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

Abstract

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.

Comparison between filgrastim and lenograstim plus chemotherapy for mobilization of PBPCs

“…[27][28][29] Other studies have shown that G-CSF is degraded by serum enzymes, particularly elastases. Carter et al 30 showed that glycosylation reduces this elastase-dependent inactivation. It may thus prolong G-CSF's activity and make it more effective.…”

Over expression and analysis of O-glycosylated recombinant human granulocyte colony stimulating factor in Pichia pastoris using Agilent 2100 Bioanalyzer

“…Digestion study of rhGCSF by hNE was performed according to Carter et al (2004a). Briefly 50 g ml −1 of purified rhGCSF protein preparations were incubated with 2 g ml −1 of hNE for 45 min at 37 • C with and without 2 mM PMSF, a known hNE inhibitor for comparison purposes.…”

“…It is mainly produced by monocytes and macrophages (Bahrami et al, 2007) and stimulates proliferation, activation and differentiation of committed precursor cells of neutrophil lineage and mature neutrophils (Fallah et al, 2003). It is effectively used for the treatment of various forms of neutropenia, leukopenia, AIDS and bone marrow transplantation (Carter et al, 2004a).…”

Neutrophil elastase in cyclic and severe congenital neutropenia

“…95 Another possibility is that mislocalized NE retains some proteolytic activity and inappropriately attacks an opportunistic substrate it would not ordinarily encounter. Potential substrates, each capable of being proteolyzed by NE, located in the vicinity of the plasma membrane, and capable of regulating hematopoiesis, include G-CSF, [96][97][98] the G-CSF receptor, 97 the c-KIT receptor, 99 and Notch proteins. 95 Regardless of the precise molecular mechanism, mislocalized (or misfolded) NE must somehow drastically reduce the production of neutrophils from the progenitor cells in the bone marrow.…”