Tan Yi, Du Jun, Cai Shaoxi, Li Xiaokun, Ma Weifeng, Guo Zhigang, Chen Hongyuan, Huang Zhifeng, Xiao Jian, Cai Lu, Cai Shaohui
Department of Clinical Pharmacology, Pharmacy School of Jinan University, Guangzhou, China.
Exp Hematol. 2006 Nov;34(11):1553-62. doi: 10.1016/j.exphem.2006.07.001.
A novel C-terminal alpha-helix-defective mutant of human stromal cell-derived factor-1 (SDF-1), hSDF-154, was designed and produced in order to develop an optimal CXC chemokine receptor 4 (CXCR4) antagonist.
Human native SDF-1 and alpha-helix defective SDF-1 (hSDF-154) were cloned from human bone marrow stromal cells by reverse transcription polymerase chain reaction, inserted into vector pET-30a(+), and transformed into Escherichia coli strain BL21(DE3). The recombinant hSDF-154 was purified and refolded under optimized conditions and its functional characteristics were compared with the native form of SDF-1. Functional evaluation includes migration of Jurkat and MOLT4 cells assessed by chemotaxis assay, intracellular calcium influx in these cells measured by flow cytometry, extracellular signal-regulated kinase (ERK) phosphorylation analyzed by Western blot assay, receptor binding affinity examined by sequential concentrations of unlabeled SDF-1alpha, hSDF-154 competition with (125)I- SDF-1alpha, and internalization of CXCR4 on the cell surface detected by flow cytometry.
hSDF-154 had significantly decreased chemotaxic ability, such as cell migration, as compared to the native hSDF-1. hSDF-154 failed to trigger CXCR4 to induce transient calcium influx and ERK phosphorylation. However, both hSDF-154 and the native hSDF-1 have similar binding affinity to CXCR4 and a similar ability to induce CXCR4 internalization.
These results indicate that hSDF-154, which has a defective C-terminal alpha-helix, a normal N-terminus, and a normal central beta-strand scaffold structure, retains normal binding affinity to CXCR4 and normal induction of CXCR4 internalization, but fails to activate CXCR4-mediated cellular signaling and chemotaxis. Therefore, the C-terminal alpha-helix of hSDF-1 plays a critical role for CXCR4 stimulation. The hSDF-154, which efficiently binds to and induces internalization of CXCR4 without activating CXCR4-related intracellular signaling and cell migration, may serve as an optimal CXCR4 antagonist.
设计并制备一种新型的人基质细胞衍生因子-1（SDF-1）的C末端α螺旋缺陷突变体hSDF-154，以开发一种最佳的CXC趋化因子受体4（CXCR4）拮抗剂。
通过逆转录聚合酶链反应从人骨髓基质细胞中克隆人天然SDF-1和α螺旋缺陷型SDF-1（hSDF-154），将其插入载体pET-30a(+)，并转化到大肠杆菌BL21(DE3)菌株中。重组hSDF-154在优化条件下进行纯化和复性，并将其功能特性与天然形式的SDF-1进行比较。功能评估包括通过趋化性测定评估Jurkat和MOLT4细胞的迁移，通过流式细胞术测量这些细胞中的细胞内钙流入，通过蛋白质印迹分析分析细胞外信号调节激酶（ERK）磷酸化，通过未标记的SDF-1α的系列浓度检测受体结合亲和力，hSDF-154与(125)I-SDF-1α的竞争，以及通过流式细胞术检测细胞表面CXCR4的内化。
与天然hSDF-1相比，hSDF-154的趋化能力显著降低，如细胞迁移。hSDF-154未能触发CXCR4诱导瞬时钙流入和ERK磷酸化。然而，hSDF-154和天然hSDF-1对CXCR4具有相似的结合亲和力和相似的诱导CXCR4内化的能力。
这些结果表明，hSDF-154具有缺陷的C末端α螺旋、正常的N末端和正常的中央β链支架结构，对CXCR4保留正常的结合亲和力和正常的CXCR4内化诱导能力，但未能激活CXCR4介导的细胞信号传导和趋化作用。因此，hSDF-1的C末端α螺旋对CXCR4刺激起关键作用。hSDF-154能有效结合并诱导CXCR4内化，而不激活CXCR4相关的细胞内信号传导和细胞迁移，可能作为一种最佳的CXCR4拮抗剂。
