Thiagarajan P, Le A, Snuggs M B, VanWinkle B
Department of Medicine, University of Texas School of Medicine, Houston 77030, USA.
Cell Adhes Commun. 1996 Nov;4(4-5):317-25. doi: 10.3109/15419069609010775.
Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E. coli. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 micrograms/well compared to plasma-derived vitronectin which reached at 0.2 micrograms/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.
玻连蛋白是一种存在于内皮下基质中的主要细胞黏附分子，可介导多种细胞的黏附和铺展。玻连蛋白的羧基末端具有糖胺聚糖结合的共有序列。为了确定该结构域的功能作用，我们通过对血浆来源的玻连蛋白进行甲酸裂解，产生了缺乏糖胺聚糖结合结构域的玻连蛋白片段。此外，我们还在大肠杆菌中生成了类似的玻连蛋白重组片段作为谷胱甘肽S-转移酶融合蛋白。测试了这些片段支持人脐静脉内皮细胞黏附的能力。这些片段促进了内皮细胞的黏附，与血浆来源的玻连蛋白在0.2微克/孔时达到半数最大活性相比，它们在2-5微克/孔时达到半数最大活性。然而，黏附于这些片段的细胞并未形成结构良好的黏着斑和肌动蛋白应力纤维。此外，在改良的Boyden小室试验中，与完整的血浆来源的玻连蛋白相比，这些片段对内皮细胞迁移的趋化作用较弱。目前的研究表明，玻连蛋白的羧基末端糖胺聚糖结合结构域对于内皮细胞在玻连蛋白基质上的正确细胞骨架组织和迁移至关重要。
