Oulu Centre for Cell-Matrix Research, Department of Medical Biochemistry and Molecular Biology, Institute of Biomedicine, University of Oulu, 90014 Oulu, Finland.
J Biol Chem. 2010 Feb 19;285(8):5258-65. doi: 10.1074/jbc.M109.033787. Epub 2009 Dec 29.
The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.
胶原 XV 的 C 端末端，restin，一直是几项研究的焦点，但全长胶原 XV 的功能仍然未知。我们在此描述了胶原 XV 的生产、纯化和功能的研究，以及对其 N 端单克隆抗体的生产。全长人胶原 XV 使用杆状病毒在昆虫细胞中生产，并从细胞培养液中纯化。产量为 15 毫克/升细胞培养液。胶原 XV 被证明是三聚体，其胶原区有二硫键。旋转阴影电子显微镜显示出具有 241.8nm 平均长度的棒状分子，并且在一端具有球形结构域。通过 N 端抗体结合证实了球形结构域是 N 端。这些分子在构象上表现出灵活性，可能是由于其胶原区的许多中断。在细胞黏附测定中测试了胶原 XV 作为细胞底物的能力，结果表明细胞不与胶原 XV 涂层表面结合。然而，当添加到成纤维细胞和纤维肉瘤细胞的培养基中时，胶原 XV 迅速结合到它们的纤维连接蛋白网络上。固相测定表明胶原 XV 结合纤维连接蛋白、层粘连蛋白和 vitronectin，并且结合纤维连接蛋白的胶原/明胶结合结构域。未检测到与原纤维胶原、纤维相关胶原或 decorin 的结合。有趣的是，当存在于含有纤维连接蛋白的基质中时，胶原 XV 被发现抑制纤维肉瘤细胞的黏附和迁移。
