From the Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155 and.
J Biol Chem. 2014 Feb 21;289(8):4941-51. doi: 10.1074/jbc.M113.530808. Epub 2013 Dec 29.
Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.
胶原蛋白与纤连蛋白的相互作用对于细胞外基质的组装和细胞过程的调节非常重要。使用展开的片段鉴定了胶原蛋白中的纤连蛋白结合区，但不清楚天然蛋白是否以相同的原始序列与纤连蛋白结合。利用重组细菌胶原蛋白来表征纤连蛋白结合的序列要求。通过将人胶原蛋白中的假定纤连蛋白结合区插入细菌胶原蛋白序列中来生成嵌合胶原蛋白。插入足够长度的人序列赋予了纤连蛋白亲和力。最小序列要求被确定为靠近独特胶原酶切割位点的 6 个三联体序列，在三螺旋和变性状态下是相同的。嵌合胶原蛋白的变性增加了其与纤连蛋白的亲和力，就像哺乳动物胶原蛋白一样。结合纤连蛋白的重组胶原蛋白不含羟脯氨酸，表明羟脯氨酸不是结合所必需的。然而，与 II 型胶原蛋白相比，其缺失可能部分解释了天然嵌合蛋白的高亲和力和变性蛋白的低亲和力。在具有纤连蛋白亲和力的嵌合胶原蛋白上培养的巨核细胞显示出改善的黏附和分化，这表明在生物医学应用中生成生物活性材料的一种策略。
