title: Glycoengineering the N-acyl side chain of sialic acid of human erythropoietin affects its resistance to sialidase.
abstract: During the last years, the use of therapeutic glycoproteins has increased strikingly. Glycosylation of recombinant glycoproteins is of major importance in biotechnology, as the glycan composition of recombinant glycoproteins impacts their pharmacological properties. The terminal position of N-linked complex glycans in mammals is typically occupied by sialic acid. The presence of sialic acid is crucial for functionality and affects the half-life of glycoproteins. However, glycoproteins in the bloodstream become desialylated over time and are recognized by the asialoglycoprotein receptors via the exposed galactose and targeted for degradation. Non-natural sialic acid precursors can be used to engineer the glycosylation side chains by biochemically introducing new non-natural terminal sialic acids. Previously, we demonstrated that the physiological precursor of sialic acid (i.e., N-acetylmannosamine) can be substituted by the non-natural precursors N-propanoylmannosamine (ManNProp) or N-pentanoylmannosamine (ManNPent) by their simple application to the cell culture medium. Here, we analyzed the glycosylation of erythropoietin (EPO). By feeding cells with ManNProp or ManNPent, we were able to incorporate N-propanoyl or N-pentanoyl sialic acid in significant amounts into EPO. Using a degradation assay with sialidase, we observed a higher resistance of EPO to sialidase after incorporation of N-propanoyl or N-pentanoyl sialic acid.
authors.affiliation: Institute of Physiological Chemistry, Martin-Luther University of Halle-Wittenberg, Hollystr. 1, D-06114 Halle, Germany.
authors.forename: Anselm
authors.initials: A
authors.lastname: Werner
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Horstkorte
authors.forename: Dagobert
authors.initials: D
authors.lastname: Glanz
authors.forename: Karina
authors.initials: K
authors.lastname: Biskup
authors.forename: Véronique
authors.initials: V
authors.lastname: Blanchard
authors.forename: Markus
authors.initials: M
authors.lastname: Berger
authors.forename: Kaya
authors.initials: K
authors.lastname: Bork
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D003016:Clostridium perfringens / Q000201:enzymology; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006595:Hexosamines / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis* / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D006595:Hexosamines; C116506:N-pantanoylmannosamine; D011994:Recombinant Proteins; D004921:Erythropoietin; C076271:N-propanoylmannosamine; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid