Interaction of Recombinant Myocilin with the Matricellular Protein SPARC: Functional Implications
Interaction of Recombinant Myocilin with the Matricellular Protein SPARC: Functional Implications
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DOI：
10.1167/iovs.09-4866
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发表时间：
2011-01-01
影响因子：
4.4
通讯作者：
Escribano, Julio
中科院分区：
文献类型：
--
作者：
Aroca-Aguilar, Jose-Daniel;Sanchez-Sanchez, Francisco;Escribano, Julio
目的。肌素是一种功能未知的细胞外糖蛋白，与青光眼有关。Calpain II在蛋白的连接区域内切割重组心肌蛋白，将c端olfactomedin结构域从n端结构域释放出来。作者先前报道过，心肌蛋白与hevin的c端区相互作用，hevin是一种属于SPARC家族的基质细胞蛋白。本研究旨在探讨心肌素与sparc的相互作用。利用酵母双杂交系统评价蛋白-蛋白相互作用。通过镍螯合高效液相色谱法纯化重组蛋白和重组蛋白在HEK-293T细胞中的共表达，证实了这种正相互作用。通过免疫荧光显微镜、Western blot和基于阵列的基因分析鉴定了眼组织中心肌蛋白、SPARC和hevin的共表达。酵母双杂交分析表明，心肌蛋白与SPARC和hevin中高度保守的c端胞外钙结合（EC）结构域相互作用。固相结合实验证实了这些相互作用，并表明心肌素及其c端嗅素片段与SPARC和含有SPARC EC结构域的肽非共价相互作用。全长心肌与SPARC及其EC结构域的相互作用比心肌c端片段具有更高的亲和力。两种重组蛋白在HEK-293T细胞中的共表达也表明了它们在细胞内的相互作用。重组心肌蛋白和SPARC通过它们的c端结构域相互作用。这些数据表明，心肌蛋白的蛋白水解过程调节了这种相互作用，以及心肌蛋白与其他细胞外基质和基质细胞蛋白的相互作用，进一步支持了这种蛋白水解裂解的功能作用。（中国眼科杂志，2011;52:179-189）DOI: 10.1167/iovs.09-4866
PURPOSE. Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC.METHODS. Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immuno-florescence microscopy, Western blot, and array-based gene profiling.RESULTS. Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction.CONCLUSIONS. Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage. (Invest Ophthalmol Vis Sci. 2011; 52: 179-189) DOI: 10.1167/iovs.09-4866