O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs

Glyco-engineering of host cells is used to increase efficacy, decrease immunogenicity and increase circulatory half-lives of protein biopharmaceuticals. The effect of transiently expressed O-glycan core chain glycosyltransferases on O-glycan biosynthesis pathways in CHO cells is reported. Liquid chromatography-mass spectrometry and Western blotting were used to map the O-glycome of a mucin-type fusion protein transiently co-transfected with beta 1,3-N-acetylglucosaminyltransferase 3 (extended Cl beta 3GnT3), core 2 beta 1,6-N-acetylglucosaminyltransferase I (C2 beta 3GnT1) or core 3 beta 1,3-N-acetylglucosaminyltransferase 6 (C3 beta 3GnT6) in CHO cells. Extended core 1 (GlcNAc beta 1, 3Gal beta 1, 3GalNAc) and core 3 (G1cNAc beta 1,3GalNAc), and increased expression of core 2 [Gal beta 1,3(GlcNAc beta 1,6)GalNAc], O-glycans were generated on P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL1/mIgG2b). Endogenous poly-N-acetyllactosamine (poly-LacNAc) synthase elongated extended core 1 and core 3 generating O-glycans with up to five LacNAc repeats. Low amounts of core 3 O-glycans appeared upon extended C1 beta 3GnT3 expression. The alpha 2,6-sialylated type 2 chain was detected upon co-transfection with the beta-galactoside alpha 2,6-sialyltransferase I. N-acetylglucosamine-6-O-sulfotransferase 2 transferred sulfate to carbon 6 of GlcNAc in poly-LacNAc sequences. CHO cells with its known O-glycan repertoire can be used to express recombinant mucin-type proteins together with selected glycosyltransferases in order to recreate carbohydrate determinants on defined O-glycan chains. They will become important tools for assessing the core chain-dependent binding activity of carbohydrate-binding proteins. (C) 2015 Elsevier B.V. All rights reserved.