The C‐terminal Ca2+‐binding domain of SPARC confers anti‐spreading activity to human urothelial cells
The C‐terminal Ca2+‐binding domain of SPARC confers anti‐spreading activity to human urothelial cells
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DOI：
10.1002/jcp.20462
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发表时间：
2006-01
影响因子：
5.6
通讯作者：
Catherine F. Delostrinos-;A. E. Hudson;W. C. Feng;Jeffrey Kosman;J. Bassuk
中科院分区：
文献类型：
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作者：
Catherine F. Delostrinos-;A. E. Hudson;W. C. Feng;Jeffrey Kosman;J. Bassuk
富含半胱氨酸（Cys）的酸性分泌蛋白的抗扩散活性已被分配给C末端第三结构域，一个富含α螺旋的区域。这种“细胞外钙结合”（EC）结构域包含两个EF-手，每个手与一个Ca 2+离子配位，形成螺旋-环-螺旋结构，不仅驱动蛋白质的构象，而且是生物活性所必需的。重组（r）EC，在E.在C末端融合His六聚体，并在变性条件下通过镍螯合物亲和色谱法分离。通过同时（i）去除变性条件、（ii）催化二硫键异构化和（iii）启动Ca 2+依赖性重折叠的程序使rEC-His复性。固有色氨酸荧光和圆二色光谱表明，rEC-His表现出与已知晶体结构一致的Ca 2+依赖性构象。铺展试验证实，rEC-His通过其抑制从移行上皮增殖的新鲜平板培养的人尿路上皮细胞铺展的能力而具有生物活性。当作为浓度或时间的函数测量时，rEC-His和rEC-His表现出高度相似的抗扩散活性。与野生型和EC重组蛋白相比，EF-hand 2的Z位置的点取代突变体rfl（E268 F）-His未能表现出α螺旋二级结构的Ca 2+依赖性变化和抗扩散活性。收集的数据提供了证据，证明负责抗扩散活性的EAF基序依赖于EF-hand 2的Z位置处的Glu残基与Ca 2+的配位，并提供了关于粘附力如何在尿路上皮细胞的细胞外基质内平衡的见解。© 2005 Wiley利斯公司
The anti‐spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C‐terminal third domain, a region rich in α‐helices. This “extracellular calcium‐binding” (EC) domain contains two EF‐hands that each coordinates one Ca2+ ion, forming a helix‐loop‐helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C‐terminus to a His hexamer and isolated under denaturing conditions by nickel‐chelate affinity chromatography. rEC‐His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+‐dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC‐His exhibited a Ca2+‐dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC‐His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC‐His and rSPARC‐His exhibited highly similar anti‐spreading activities when measured as a function of concentration or time. In contrast to the wild‐type and EC recombinant proteins, rSPARC(E268F)‐His, a point substitution mutant at the Z position of EF‐hand 2, failed to exhibit both Ca2+‐dependent changes in α‐helical secondary structure and anti‐spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti‐spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF‐hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. © 2005 Wiley‐Liss, Inc.