Joint study on cell adhesion molecules in development

在脊椎动物胚胎发生中，细胞粘附分子在发育和形态发生中起着重要作用。我们以前报道过钙粘蛋白介导的Ca^<2+>依赖的细胞-细胞粘附系统是唯一在蛙早期胚胎发育中起作用的细胞粘附系统。最近，我们发现这种依赖Ca^<2+>的细胞间粘附系统的功能被抗人血型B的单克隆抗体完全破坏。抗体的Fab B片段或纯化的血型B活性物质也破坏了Ca^2+依赖的细胞粘附，并且钙粘蛋白介导的细胞粘附的抑制不是由于抗体引起的空间位阻。在糖脂和糖蛋白上检测到血型B活性决定簇。我们确定了囊胚细胞中发现的血型B活性五糖基神经酰胺的完整结构。钙粘蛋白上未发现糖表位。本实验室部分纯化了具有B血型表位的糖蛋白。通过测量两种分子之间的共振能量转移，我们发现，人类血型B活性决定簇与蝌蚪中的E-钙粘蛋白和囊胚中的XB-钙粘蛋白共定位。因此，人血型B决定簇很可能与钙粘蛋白结合，并通过与钙粘蛋白形成超分子复合物来调节钙粘蛋白的功能。为了分析胚胎发生中这种相互作用的性质，我们制备了显性负性形式的XB-钙粘蛋白，并使其在早期胚胎发生中表达。显性负性钙粘蛋白的表达显著影响蛙胚胎的形态发生和发育，严重破坏了B血型活性决定簇与钙粘蛋白的相互作用。我们现在正在分析这些胚胎中血型B活性决定簇的分布和功能。通过使用新改进的技术，也正在尝试用反义寡核苷酸敲除母体钙粘蛋白库。

In vertebrate embryogenesis, cell adhesion molecules play fundamental roles in development and morphogenesis. We previously reported that cadherin mediated Ca^<2+> dependent cell-cell adhesion system is the only cell adhesion system functional in early frog embryogenesis. Recently, we found that the function of this Ca^<2+> dependent cell-cell adhesion system is completely disrupted by an antihuman blood group B monoclonal antibody. Fab fragments of the antibody or purified blood group B active substances also disrupted Ca^<2+> dependent cell adhesion, and the inhibition of cadherin-mediated cell adhesion is not due to the steric hindrance caused by the antibody. The blood group B active determinant was detected on glycolipids and on glycoproteins. We determined the complete structure of the blood group B active pentaglycosylceramides found in blastula cells. The glycoepitoe was not found on cadherins. The glycoproteins with blood group B epitopes were partially purified in our laboratory. By measuring resonance energy transfer between two molecules, we showed that the human blood group B active determinants colocalize with E-cadherins in tadpoles, and with XB-cadherins in blastulae. Thus, it is highly possible that human blood type B determinant associates with cadherin and is regulating cadherin functions by forming supramolecular complex with them. To analyze the nature of this interaction in embryogenesis, we prepared dominant negative forms of XB-cadherins and made them expressed in early embryogenesis. The morphogenesis and development of the frog embryos were markedly affected by expressing dominant negative cadherins, and the interactions of blood group B active determinant and cadherins were severely disrupted. We are now analyzing the distribution and function of blood group B active determinants in these embryos. Knocking out of the maternal pool of cadherins by antisense oligonucleotides is also being tried by using newly refined technique.