Hashimoto-Uoshima M, Yan Y Z, Schneider G, Aukhil I
Department of Periodontics, University of North Carolina, Chapel Hill, NC 27599-7450, USA.
J Cell Sci. 1997 Sep;110 ( Pt 18):2271-80. doi: 10.1242/jcs.110.18.2271.
Fibronectin has a complex pattern of alternative splicing at the pre-mRNA level leading to the expression of different isoforms. The alternatively spliced domains EIIIB and EIIIA are known to be prominently expressed during development and wound healing. While the other spliced domain (CS-segment) is known to promote cell adhesion in a cell type specific manner, the biological functions of the spliced domains EIIIB and EIIIA are not well understood. In the present study, we have prepared expression proteins of specific domains of human fibronectin using a prokaryotic expression system and used the purified fragments to test their ability to support adhesion and spreading of cultured cells. Fragments from type-III domains #7 to #12 were prepared in various combinations to include or exclude the spliced domains EIIIB and EIIIA. The results indicate that cultured NIL fibroblasts adhere to many of the fragments tested. However, the cell adhesion and spreading are enhanced, especially at lower concentrations, to fragments including the domain EIIIB. The inclusion of domain EIIIA led to a decrease in the adhesion of cells and those that adhered did not spread well. When tested in a centrifugal cell adhesion assay, fragments including domain EIIIB resisted the detaching forces and stayed adhered. Fragments that included domain EIIIA were unable to resist the detaching centrifugal forces to the same extent as the fragments that included domain EIIIB alone. These results suggest that the spliced domain EIIIB may be serving important biological functions in enhancing cell adhesion and spreading. This is likely to be mediated by conformational effects because domain EIIIB alone neither exhibited any adhesive activity nor competed in inhibiting adhesion to fragments #7-10.
纤连蛋白在mRNA前体水平具有复杂的可变剪接模式，导致不同亚型的表达。已知可变剪接结构域EIIIB和EIIIA在发育和伤口愈合过程中显著表达。虽然另一个剪接结构域（CS片段）已知以细胞类型特异性方式促进细胞黏附，但剪接结构域EIIIB和EIIIA的生物学功能尚不清楚。在本研究中，我们使用原核表达系统制备了人纤连蛋白特定结构域的表达蛋白，并使用纯化的片段测试它们支持培养细胞黏附和铺展的能力。制备了来自III型结构域#7至#12的各种组合片段，以包含或排除剪接结构域EIIIB和EIIIA。结果表明，培养的NIL成纤维细胞能黏附于许多测试片段。然而，细胞黏附和铺展在较低浓度下尤其增强，对于包含结构域EIIIB的片段。包含结构域EIIIA导致细胞黏附减少，且黏附的细胞铺展不佳。在离心细胞黏附试验中测试时，包含结构域EIIIB的片段能抵抗分离力并保持黏附。包含结构域EIIIA的片段不能像仅包含结构域EIIIB的片段那样在相同程度上抵抗分离离心力。这些结果表明，剪接结构域EIIIB可能在增强细胞黏附和铺展中发挥重要生物学功能。这可能是由构象效应介导的，因为单独的结构域EIIIB既未表现出任何黏附活性，也未在抑制对片段#7 - 10的黏附中产生竞争。
