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

作者：Lindberg Linda; Liu Jining; Gaunitz Stefan; Nilsson Anki; Johansson Tomas; Karlsson Niclas G; Holgersson Jan^*

来源：Glycobiology, 2013, 23(6): 720-735.

DOI：10.1093/glycob/cwt011

• blood group ABH antigens
• glycoengineering
• immunoadsorption
• mucins

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 (beta 1,6GlcNAc-T1) or core 3 (beta 1,3GlcNAc-T6 and beta 1,3Gal-T5) enzymes together with alpha 1,2Fuc-T1 or alpha 1,2Fuc-T2 and the A or B gene-encoded alpha 1,3GalNAcT or alpha 1,3Gal-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 (Gal beta 3GlcNAc), type 2 (Gal beta 4GlcNAc) and type 3 (Gal beta 3GalNAc alpha) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Gal beta 3GalNAc) to core 3 (GlcNAc beta 3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAc beta 6GalNAc) O-glycans was incomplete as was the alpha 2-fucosylation of the core 1 chain. Sialylation may prevent these bio-synthetic 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.

• 出版日期2013-6