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Originally published online as doi:10.1189/jlb.0803378 on December 4, 2003

Published online before print December 4, 2003
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(Journal of Leukocyte Biology. 2004;75:515-522.)
© 2004 by Society for Leukocyte Biology

The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation

Clive R. D. Carter1, Katie M. Whitmore and Robin Thorpe

Division of Immunobiology, National Institute for Biological Standards and Control, Herts, United Kingdom

1 Correspondence: Division of Imunobiology, NIBSC, Blanch Lane, South Mimms, Potters Bar, Herts, EN6 3QG, UK. E-mail: ccarter{at}nibsc.ac.uk

It has been reported recently that granulocyte-colony stimulating factor (G-CSF) is degraded upon exposure to human neutrophil elastase (HNE), and this has a negative effect on the ability of the cytokine to promote the in vitro proliferation and maturation of CD34+ cells. This has important implications on the possible in vivo role of elastase in providing negative feedback to granulopoiesis by the direct antagonism of G-CSF. The cytokine used in that study was expressed in Escherichia coli [and was nonglycosylated (NG)], unlike the naturally occurring cytokine, which is an O-linked glycoprotein. As a Chinese hamster ovary-derived (glycosylated) cytokine is available, we compared the susceptibility of NG and glycosylated G-CSF to elastase degradation by incubating the cytokines with HNE and assessing its impact by sodium dodecyl sulfate gel electrophoresis and bioassay. We confirmed the ability of elastase to degrade NG G-CSF in a time- and concentration-dependent manner and found this was associated with a reduction in biological activity of the cytokine. Glycosylated G-CSF, however, was more resistant to elastase degradation, although prolonged exposure did lead to degradation and decreased biological activity. The significance of sugar residues on glycosylated G-CSF in providing protection against the effects of elastase was investigated using enzymatically deglycosylated G-CSF and a mutated form of the G-CSF molecule that was expressed in yeast but was NG. The possible role of HNE in serum-induced inactivation of NG G-CSF was also considered.

Key Words: glycosylation • proteases • deglycosylation • granulopoiesis • bioassay




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