Julier Ziad, Martino Mikaël M, de Titta Alexandre, Jeanbart Laura, Hubbell Jeffrey A
Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.
1] Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland [2] World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
Sci Rep. 2015 Feb 24;5:8569. doi: 10.1038/srep08569.
Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8(+) T cell responses in two murine cancer models.
纤连蛋白（FN）是一种细胞外基质（ECM）蛋白，包含许多具有不同功能的III型纤连蛋白（FNIII）重复序列。含有额外A结构域（FNIII EDA）的可变剪接FN变体位于FNIII 11和FNIII 12之间，在损伤部位、慢性炎症部位和实体瘤中表达。尽管其功能尚不完全清楚，但已知FNIII EDA可作用于Toll样受体4（TLR4）。在这里，通过产生各种含有FNIII EDA的FN片段，我们发现FNIII EDA的免疫活性取决于其在FN链内的局部分子内环境。将分离的FNIII EDA与其相邻的FNIII重复序列（FNIII 9 - 10 - 11）进行N端延伸增强了其作用于TLR4的活性，而与天然的FNIII 12 - 13 - 14肝素结合结构域进行C端延伸则消除了该活性。此外，我们发现弹性蛋白酶2切割位点存在于FNIII EDA和FNIII 12之间。在用弹性蛋白酶2切割FNIII 12 - 13 - 14结构域后，C端延伸的FNIII EDA的活性可以恢复。由于FN天然结合于ECM，我们将含有FNIII EDA的FN片段与抗原肽一起固定在纤维蛋白基质模型中。在两种小鼠癌症模型中，这种基质显示出可刺激细胞毒性CD8(+) T细胞反应。
