Leong S R, Lowman H B, Liu J, Shire S, Deforge L E, Gillece-Castro B L, McDowell R, Hébert C A
Department of Immunology, Genentech, Inc., South San Francisco, California 94080, USA.
Protein Sci. 1997 Mar;6(3):609-17. doi: 10.1002/pro.5560060310.
Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants.
趋化因子白细胞介素-8（IL-8）的共价单链二聚体已被设计用于模拟溶液中IL-8的二聚体形式，并促进IL-8异源二聚体变体的产生。物理研究表明，使用简单的肽接头连接两个亚基，在允许受体结合和激活的同时，会导致连接的二聚体发生自缔合。然而，在连接的亚基之间添加单个二硫键交联可防止这种多聚体形成，产生一种二聚体分子量的物质。交联的单链二聚体与IL-8中性粒细胞受体CXCR1和CXCR2以及DARC结合，没有肽接头的双二硫键连接的二聚体也是如此。此外，通过趋化性或β-葡萄糖醛酸酶释放测量，中性粒细胞对这些二聚体的反应与野生型IL-8引发的反应相似，这证明这些生物学相关活性不需要二聚体物种的解离。最后，通过构建单链异源二聚体突变体，我们表明只有第一个亚基的ELR基序是单链变体。
