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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

Journal article
Authors L. Lindberg
J. N. Liu
S. Gaunitz
A. Nilsson
T. Johansson
Niclas G. Karlsson
Jan Holgersson
Published in Glycobiology
Volume 23
Issue 6
Pages 720-735
ISSN 0959-6658
Publication year 2013
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 720-735
Language en
Links dx.doi.org/10.1093/glycob/cwt011
Keywords blood group ABH antigens, glycoengineering, immunoadsorption, mucins, group alpha(1,2)fucosyltransferase gene, antigen-specific, immunoadsorption, human-erythrocyte-membranes, carbohydrate, interactions, kidney-transplantation, molecular-cloning, expression, antibodies, beta-1,3-galactosyltransferase, glycosyltransferases
Subject categories Basic Medicine

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 G2b (PSGL-1/mIgG2b) 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/mIgG2b 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.

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