NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony-stimulating factor

Human granulocyte-colony-stimulating factor (G-CSF) is a hematopoietic growth factor that plays a major role in the stimulation of the proliferation and maturation of granulocyte neutrophil cells. With the recent increased understanding of its biological properties in vivo together with available preparations of recombinant human G-CSF, this growth factor has become an essential agent for clinical applications. The presence of an O-linked carbohydrate chain at position 133 greatly improves the physical stability of the protein. To clarify the molecular basis for the stabilisation effect of saccharide moieties on human G-CSF, the whole glycoprotein expressed in CHO cells has been investigated by means of two H-1-NMR-spectroscopy and two H-1-detected-heteronuclear H-1-C-13 experiments at natural abundance, and compared with the non-glycosylated form. The present NMR study reports assignments of H-1 and C-13 resonances of the bound saccharidic chain NeuNAc(alpha 2-3)Gal(beta 1-3)[NeuNAc(alpha 2-6)]GalNAc, where NeuNAc repre sents N-acetylneuraminic acid, and demonstrates the alpha-anomeric configuration of the N-acetylgalaclosamine-threonine linkage. It also provides results suggesting that the carbohydrate moiety reduces the local mobility around the glycosylation site. which could be responsible for the stabilising effect observed on the glycoprotein.