The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation
The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation
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DOI：
10.1189/jlb.0803378
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发表时间：
2004-03-01
影响因子：
5.5
通讯作者：
Thorpe, R
中科院分区：
文献类型：
--
作者：
Carter, CRD;Whitmore, KM;Thorpe, R
最近有报道称，粒细胞集落刺激因子(G-CSF)在人中性粒细胞弹性蛋白酶(HNE)作用下会发生降解，从而影响其促进CD34(+)细胞体外增殖和成熟的能力。这对弹力酶通过直接拮抗G-CSF对粒细胞生成提供负反馈的可能的体内作用具有重要意义。这项研究中使用的细胞因子在大肠杆菌中表达[并且是非糖基化的(NG)]，与自然产生的细胞因子不同，自然产生的细胞因子是一种O-连接的糖蛋白。由于中国仓鼠卵巢来源的(糖基化的)细胞因子是可用的，我们比较了NG和糖基化G-CSF对弹性蛋白酶降解的敏感性，方法是将这些细胞因子与HNE孵育，并通过十二烷基硫酸钠凝胶电泳法和生物测定来评估其影响。我们证实了弹性蛋白酶以时间和浓度依赖的方式降解NG G-CSF的能力，并发现这与细胞因子的生物活性降低有关。然而，糖基化G-CSF对弹性酶降解的抵抗力更强，尽管长时间暴露确实会导致降解和生物活性降低。利用酶去糖化的G-CSF和在酵母中表达但为NG的G-CSF分子的突变形式，研究了糖基化G-CSF上的糖残基在提供对弹性蛋白酶作用的保护方面的意义。此外，还探讨了HNE在血清诱导的NG G-CSF失活中的作用。
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.