Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation.
Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation.
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小鼠粒细胞巨噬细胞集落刺激因子羧基末端残基的缺失会导致生物活性丧失和糖基化改变。
DOI：
10.1016/0003-9861(90)90022-q
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发表时间：
1990
影响因子：
3.9
通讯作者：
Prystowsky,MB
中科院分区：
文献类型：
--
作者：
LaBranche,CC;Clark,SC;Johnson,GD;Ornstein,D;Sabath,DE;Tushinski,R;Paetkau,V;Prystowsky,MB
制备了一种原代结构与天然基因GM-CSF羧基末端11个氨基酸不同的小鼠粒细胞-巨噬细胞集落刺激因子（GM-CSF）缺失突变体。4个氨基酸残基发生突变，包括Cys-118在内的7个末端残基缺失。转染这种缺失突变体（GM-CSF(del)）的COS-1细胞的上清显示，在体外刺激骨髓干细胞增殖和分化为粒细胞和巨噬细胞的能力下降了3000倍。使用从转染细胞中提取的poly(A)+RNA进行Northern blot分析，显示GM-CSF和GM-CSF（del）的积累量相等。转染全长GM-CSF后，用兔抗rgm - csf抗血清免疫沉淀代谢标记的上清蛋白，主要产生23-kDa的完全糖基化形式，少量的29-kDa形式和18-kDa非n-糖基化形式。转染GM-CSF（del）突变体和免疫沉淀在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示一个大的弥散带，分子量约为29 kDa。用n -聚糖酶消化免疫沉淀的29-kDa物种，将29-kDa形式转化为大约23和18 kDa的两种形式，这表明缺失突变蛋白分子量的增加是由高糖基化引起的。将tunicamycin添加到转染了GM-CSF（del）的细胞的培养基中也产生了一个约18 kDa的非n-糖化种，但其分泌水平明显低于未经tunicamycin处理的细胞中29 kDa的高糖化GM-CSF（del）蛋白或未经tunicamycin处理的细胞中18 kDa的非n-糖化全长GM-CSF。由于最近的扫描缺失分析表明，在Cys-118附近有一个关键的活性区域，并且Cys-118是最大活性所必需的，我们得出结论，Cys-118残基是GMCSF适当的糖基化和最大生物活性所必需的。
A deletion mutant of murine granulocyte—Macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del)) showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophagesin vitro. Northern blot analysis using poly(A)+RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate—Polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species withN-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycintreated cells or the 18-kDa non-N-glycosylated fulllength GM-CSF from tunicamycin-treated cells. Since very recent scanning—deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GMCSF.
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DOI：
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发表时间：
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期刊：
The Journal of biological chemistry
影响因子：
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作者：
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影响因子：
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作者：
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