Käpylä Jarmo, Jäälinoja Juha, Tulla Mira, Ylöstalo Joni, Nissinen Liisa, Viitasalo Tiina, Vehviläinen Piia, Marjomäki Varpu, Nykvist Petri, Säämänen Anna-Marja, Farndale Richard W, Birk David E, Ala-Kokko Leena, Heino Jyrki
Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä FI-40014, Finland.
J Biol Chem. 2004 Dec 3;279(49):51677-87. doi: 10.1074/jbc.M409412200. Epub 2004 Sep 21.
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.
IX型胶原蛋白是三股螺旋中存在间断的原纤维相关胶原蛋白。在人体软骨中，它覆盖胶原纤维，但其假定的细胞受体尚不清楚。对人类胎儿组织进行的逆转录聚合酶链反应分析表明，基于其分布情况，所有四种胶原受体整合素，即α1β1、α2β1、α10β1和α11β1，都可能是IX型胶原蛋白的受体。此外，原代软骨细胞和软骨肉瘤细胞同时表达这四种整合素。软骨肉瘤细胞以及转染后仅表达α1β1、α2β1或α10β1整合素作为其唯一胶原受体的中国仓鼠卵巢细胞，在人重组IX型胶原蛋白上显示出快速附着和铺展，表明它是一种有效的细胞粘附蛋白。为了进一步研究对IX型胶原蛋白的识别，我们在大肠杆菌中生产了重组αI结构域。对于这四个αI结构域中的每一个，IX型胶原蛋白都是最佳的胶原配体之一，与迄今为止测试的所有其他胶原亚型相比，IX型胶原蛋白具有特殊性。α1I-IX型胶原蛋白复合物和α2I-IX型胶原蛋白复合物的旋转阴影电子显微镜图像仅揭示了一个位于COL3结构域中靠近它与COL2结构域之间扭结处的结合位点。α2I对IX型胶原蛋白的识别被认为代表了一种新机制，原因有两个。第一，IX型胶原蛋白没有GFOGER基序，并且所确定的结合区域缺乏任何相似序列。第二，α2I结构域突变D219R和H258V，这两个突变都降低了与I型胶原蛋白和GFOGER的结合，但对其与IX型胶原蛋白的结合有非常不同的影响。D219R没有影响，而H258V阻止了IX型结合。因此，我们的结果表明，与其他胶原蛋白相比，IX型胶原蛋白具有独特的细胞粘附特性，并且它为细胞粘附到软骨基质提供了一种新机制。
