Syed Shabih-e-Hassnain, Trinnaman Brian, Martin Stephen, Major Sarah, Hutchinson Jon, Magee Anthony I
Division of Membrane Biology, The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Biochem J. 2002 Mar 1;362(Pt 2):317-27. doi: 10.1042/0264-6021:3620317.
Desmocollins (Dscs) and desmogleins (Dsgs) are cell-adhesion molecules involved in the formation of desmosome cell-cell junctions and share structural similarities to classical cadherins such as E-cadherin. In order to identify and provide quantitative information on the types of protein-protein interactions displayed by the type 2 isoforms and investigate the role of Ca(2+) in this process, we have developed an Escherichia coli expression system to generate recombinant proteins containing the first two extracellular domains, namely Dsg2(1-2) and Dsc2(1-2). Analytical ultracentrifugation, chemical cross-linking, CD, fluorescence and BIAcore have been used to provide the first direct evidence of Ca(2+) binding to desmosomal cadherins. These studies suggest that Dsc2(1-2) not only exhibits homophilic interactions in solution, but can also form heterophilic interactions with Dsg2(1-2). The latter, on the other hand, shows much weaker homophilic association. Our results further demonstrate that heterophilic interactions are Ca(2+)-dependent, whereas the Ca(2+)-dependence of homophilic association is less clear. Our data indicate that the functional properties of Dsc2(1-2) are more similar to those of classical cadherins, consistent with the observation that Dsc shares a higher level of sequence homology with classical cadherins than does Dsg. In addition to corroborating the conclusions of previously reported transfection studies which suggest the formation of lateral heterodimers and homodimers, our results also provide direct quantitative information on the strength of these interactions which are essential for understanding the adhesion mechanism.
桥粒芯蛋白(Dscs)和桥粒芯胶粘蛋白(Dsgs)是参与桥粒细胞间连接形成的细胞粘附分子,与经典钙粘蛋白如E-钙粘蛋白具有结构相似性。为了鉴定2型异构体所展示的蛋白质-蛋白质相互作用类型并提供定量信息,并研究Ca(2+)在此过程中的作用,我们开发了一种大肠杆菌表达系统,以产生包含前两个细胞外结构域的重组蛋白,即Dsg2(1-2)和Dsc2(1-2)。分析超速离心、化学交联、圆二色光谱、荧光和生物传感器技术已被用于提供Ca(2+)与桥粒钙粘蛋白结合的首个直接证据。这些研究表明,Dsc2(1-2)不仅在溶液中表现出嗜同性相互作用,还能与Dsg2(1-2)形成嗜异性相互作用。另一方面,后者的嗜同性结合要弱得多。我们的结果进一步证明,嗜异性相互作用依赖于Ca(2+),而嗜同性结合的Ca(2+)依赖性则不太明确。我们的数据表明,Dsc2(1-2)的功能特性与经典钙粘蛋白更相似,这与观察到的Dsc与经典钙粘蛋白的序列同源性水平高于Dsg一致。除了证实先前报道的转染研究的结论,即提示形成侧向异二聚体和同二聚体之外,我们的结果还提供了关于这些相互作用强度的直接定量信息,这对于理解粘附机制至关重要。