Wong Michael J, Malapitan Irish A, Sikorski Barbara A, Jongstra Jan
Toronto Western Research Institute, Cell and Molecular Biology Division and the Department of Immunology, University of Toronto, Toronto, ON, Canada M5T 2S8.
Biochim Biophys Acta. 2003 Sep 23;1642(1-2):17-24. doi: 10.1016/s0167-4889(03)00082-x.
The leukocyte specific protein 1 or LSP1 is a multi functional protein involved in such divers biological processes as the regulation of neutrophil motility, chemotaxis, adhesion and membrane immunoglobulin M (mIgM) mediated apoptosis of B-lymphocytes. The 330-amino-acid mouse LSP1 protein contains a high-affinity F-actin binding site and in intact cells localizes to the F-actin filament containing cytoskeleton. Here we use a high-speed F-actin co sedimentation assay and transfection experiments in the LSP1- T-lymphoma cell line BW5147 to show that LSP1 interacts with F-actin and the cytoskeleton through residues downstream of amino acid residue 230. We then designed a novel cell-free cytoskeleton binding assay in which a set of GST-LSP1 fusion proteins are allowed to bind to the cytoskeleton in NP-40 soluble lysates of BW5147 cells and are recovered in the low-speed detergent insoluble pellet. Using this assay the cytoskeleton binding site of mouse LSP1 maps to the 300-330 interval. These results will allow the design of LSP1 mutants that do not bind to the cytoskeleton to determine the importance of LSP1 cytoskeleton binding for the diverse functions of LSP1.
白细胞特异性蛋白1（LSP1）是一种多功能蛋白，参与多种生物学过程，如中性粒细胞运动、趋化性、黏附以及膜免疫球蛋白M（mIgM）介导的B淋巴细胞凋亡的调节。330个氨基酸的小鼠LSP1蛋白含有一个高亲和力的F-肌动蛋白结合位点，在完整细胞中定位于含F-肌动蛋白丝的细胞骨架。在此，我们利用高速F-肌动蛋白共沉降分析以及在LSP1-T淋巴瘤细胞系BW5147中的转染实验，表明LSP1通过氨基酸残基230下游的残基与F-肌动蛋白和细胞骨架相互作用。然后，我们设计了一种新型的无细胞细胞骨架结合分析方法，其中一组GST-LSP1融合蛋白被允许与BW5147细胞NP-40可溶性裂解物中的细胞骨架结合，并在低速去污剂不溶性沉淀中回收。利用该分析方法，小鼠LSP1的细胞骨架结合位点定位在300 - 330区间。这些结果将有助于设计不与细胞骨架结合的LSP1突变体，以确定LSP1细胞骨架结合对其多种功能的重要性。
