Direct detection of collagenous proteins by fluorescently labeled collagen mimetic peptides.
Direct detection of collagenous proteins by fluorescently labeled collagen mimetic peptides.
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DOI：
10.1021/bc3005842
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发表时间：
2013-01-16
影响因子：
4.7
通讯作者：
Yu, S. Michael
中科院分区：
文献类型：
--
作者：
Li, Yang;Ho, Daniel;Meng, Huan;Chan, Tania R.;An, Bo;Yu, Hanry;Brodsky, Barbara;Jun, Albert S.;Yu, S. Michael
尽管纤维状胶原蛋白是哺乳动物细胞外基质的主要结构成分，但胶原蛋白过量产生与许多人类疾病有关，包括癌症和纤维化。在生物医学研究中，胶原蛋白通常通过蛋白质印迹和免疫组织化学来鉴定；然而，这些分析中使用的抗胶原抗体很难制备，如果胶原蛋白在分析过程中变性，它们对胶原蛋白的亲和力可能会降低。此前，我们发现单链胶原模拟肽[CMPs，序列：(GlyProHyp)9]可以通过三螺旋杂交与变性胶原链结合。在这里，我们提出了使用与常见荧光团（例如羧基荧光素）缀合的简单 CMP 的胶原蛋白特异性染色方法，该方法可以直接检测 SDS-PAGE 和各种哺乳动物组织切片中的胶原蛋白和类胶原蛋白。通过用荧光标记的 CMP 直接对 SDS-PAGE 凝胶进行染色，可以检测到完整的（I 型、II 型和 IV 型）和 MMP-1 切割的胶原蛋白（I 型）链以及补体因子 C1q。光学可视化含有少至 5 ng 的胶原带，而纤连蛋白、层粘连蛋白和来自哺乳动物细胞裂解物的蛋白质集合没有观察到染色。 CMP 无法对胶原蛋白样细菌蛋白进行染色，该蛋白含有大量带电氨基酸，据信这些氨基酸可以代替 Hyp 稳定三螺旋。我们还表明，荧光标记的 CMP 可以比抗胶原蛋白 I 抗体更有效地特异性地显示固定组织切片（例如皮肤、角膜和骨骼）中的胶原蛋白，并且可以轻松识别纤维化肝组织中的病理状况。
Although fibrous collagens are major structural components of extracellular matrix in mammals, collagen overproduction is associated with many human diseases including cancers and fibrosis. Collagen is typically identified in biomedical research by western blot and immunohistochemistry; however anti-collagen antibodies employed in these analyses are difficult to prepare and their affinities to collagen can diminish if collagen becomes denatured during analyses. Previously, we discovered that single-stranded collagen mimetic peptides [CMPs, sequence: (GlyProHyp)9] can bind to denatured collagen chains by triple helix hybridization. Here we present collagen-specific staining methods using simple CMPs conjugated to common fluorophores (e.g. carboxyfluorescein), which allow direct detection of collagens and collagen-like proteins in SDS-PAGE and in various mammalian tissue sections. By directly staining SDS-PAGE gels with fluorescently labeled CMPs, both intact (type I, II, and IV) and MMP-1 cleaved collagen (type I) chains as well as complement factor C1q were detected. Collagen bands containing as little as 5 ng were optically visualized while no staining was observed for fibronectin, laminin, and a collection of proteins from mammalian cell lysate. The CMP was unable to stain collagen-like bacterial protein which contains numerous charged amino acids that are believed to stabilize triple helix in place of Hyp. We also show that fluorescently labeled CMPs can specifically visualize collagens in fixed tissue sections (e.g., skin, cornea, and bone) more effectively than anti-collagen I antibody, and allow facile identification of pathologic conditions in fibrotic liver tissues.
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