Alcover A, Hervé F, Boursier J P, Spagnoli G, Olive D, Mariuzza R A, Acuto O
Laboratoires d'Immunologie Moleculaire, Institut Pasteur, Paris, France.
Mol Immunol. 1993 Jan;30(1):55-67. doi: 10.1016/0161-5890(93)90426-c.
We have generated a soluble form of the CD8 molecule consisting of the entire extracellular domains of the human alpha chain, by expressing a mutated CD8 alpha cDNA in SF9 cells infected with a recombinant baculovirus. The truncated molecule was secreted into the medium mostly as a disulfide-linked homodimer in which a single cysteine residue in the hinge-like region (Cys143) was sufficient to assure covalent bonding. Soluble CD8 purified to homogeneity appears to be monodisperse as assessed by gel filtration analysis and contains only O-linked carbohydrates. To determine whether recombinant CD8 can interact with MHC class I molecules, we developed an assay that measures binding of MHC class I-bearing cell lines to purified CD8 adsorbed to plastic plates. The level of binding of cells to immobilized CD8 depended on the amount of CD8 bound to the plate and correlated with the levels of cell surface MHC class I expression. The binding was specifically inhibited by monoclonal antibodies directed either against CD8 or MHC class I molecules. This assay therefore provides a way to measure CD8 binding to MHC class I independently of other cell-cell interactions and should allow direct structure-function studies.
我们通过在感染重组杆状病毒的SF9细胞中表达突变的CD8α cDNA，生成了一种由人α链的整个胞外域组成的可溶性CD8分子形式。截短的分子主要以二硫键连接的同二聚体形式分泌到培养基中，其中铰链样区域中的单个半胱氨酸残基（Cys143）足以确保共价结合。通过凝胶过滤分析评估，纯化至同质的可溶性CD8似乎是单分散的，并且仅含有O-连接的碳水化合物。为了确定重组CD8是否能与MHC I类分子相互作用，我们开发了一种检测方法，该方法可测量携带MHC I类的细胞系与吸附在塑料板上的纯化CD8的结合。细胞与固定化CD8的结合水平取决于与板结合的CD8量，并与细胞表面MHC I类表达水平相关。该结合被针对CD8或MHC I类分子的单克隆抗体特异性抑制。因此，该检测方法提供了一种独立于其他细胞间相互作用来测量CD8与MHC I类结合的方法，并且应该允许进行直接的结构-功能研究。
