
authors.forename: John
authors.initials: J
authors.lastname: Bukowski
authors.forename: Daniel
authors.initials: D
authors.lastname: Hopkins
authors.forename: Shaina
authors.initials: S
authors.lastname: Schwartz
authors.forename: Min
authors.initials: M
authors.lastname: Du
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Sharkey
authors.forename: Piotr
authors.initials: P
authors.lastname: Bobrowicz
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
mesh_terms: D008358:Mannose / Q000096:biosynthesis*; D060847:Metabolic Engineering / Q000379:methods*; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D015202:Protein Engineering; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D043323:alpha-Mannosidase; D008358:Mannose
keywords: O-glycosylation; Pichia pastoris; PomGnT1; glycoengineered; mannosidase