title: Cloning and characterizing mutated human stromal cell-derived factor-1 (SDF-1): C-terminal alpha-helix of SDF-1alpha plays a critical role in CXCR4 activation and signaling, but not in CXCR4 binding affinity.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.affiliation: Department of Clinical Pharmacology, Pharmacy School of Jinan University, Guangzhou, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tan
authors.forename: Jun
authors.initials: J
authors.lastname: Du
authors.forename: Shaoxi
authors.initials: S
authors.lastname: Cai
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
authors.forename: Weifeng
authors.initials: W
authors.lastname: Ma
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Guo
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Chen
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Huang
authors.forename: Jian
authors.initials: J
authors.lastname: Xiao
authors.forename: Lu
authors.initials: L
authors.lastname: Cai
authors.forename: Shaohui
authors.initials: S
authors.lastname: Cai
mesh_terms: D002460:Cell Line; D054377:Chemokine CXCL12 / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D006801:Humans; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000502:physiology; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D017154:Stromal Cells / Q000378:metabolism
chemical_list: C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019718:Receptors, CXCR4; D011994:Recombinant Proteins