Meyerson H J, Huang J H, Fayen J D, Tsao H M, Getty R R, Greenspan N S, Tykocinski M L
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
J Immunol. 1996 Jan 15;156(2):574-84.
The contribution of the CD8 alpha.IgV homologue domain to class I MHC binding was evaluated using a series of chimeric human CD8 alpha:Fc polypeptides incorporating alternative CD8 alpha extracellular domain components. Using a nonisotopic cellfree physical binding assay, those Fc chimeras encompassing the CD8 alpha.IgV homologue domain only (dissociated from the 48-amino acid CD8 alpha connecting peptide) were shown to retain the capacity of the complete CD8 alpha extracellular domain to bind to a recombinant soluble class I MHC alpha 3 domain unit or to intact class I MHC. The specificity of the CD8 alpha:class I MHC alpha 3 domain interaction was verified by mAb and soluble polypeptide blocking experiments. Furthermore, co-precipitation of an Fc chimera incorporating only the CD8 alpha.IgV homologue domain and a recombinant soluble class I MHC alpha 3 domain unit was accomplished. In addition, a glycosylphosphatidylinositol (GPI)-modified variant of the CD8 alpha.IgV homologue domain was generated via chimerization with the GPI signal sequence from decay-accelerating factor. GPI anchorage for this truncated CD8 alpha polypeptide was verified, and its capacity to promote intercellular adhesion through class I MHC binding was shown in a cell:cell binding assay. The findings indicate that the CD8 alpha.IgV homologue domain acts as an independent structural unit when dissociated from the CD8 alpha connecting peptide, and in so doing retains class I MHC binding capacity. This further establishes the principle that Ig superfamily domains from receptor:counter-receptor pairs can interact with each other as isolated units, providing an experimental path for tailoring therapeutically useful IgSF protein derivatives.
使用一系列包含不同CD8α胞外域成分的嵌合人CD8α:Fc多肽，评估CD8α免疫球蛋白V（IgV）同源结构域对I类主要组织相容性复合体（MHC）结合的贡献。通过非同位素无细胞物理结合试验，发现那些仅包含CD8α IgV同源结构域（与48个氨基酸的CD8α连接肽解离）的Fc嵌合体保留了完整CD8α胞外域与重组可溶性I类MHC α3结构域单元或完整I类MHC结合的能力。通过单克隆抗体（mAb）和可溶性多肽阻断实验验证了CD8α与I类MHC α3结构域相互作用的特异性。此外，实现了仅包含CD8α IgV同源结构域的Fc嵌合体与重组可溶性I类MHC α3结构域单元的共沉淀。另外，通过与衰变加速因子的糖基磷脂酰肌醇（GPI）信号序列嵌合，产生了CD8α IgV同源结构域的GPI修饰变体。验证了该截短的CD8α多肽的GPI锚定，并在细胞间结合试验中显示了其通过I类MHC结合促进细胞间粘附的能力。这些发现表明，CD8α IgV同源结构域与CD8α连接肽解离时可作为一个独立的结构单元，并且这样做时保留了I类MHC结合能力。这进一步确立了受体 - 反受体对中的免疫球蛋白超家族（IgSF）结构域可以作为分离的单元相互作用的原则，为定制具有治疗用途的IgSF蛋白衍生物提供了一条实验途径。
