O-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity.
Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein carrying one O-linked sugar chain. To clarify the role of the oligosaccharide in hG-CSF, some biological and physicochemical properties of the deglycosylated hG-CSF and the intact factor were compared. Recombinant hG-CSF produced in transfected Chinese hamster ovary cells was sequentially digested with neuraminidase and endo-alpha-N-acetylgalactosaminidase. The deglycosylated hG-CSF was one-third as active as the intact form in the colony-forming assay, but it was almost as active as the intact hG-CSF in the cell proliferation assay using NFS-60 cells (NFS-60 bioassay). Inactivation of the deglycosylated hG-CSF was also found by NFS-60 bioassay after incubation for 2 days at pH values from 7 to 8 and at 37 degrees C. This inactivation was accompanied by polymerization of the factor which did not occur with the glycosylated factor. Circular dichroic and calorimetric analyses demonstrated that the deglycosylated hG-CSF is more sensitive to heat denaturation than the intact form and that the inactivation of both forms of hG-CSF was accompanied by conformational change of the proteins. From these results, it was concluded that the O-linked sugar chain of hG-CSF contributes to the stability of the factor by suppressing polymerization and/or its conformational changes.
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan.
M
M
Oh-eda
M
M
Hasegawa
K
K
Hattori
H
H
Kuboniwa
T
T
Kojima
T
T
Orita
K
K
Tomonou
T
T
Yamazaki
N
N
Ochi
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002942:Circular Dichroism; D003115:Colony-Stimulating Factors / Q000378:metabolism / Q000494:pharmacology; D004355:Drug Stability; D006023:Glycoproteins; D016179:Granulocyte Colony-Stimulating Factor; D006801:Humans; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013816:Thermodynamics; D014162:Transfection
D000596:Amino Acids; D003115:Colony-Stimulating Factors; D006023:Glycoproteins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor