Joint study on cell adhesion molecules in development

在脊椎动物胚胎发育过程中，细胞黏附分子在发育和形态发生中起着重要的作用。我们曾报道钙粘附素介导的细胞-细胞黏附系统是青蛙胚胎发育早期唯一具有功能的细胞黏附系统。最近，我们发现这种依赖于钙离子的细胞-细胞黏附系统的功能被一种抗人B血型的单抗完全破坏。抗体的Fab片段或纯化的B血型活性物质也可破坏钙依赖的细胞黏附，而对钙粘素介导的细胞黏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B血型活性决定簇。我们确定了在胚泡细胞中发现的B血型活性五糖神经酰胺的完整结构。在钙粘附素上没有发现糖表位。本实验室对具有B血型表位的糖蛋白进行了部分纯化。通过测量两个分子之间的共振能量转移，我们发现人B血型活性决定簇在蝌蚪中与E-钙粘附素共存，在胚泡中与XB-钙粘附素共存。因此，很有可能人类B型血型决定簇与钙粘附素结合，并通过与它们形成超分子复合体来调节钙粘附素的功能。为了分析这种相互作用在胚胎发生中的本质，我们制备了XB-钙粘附素的显性负性形式，并使它们在早期胚胎发育中表达。显性负性钙粘附素的表达显著影响青蛙胚胎的形态发生和发育，B血型活性决定簇与钙粘附素的相互作用受到严重干扰。我们现在正在分析B血型活性决定因素在这些胚胎中的分布和功能。通过反义寡核苷酸敲除母体钙粘附素池也正在尝试使用新的精炼技术。

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.