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 β1,3-N-acetylglucosaminyltransferase 3 (extended C1 β3GnT3), core 2 β1,6-N-acetylglucosaminyltransferase I (C2 β3GnT1) or core 3 β1,3-N-acetylglucosaminyltransferase 6 (C3 β3GnT6) in CHO cells. Extended core 1 (GlcNAcβ1,3Galβ1,3GalNAc) and core 3 (GlcNAcβ1,3GalNAc), and increased expression of core 2 [Galβ1,3(GlcNAcβ1,6)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 β3GnT3 expression. The α2,6-sialylated type 2 chain was detected upon co-transfection with the β-galactoside α2,6-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.
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-413 45 Gothenburg, Sweden.
Jining
J
Liu
Department of Medical Biochemistry, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-405 30 Gothenburg, Sweden. Electronic address: chunsheng.jin@medkem.gu.se.
Chunsheng
C
Jin
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-413 45 Gothenburg, Sweden.
Reeja Maria
RM
Cherian
Department of Medical Biochemistry, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
Niclas G
NG
Karlsson
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-413 45 Gothenburg, Sweden.
Jan
J
Holgersson
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics*; D016695:Glycosyltransferases; D009077:Mucins / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D009077:Mucins; D011134:Polysaccharides; D011994:Recombinant Proteins; D016695:Glycosyltransferases
CHO; Core structure; Glycosyltransferase; Mucin; O-glycans