Gilbert M, Shaw W J, Long J R, Nelson K, Drobny G P, Giachelli C M, Stayton P S
Department of Bioengineering and Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
J Biol Chem. 2000 May 26;275(21):16213-8. doi: 10.1074/jbc.M001773200.
Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.
细胞外基质蛋白在骨重塑过程中控制成骨细胞和破骨细胞的活性方面发挥着关键作用。这些骨特异性细胞外基质蛋白包含介导细胞黏附的氨基酸序列，并且许多骨特异性基质蛋白还含有与矿物质表面相互作用的酸性结构域，这些酸性结构域可能会使信号结构域定向。在此，我们报告一种融合肽设计，该设计基于这种在生物矿物表面展示信号肽序列的天然方法。唾液富组蛋白含有一个15个氨基酸的羟基磷灰石结合结构域（N15），该结构域在溶液中呈松散螺旋状。为了测试N15是否能够使活性肽序列在羟基磷灰石上定向，将骨桥蛋白的RGD及其侧翼残基融合到C末端。融合肽与羟基磷灰石紧密结合，并且当固定在羟基磷灰石表面时，N15 - PGRGDS肽介导了Moα(v)黑色素瘤细胞的剂量依赖性黏附。对整合素分选的Moα(v)亚群进行的实验表明，α(v)β(3)整合素是融合肽的主要受体靶点。固态核磁共振实验表明，水合融合肽的RGD部分在羟基磷灰石表面具有高度的动态性。因此，这种融合肽框架可能为将生物活性序列固定在羟基磷灰石上用于生物材料、组织工程和疫苗应用提供一种直接的设计方法。
