OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
OBJECTIVE: A novel C-terminal alpha-helix-defective mutant of humanstromal cell-derived factor-1 (SDF-1), hSDF-154, was designed and produced in order to develop an optimal CXC chemokine receptor 4 (CXCR4) antagonist. MATERIALS AND METHODS: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. RESULTS: 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. CONCLUSION: 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.
Authors: Yi Tan; Yan Li; Jian Xiao; Hongwei Shao; Chuanlin Ding; Gavin E Arteel; Keith A Webster; Jun Yan; Hong Yu; Lu Cai; Xiaokun Li Journal: Cardiovasc Res Date: 2009-02-05 Impact factor: 10.787
Authors: Mohammad Abul Kashem; Xiaoou Ren; Hongzhao Li; Binhua Liang; Lin Li; Francis Lin; Francis A Plummer; Ma Luo Journal: Front Cell Dev Biol Date: 2020-07-03