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 1H-NMR-spectroscopy and two 1H-detected-heteronuclear 1H-13C experiments at natural abundance, and compared with the non-glycosylated form. The present NMR study reports assignments of 1H and 13C resonances of the bound saccharidic chain NeuNAc(alpha2-3)Gal(beta1-3)[NeuNAc(alpha2-6)]GalNAc, where NeuNAc represents N-acetylneuraminic acid, and demonstrates the alpha-anomeric configuration of the N-acetylgalactosamine-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.
Gervais
V
V
European Molecular Biology Laboratory, Heidelberg, Germany.
Zerial
A
A
Oschkinat
H
H
D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011489:Protein Denaturation; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor