Lybarger Lonnie, Yu Y Y Lawrence, Miley Michael J, Fremont Daved H, Myers Nancy, Primeau Tina, Truscott Steven M, Connolly Janet M, Hansen Ted H
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2003 Jul 18;278(29):27105-11. doi: 10.1074/jbc.M303716200. Epub 2003 May 5.
Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.
主要组织相容性复合体I类分子可以表达为单链多肽，其中抗原肽、β2-微球蛋白和重链通过柔性接头连接。这些分子，即单链三聚体（SCT），与天然（非共价连接）I类分子相比，在细胞表面具有显著的稳定性。在本研究中，我们采用基于结构的方法设计了小鼠I类分子Kb的F口袋变体SCT，该变体呈现卵清蛋白的SIINFEKL表位。SCT中重链残基Tyr84（Y84A）的突变导致共价连接肽的血清学和细胞溶解性CD8 T细胞识别增强，这是因为肽C末端延伸的接头得到了更好的容纳。这些SCT表现出显著的细胞表面稳定性，我们推测这是由于它们能够持续有效地重新结合共价连接的肽。此外，我们证明SCT技术可应用于使用在大肠杆菌中表达的重组SCT构建四聚体。基于SCT的四聚体可用于计数识别易于解离的肽-I类复合物的T细胞和自然杀伤细胞。
