Cloning and characterizing mutated human stromal cell-derived factor-1 (SDF-1): C-terminal α-helix of SDF-1α plays a critical role in CXCR4 activation and signaling, but not in CXCR4 binding affinity

Tan, Yi; Du, Jun; Cai, Shaoxi; Li, Xiaokun; Ma, Weifeng; Guo, Zhigang; Chen, Hongyuan; Huang, Zhifeng; Xiao, Jian; Cai, Lu; Cai, Shaohui

doi:10.1016/j.exphem.2006.07.001

« Previous Next »Experimental Hematology
Volume 34, Issue 11 , Pages 1553-1562, November 2006

Cloning and characterizing mutated human stromal cell-derived factor-1 (SDF-1): C-terminal α-helix of SDF-1α plays a critical role in CXCR4 activation and signaling, but not in CXCR4 binding affinity

Yi Tan
- Department of Clinical Pharmacology, Pharmacy School of Jinan University, Guangzhou, China
- College of Bioengineering, Key Laboratory for Biomechanics and Tissue Engineering of the State Ministry of Education, Chongqing University, Chongqing, China
- Bioengineering Medicine Research Center of the State Ministry of Education, Jinan University, Guangzhou, China
- School of Pharmaceutical Sciences, Wenzhou Medical College, Wenzhou, China
Jun Du
- School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
Shaoxi Cai
- College of Bioengineering, Key Laboratory for Biomechanics and Tissue Engineering of the State Ministry of Education, Chongqing University, Chongqing, China
Xiaokun Li
- Bioengineering Medicine Research Center of the State Ministry of Education, Jinan University, Guangzhou, China
- School of Pharmaceutical Sciences, Wenzhou Medical College, Wenzhou, China
Weifeng Ma
- College of Bioengineering, Key Laboratory for Biomechanics and Tissue Engineering of the State Ministry of Education, Chongqing University, Chongqing, China
Zhigang Guo
- College of Bioengineering, Key Laboratory for Biomechanics and Tissue Engineering of the State Ministry of Education, Chongqing University, Chongqing, China
Hongyuan Chen
- College of Bioengineering, Key Laboratory for Biomechanics and Tissue Engineering of the State Ministry of Education, Chongqing University, Chongqing, China
Zhifeng Huang
- School of Pharmaceutical Sciences, Wenzhou Medical College, Wenzhou, China
Jian Xiao
- Department of Clinical Pharmacology, Pharmacy School of Jinan University, Guangzhou, China
- School of Pharmaceutical Sciences, Wenzhou Medical College, Wenzhou, China
Lu Cai
- School of Pharmaceutical Sciences, Wenzhou Medical College, Wenzhou, China
- Departments of Medicine, Radiation Oncology and Pharmacology and Toxicology, University of Louisville, Ky., USA
- Offprint requests to: Lu Cai, M.D., Ph.D., Departments of Medicine, Radiation Oncology and Pharmacology and Toxicology, University of Louisville, 511 South Floyd Street, MDR 533, Louisville, KY 40202
Shaohui Cai
- Department of Clinical Pharmacology, Pharmacy School of Jinan University, Guangzhou, China
- Offprint requests to: Shaohui Cai, M.D., Ph.D., Departments of Medicine, Radiation Oncology and Pharmacology and Toxicology, University of Louisville, 511 South Floyd Street, MDR 533, Louisville, KY 40202

Received 9 June 2006; received in revised form 28 June 2006; accepted 10 July 2006.

Objective

A novel C-terminal α-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 α-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-1α, hSDF-154 competition with ¹²⁵I- SDF-1α, 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 α-helix, a normal N-terminus, and a normal central β-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 α-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.