Kobayashi Naoyuki, Kostka Günter, Garbe Jörg H O, Keene Douglas R, Bächinger Hans Peter, Hanisch Franz-Georg, Markova Dessislava, Tsuda Takeshi, Timpl Rupert, Chu Mon-Li, Sasaki Takako
Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
J Biol Chem. 2007 Apr 20;282(16):11805-16. doi: 10.1074/jbc.M611029200. Epub 2007 Feb 26.
Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of approximately 20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins.
纤连蛋白是一族由五个细胞外基质蛋白组成的蛋白家族，其特征在于具有串联排列的表皮生长因子样结构域和一个C末端纤连蛋白型模块。它们广泛分布，并且常与脉管系统和弹性组织相关联。在本研究中，我们在哺乳动物细胞中表达了最近鉴定出的三个家族成员，即纤连蛋白-3、纤连蛋白-4和纤连蛋白-5，作为重组蛋白。经过旋转投影和电子显微镜观察，纯化后的蛋白呈现出约20纳米的短杆状结构，一端有一个小球体。我们纯化出了两种形式的小鼠纤连蛋白-3，并对较大形式的O-聚糖谱进行了表征。针对纯化蛋白产生的多克隆抗体与其他家族成员没有任何交叉反应，用于评估小鼠组织中纤连蛋白的水平和定位。我们将它们的结合相互作用、细胞黏附特性和组织定位与之前已表征的纤连蛋白-1和-2进行了平行分析。纤连蛋白-2和-5与原弹性蛋白的结合很强，纤连蛋白-4和-1的结合适中，而纤连蛋白-3的结合相对较弱。纤连蛋白-4与IV型胶原和巢蛋白-2表现出明显的相互作用，与XV型胶原的内皮抑素结构域有适度结合，而纤连蛋白-3和-5则没有。在测试的各种细胞系中，除了小鼠纤连蛋白-2外，所有纤连蛋白均未观察到细胞黏附活性。在软骨膜和肺的各个区域均发现了所有五种纤连蛋白。免疫电子显微镜将纤连蛋白-4和-5定位在原纤维微原纤维的不同位置。我们的研究表明，这些结构相关的蛋白具有独特和冗余的功能。
