Ağbaş A, Sarras M P
Department of Anatomy & Cell Biology, University of Kansas Medical Center, Kansas City 66160.
Cell Adhes Commun. 1994 Apr;2(1):59-73. doi: 10.3109/15419069409014202.
The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [3H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreening studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-beta 1 integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified tow major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of > 200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian.
本研究旨在鉴定普通水螅潜在的细胞表面细胞外基质结合蛋白并对其进行功能表征。使用[3H] - 层粘连蛋白作为探针，对解离的混合水螅细胞制剂进行放射受体分析表明，每个细胞的层粘连蛋白结合位点平均数量约为10,000个，解离常数为1.49 nM。这些结合位点可以被未标记的层粘连蛋白以剂量依赖的方式取代，也可以被高浓度（500 nM）的未标记纤连蛋白取代。未观察到IV型胶原和I型胶原的取代作用。用一系列针对哺乳动物细胞外基质（ECM）结合蛋白产生的抗体进行免疫筛选研究表明，抗β1整合素单克隆抗体mAb JG22存在潜在的细胞表面结合位点。细胞黏附研究表明，mAb JG22阻断了水螅细胞与层粘连蛋白的结合，但不影响它们与纤连蛋白、IV型胶原或I型胶原的结合。光镜和电镜免疫细胞化学研究表明，mAb JG22定位于外胚层和内胚层上皮细胞的基底质膜。免疫沉淀研究在还原条件下鉴定出两条主要条带，质量约为196 kDa和150 kDa，在非还原条件下鉴定出两条质量> 200 kDa的条带。功能研究表明，mAb JG22可以可逆地阻断水螅细胞聚集体的形态发生，并可以阻断水螅移植中体内间质细胞的迁移。这些观察结果表明，水螅具有细胞表面ECM成分结合位点，在这种简单的刺胞动物发育过程中具有重要的功能。
