Functional mapping of cytotactin: proteolytic fragments active in cell-substrate adhesion.
Functional mapping of cytotactin: proteolytic fragments active in cell-substrate adhesion.
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DOI：
10.1083/jcb.107.6.2329
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发表时间：
1988-12
期刊：
影响因子：
--
通讯作者：
Edelman GM
中科院分区：
文献类型：
--
作者：
Friedlander DR;Hoffman S;Edelman GM
Cytokine是一种细胞外基质糖蛋白，在发育过程中分布受限。在电子显微镜图像中，它看起来像一个六臂体，从中央核心延伸出六条臂。Cytocytoin与其他细胞外基质蛋白结合，包括硫酸软骨素蛋白聚糖（CTB蛋白聚糖）和纤连蛋白。虽然细胞生长素与包括成纤维细胞和神经元在内的多种细胞结合，但在某些情况下，它会导致培养中的细胞聚集并抑制它们的迁移。为了将细胞生长素对细胞行为的各种影响与其结合区域联系起来，我们研究了其支持细胞-基质粘附的能力，并将其细胞结合功能映射到其结构上。在细胞-基质粘附试验中，成纤维细胞与cytokine结合，但保持圆形。相反，它们都附着在纤连蛋白上并在纤连蛋白上扩散。在该测定中，神经元和神经胶质细胞均不与细胞色素结合。然而，在通过离心启动细胞-基质接触的测定中，神经元和神经胶质细胞与cytotoxin结合良好;这种结合被特异性抗cytotoxin抗体阻断。结果表明，神经元和神经胶质细胞可以结合到cytotactin包被的基板，这些细胞，像成纤维细胞，具有细胞因子的细胞表面配体。在应用有限的蛋白水解和分级分离的方法后，这些测定被用于将细胞色素蛋白的结合功能映射到其结构上。有限的蛋白水解产生的片段被分成两个主要的池：一个（馏分I）含有二硫键连接的低聚物的100 kD片段和两个次要的相关片段，第二（馏分II）含有单体90-和65-kD片段。组分II中的90-和65-kD片段彼此密切相关，并且在结构和免疫学上不同于组分I中的片段。只有组分I中的单克隆抗体M1，它结合到位于近端部分的武器的hexabrachion和由一个多克隆抗体制备的75 kD的CNBr片段的完整的cytotoxin的表位识别。A单克隆抗体（1D 8）和多克隆抗体制备的35 kD CNBr片段的cytotoxin只承认组分II中存在的。在细胞结合实验中，成纤维细胞、神经元和神经胶质各自粘附到用组分II包被的基底上，但不粘附到用组分I包被的基底上。Fab片段的抗体的35 kD的溴化氰片段强烈抑制细胞的结合cytotoxin，支持的结论，即馏分II包含一个细胞结合区。此外，该抗体的Fab片段可抑制cytokine与CTB蛋白的结合。
Cytotactin is an extracellular matrix glycoprotein with a restricted distribution during development. In electron microscopic images, it appears as a hexabrachion with six arms extending from a central core. Cytotactin binds to other extracellular matrix proteins including a chondroitin sulfate proteoglycan (CTB proteoglycan) and fibronectin. Although cytotactin binds to a variety of cells including fibroblasts and neurons, in some cases it causes cells in culture to round up and it inhibits their migration. To relate these various effects of cytotactin on cell behavior to its binding regions, we have examined its ability to support cell-substrate adhesion and have mapped its cell- binding function onto its structure. In a cell-substrate adhesion assay, fibroblasts bound to cytotactin but remained round. In contrast, they both attached and spread on fibronectin. Neither neurons nor glia bound to cytotactin in this assay. In an assay in which cell-substrate contact was initiated by centrifugation, however, neurons and glia bound well to cytotactin; this binding was blocked by specific anti- cytotactin antibodies. The results suggest that neurons and glia can bind to cytotactin-coated substrates and that these cells, like fibroblasts, possess cell surface ligands for cytotactin. After applying methods of limited proteolysis and fractionation, these assays were used to map the binding functions of cytotactin onto its structure. Fragments produced by limited proteolysis were fractionated into two major pools: one (fraction I) contained disulfide-linked oligomers of a 100-kD fragment and two minor related fragments, and the second (fraction II) contained monomeric 90- and 65-kD fragments. The 90- and 65-kD fragments in fraction II were closely related to each other and were structurally and immunologically distinct from the fragments in fraction I. Only components in fraction I were recognized by mAb M1, which binds to an epitope located in the proximal portion of the arms of the hexabrachion and by a polyclonal antibody prepared against a 75-kD CNBr fragment of intact cytotactin. A mAb (1D8) and a polyclonal antibody prepared against a 35-kD CNBr fragment of cytotactin only recognized components present in fraction II. In cell- binding experiments, fibroblasts, neurons, and glia each adhered to substrates coated with fraction II, but did not adhere to substrates coated with fraction I. Fab fragments of the antibody to the 35-kD CNBr fragment strongly inhibited the binding of cells to cytotactin, supporting the conclusion that fraction II contains a cell-binding region. In addition, Fab fragments of this antibody inhibited the binding of cytotactin to CTB pr