Nykvist P, Tasanen K, Viitasalo T, Kapyla J, Jokinen J, Bruckner-Tuderman L, Heino J
Department of Biological and Environmental Science, University of Jyväskylä, FIN-40351 Jyväskylä, Finland.
J Biol Chem. 2001 Oct 19;276(42):38673-9. doi: 10.1074/jbc.M102589200. Epub 2001 Aug 20.
Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.
XVII型胶原蛋白（BP180）是一种角质形成细胞跨膜蛋白，在半桥粒中以全长蛋白形式存在，在细胞外基质中则以120 kDa的脱落胞外结构域形式存在。XVII型胶原蛋白最大的胶原结构域COL15，先前已被描述为细胞粘附结构域（塔萨宁，K.，埃布尔，J. A.，奥马利，M.，舒曼，H.，贝奇，J，屠，H.，布鲁克纳，P.，和布鲁克纳 - 图德曼，L.（2000年）《生物化学杂志》275卷，3093 - 3099页）。在本研究中，对三螺旋人重组COL15的整合素结合进行了测试。使用重组整合素α(1)I、α(2)I和α(10)I结构域的固相结合试验，以及用表达α(1)β(1)和α(2)β(1)的中国仓鼠卵巢细胞进行的细胞铺展试验表明，与其他胶原蛋白不同，COL15不被胶原蛋白受体识别。COL15结构域的变性增加了人HaCaT角质形成细胞的铺展，这些细胞在变性的COL15结构域上迁移的效率与在纤连蛋白上一样高。HaCaT细胞在COL15结构域上的铺展由α(5)β(1)和α(V)β(1)整合素介导，并且可以被RGD肽阻断。COL15结构域中的胶原α链不包含RGD基序，而是包含12个紧密相关的KGD基序，在三条α链中的每条链上各有四个。测试了与整个COL15结构域对应的22个重叠合成肽；三个都包含KGD基序的肽抑制了HaCaT细胞在变性COL15结构域上的铺展。此外，这种效应因从D突变为E（KGE而非KGD）而丧失。我们认为，XVII型胶原蛋白的COL15结构域代表一种特定的胶原结构，无法与其他胶原蛋白的细胞受体相互作用。从细胞表面脱落之后，它可能支持角质形成细胞的铺展和迁移。
