LaBranche C C, Clark S C, Johnson G D, Ornstein D, Sabath D E, Tushinski R, Paetkau V, Prystowsky M B
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104.
Arch Biochem Biophys. 1990 Jan;276(1):153-9. doi: 10.1016/0003-9861(90)90022-q.
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 macrophages in 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 with N-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-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length 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 GM-CSF.
制备了一种小鼠粒细胞-巨噬细胞集落刺激因子（GM-CSF）的缺失突变体，其在羧基末端11个氨基酸的一级结构上与天然GM-CSF不同。四个氨基酸残基发生了突变，包括Cys-118在内的七个末端残基被删除。用这种缺失突变体（GM-CSF(del)）转染的COS-1细胞的上清液在体外刺激骨髓干细胞增殖并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聚腺苷酸（poly(A)+）RNA进行的Northern印迹分析显示GM-CSF和GM-CSF(del)有等量积累。用全长GM-CSF转染，然后用兔抗rGM-CSF抗血清对代谢标记的上清液蛋白进行免疫沉淀，主要产生23 kDa的完全糖基化形式以及少量的29 kDa形式和18 kDa的非N-糖基化形式。GM-CSF(del)突变体的转染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出一条大的弥散带，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形式转化为两种约23 kDa和18 kDa的形式，这表明缺失突变体蛋白分子量的增加是由于高糖基化。向用GM-CSF(del)转染的细胞的培养基中添加衣霉素也产生了一种约18 kDa的单一非N-糖基化物种，但分泌水平明显低于未用衣霉素处理的细胞中29 kDa的高糖基化GM-CSF(del)蛋白或用衣霉素处理的细胞中18 kDa的非N-糖基化全长GM-CSF。由于最近的扫描缺失分析表明在Cys-118附近存在一个关键的活性区域，并且Cys-118对于最大活性是必需的，我们得出结论，Cys-118残基对于GM-CSF的正确糖基化和最大生物学活性是必需的。
