tailieunhanh - Báo cáo khóa học: N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary cell line

GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the defi-nition of the glycosidic moiety, including the degree of site occupancy | Eur. J. Biochem. 271 907-919 2004 FEBS 2004 doi N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary cell line Guillermina Forno1 Mariela Bollati Fogolin1 Marcos Oggero1 Ricardo Kratje1 Marina Etcheverrigaray1 Harald S. Conradt2 and Manfred Nimtz3 1 Laboratorio de Cultivos Celulares Facultad de Bioquimica y Ciencias Biologicas Universidad National del Litoral Ciudad Universitaria Santa Fe Argentina 2Protein Glycosylation and 3Structural Biology GBF - Gesellschaft fur Biotechnologische Forschung mbH Braunschweig Germany GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production migration and function. It has been produced in different cell types with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked gly-cans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms differing in the number of Hex-HexNAc units Am 365 Da were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetra-antennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10 of bian-tennary oligosaccharides all containing more than 90 proximal a1-6-linked fucose. The

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