Screening and Mechanism of Antagonist Peptide for CC Chemokine Receptor 1 (CCR1) Derived from Viral Macrophage Inflammatory Protein II
Purpose: To search for effective antagonist peptide of CC chemokine receptor 1 (CCR1), and evaluate the potential role and mechanism of peptide C18P derived from viral macrophage inflammatory protein II (vMIP-II).
Methods: Alignment, simulated peptide-cut, bioinformatics and protease digestion were used to screen and prepare antagonist peptide. Interactions between C18P and CCR1 were determined by radioligand binding assays and [35S]GTPγS binding experiment. Chemotaxis assay was utilized to assess the potency for inducing or inhibiting peripheral blood mononuclear cells (PBMCs) migration. Ligandinduced intracellular calcium mobilization was tested by flow cytometry.
Results: A peptide containing 18 amino acids (C18P) was screened. C18P bound to CCR1 with a Kd of 5.7 ng/ml and displaced 125I-labeled MIP-1α and 125I-labeled RANTES on human CCR1-transfected HEK293 cells (IC50 = 11.2 and 9.6 ng/ml, respectively) in radioligand binding studies. C18P lacked intrinsic agonist activity but strongly inhibited HCC-1 (100 nM) induced [35S]GTPγS binding (IC50 = 3.7 ug/ml), chemotaxis induced by HCC-1, MIP-1α and RANTES (IC50 = 23, 25 and 13.1 ng/ml, respectively), and intracellular calcium mobilization.
Conclusion: These results demonstrate that bioinformatics and protease digestion are feasible to screen and prepare C18P, and that C18P is a novel and specific small molecule peptide antagonist of CCR1 with therapeutic potential for preventing cell migration.
Keywords: CC Chemokine receptor 1; Simulated peptide-cut; Antagonist peptide; Viral macrophage inflammatory protein II; Bioinformatics; Protease digestion; HEK293 cells; Radioligand binding
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