Super molecular association of laminin-8 creating contact structure between capillary endothelial cells and adipocytes.

层粘连蛋白α4链是由内皮细胞和脂肪细胞合成的层粘连蛋白8（α4β1γ1）的组成部分。为了研究层粘连蛋白-8在血管生成和脂肪形成中的作用，我们将重点放在α4链的G结构域上，发现在形成G结构域的LG 1-LG 5亚结构域中，LG 4亚结构域与肝素的亲和力最高。为了确定肝素结合的功能氨基酸，我们在大肠杆菌中构建了α 4LG 4与谷胱甘肽S-转移酶的融合蛋白。coli，并在19个碱性氨基酸（K，R，H）处引入定点突变。在α 4LG 4的三维结构模型上定位功能性碱性氨基酸，发现肝素结合表面位于钙结合位点的对侧。我们还发现，添加重组α 4LG 1-LG 5对皮下注射Matrigel和碱性成纤维细胞生长因子诱导的小鼠从头脂肪形成具有抑制作用。α 4LG 4 -5和α 4LG 1-LG 3的抑制作用强于α 4LG 1-LG 5，提示层粘连蛋白-8 α4链G结构域可能在LG 3和LG 4交界处被内皮细胞和前脂肪细胞分泌后断裂，通过内皮细胞和前脂肪细胞之间的相互作用支持脂肪形成。

Laminin α4 chain is a component of laminin-8 (α4β1γ1) synthesized by endothelial cells and adipocytes. In order to study the function of laminin-8 during angiogenesis and adipogenesis, we focused on the G domain of α4 chain and found that the subdomain LG4 has highest affinity to heparin among LG1-LG5 subdomain forming the G domain. In order to specify the functional amino acid for the heparin-binding, we produced a fusion protein of α4LG4 with glutathione S-transferase in E. coli and introduced site-directed mutagenesis at 19 basic amino acids (K, R, H). Mapping of the functional basic amino acids on a 3D structure model of α4LG4 showed the heparin-binding surface locating at opposite side of the calcium-binding site. We also found that addition of recombinant α4LG1-LG5 is inhibitory to the de novo adipogenesis induced by subcutaneous injection of Matrigel and basic fibroblast growth factor into mice. α4LG4-5 and α4LG1-LG3 showed stronger inhibition than α4LG1-LG5, suggesting that the G domain of α4 chain in laminin-8 may be cleaved after secretion from endothelial cells and preadipocytes at the boundary between LG3 and LG4 for its function of supporting the adipogenesis by the interaction between endothelial cells and preadipocytes.