title: Mucin-type fusion proteins with blood group A or B determinants on defined O-glycan core chains produced in glycoengineered Chinese hamster ovary cells and their use as immunoaffinity matrices.
abstract: Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) (PSGL-1/mIgG(2b)) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG(2b) carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored.
authors: AbSorber AB, Franzéngatan 5, SE-112 51 Stockholm, Sweden.
authors: Linda
authors: L
authors: Lindberg
authors: Jining
authors: J
authors: Liu
authors: Stefan
authors: S
authors: Gaunitz
authors: Anki
authors: A
authors: Nilsson
authors: Tomas
authors: T
authors: Johansson
authors: Niclas G
authors: NG
authors: Karlsson
authors: Jan
authors: J
authors: Holgersson
mesh_terms: D000017:ABO Blood-Group System / Q000096:biosynthesis*; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D003412:Cricetulus; D006031:Glycosylation; D007074:Immunoglobulin G / Q000096:biosynthesis; D007163:Immunosorbent Techniques; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis*; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins
chemical_list: D000017:ABO Blood-Group System; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D009077:Mucins; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; D017350:N-Acetylgalactosaminyltransferases
keywords: blood group ABH antigens; glycoengineering; immunoadsorption; mucins