Structure and function of SMOC-1 and SMOC-2, two novel calcium-binding proteins associated with basement membranes

我们已经分离出两个新的基因编码分泌的模块化蛋白含有钙结合结构域的BM-40蛋白家族的结构相关，并被称为SMOC-1和SMOC-2。在真核细胞中的重组表达表明，这两种蛋白质都是具有钙依赖性构象的糖蛋白。北方印迹、RT-PCR和免疫印迹结果表明，在许多组织中广泛表达。针对SMOC-1的抗血清的免疫荧光研究和免疫金电子显微镜显示，该蛋白质是基底膜的一个组成部分，虽然它不是一个组成性的基底膜蛋白。在卵巢中，卵泡周围的基底膜缺乏SMOC-1，此处该蛋白位于卵母细胞周围的透明膜中。我们将以与SMOC-1类似的详细方式分析SMOC-2的表达模式。此外，我们将研究这两种蛋白质在胚胎发育过程中的时空表达，并确定它们的细胞起源。重组蛋白将用于在相互作用研究中鉴定结合伴侣。将通过共定位研究验证体外相互作用的相关性。SMOC-1和SMOC-2在细胞过程中的参与将通过关于受精、细胞粘附和细胞迁移的不同体外测定来解决。在该项目的后期阶段，负责相互作用或细胞功能的结构域将通过使用重组片段进行映射。将产生并分析SMOC-1或SMOC-2缺陷的小鼠。

We have isolated two novel genes encoding secreted modular proteins containing a calcium-binding domain that are structurally related to the BM-40 protein family and were termed SMOC-1 and SMOC-2. Recombinant expression in eucaryotic cells showed that both proteins are glycoproteins with a calcium-dependent conformation. Results from northern blots, RT-PCR and immunoblots demonstrated a widespread expression in many tissues. Immunofluorescence studies and immunogold electron microscopy with an antiserum directed against SMOC-1 revealed, that the protein is an integral component of basement membranes although it is not a constitutive basement membrane protein. In the ovary, basement membranes around follicles are devoid of SMOC-1, here the protein is located in the zona pellucida surrounding the oocyte. We will analyse the expression pattern of SMOC-2 in similar detail as done for SMOC-1. In addition, we will investigate the spatio-temporal expression of both proteins during embryonal development and identify their cellular origin. Recombinant proteins will be used to identify binding partners in interaction studies. The relevance of in vitro interactions will be verified by colocalization studies. Involvement of SMOC-1 and SMOC-2 in cellular processes will be addressed by different in vitro assays concerning fertilization, cell adhesion and cell migration. At a later stage of the project, the domains responsible for interactions or cellular functions will be mapped by the use of recombinant fragments. Mice deficient in either SMOC-1 or SMOC-2 will be generated and analyzed.