Deng P, Wang Y L, Pattengale P K, Rettenmier C W
Department of Pathology, Childrens Hospital of Los Angeles, USA.
Biochem Biophys Res Commun. 1996 Nov 12;228(2):557-66. doi: 10.1006/bbrc.1996.1698.
The shortest form of human macrophage colony-stimulating factor (M-CSF alpha, CSF-1(256) is expressed on the cell surface as a homodimeric type I transmembrane glycoprotein. The seven cysteine residues in CSF-1(256) form three intrachain disulfide bonds (Cys7-Cys90, Cys48-Cys139, and Cys 102-Cys146), and one interchain disulfide bond (Cys31-Cys31). To examine the role of the seven cysteine residues in CSF-1(256), we replaced each half-cystine by a serine using site-directed mutagenesis, and stably expressed the mutated genes in mouse NIH 3T3 cells. We showed that each of the seven cysteines of CSF-1(256) is essential for its biological activity. Our data further show that substitution of Cys48 or Cys139 totally blocked dimer formation and cell surface expression of CSF-1(256), and that substitution of Cys102 and Cys146 severely impaired CSF-1 dimer formation and cell surface expression. In contrast, substitution of Cys7 or Cys90 affected CSF-1 dimer formation to a lesser degree but did not significantly affect cell surface expression of CSF-1. Furthermore, disruption of the interchain disulfide bond led to efficient cell surface expression of monomeric CSF-1. All of the cell surface expressed mutant CSF-1 proteins, either dimeric or monomeric, still underwent efficient ectodomain cleavage. The electrophoretic mobilities of the cleaved dimeric ectodomains of these mutant CSF-1 proteins on SDS-PAGE exhibited distinctly different patterns as compared with the wild type. Substitution of either Cys7 or Cys90 produced the same shift, while substitution of either Cys102 or Cys146 resulted in a shift distinct from that caused by substitution of Cys7 or Cys90. These data suggest that replacement of either of a pair of intrachain half-cystine residues results in similar conformational changes, and may provide a novel method for mapping intrachain disulfide bonds in dimeric proteins.
人巨噬细胞集落刺激因子（M-CSFα，CSF-1(256)）的最短形式以同型二聚体I型跨膜糖蛋白的形式表达于细胞表面。CSF-1(256)中的七个半胱氨酸残基形成三个链内二硫键（Cys7-Cys90、Cys48-Cys139和Cys102-Cys146）以及一个链间二硫键（Cys31-Cys31）。为了研究CSF-1(256)中七个半胱氨酸残基的作用，我们利用定点诱变将每个半胱氨酸替换为丝氨酸，并在小鼠NIH 3T3细胞中稳定表达突变基因。我们发现CSF-1(256)的七个半胱氨酸中的每一个对于其生物学活性都是必不可少的。我们的数据进一步表明，Cys48或Cys139的替换完全阻断了CSF-1(256)的二聚体形成和细胞表面表达，并且Cys102和Cys146的替换严重损害了CSF-1二聚体的形成和细胞表面表达。相比之下，Cys7或Cys90的替换对CSF-1二聚体形成的影响较小，但对CSF-1的细胞表面表达没有显著影响。此外，链间二硫键的破坏导致单体CSF-1在细胞表面有效表达。所有细胞表面表达的突变型CSF-1蛋白，无论是二聚体还是单体，仍然经历有效的胞外域切割。与野生型相比，这些突变型CSF-1蛋白切割后的二聚体胞外域在SDS-PAGE上的电泳迁移率呈现出明显不同的模式。Cys7或Cys90的替换产生相同的迁移率变化，而Cys102或Cys146的替换导致的迁移率变化与Cys7或Cys90替换引起的不同。这些数据表明，一对链内半胱氨酸残基中任何一个的替换都会导致相似的构象变化，并可能为绘制二聚体蛋白中的链内二硫键提供一种新方法。
