Abstract
Cecropin B (CB) is a 35-residue natural antimicrobial peptide isolated from the immune hemolymph of Hyalophora cecropia. CB shows a broad spectrum of activity against bacteria but has little cytolytic effect. CD measurements revealed that CB adopts random structure in aqueous solution but form helical conformation in polar solvent. The solution structure of CB in 20% HFIP was studied by using NMR spectroscopy. It consists of two amphipathic alpha-helices (residues 4-21 and 25-34) connected by a proline kink region (residues 22-24). In order to undertake the structural dissection of CB, CB-N27 (residues 1-27) and CB-C17 (residues 19-35) were synthesized to investigate the membrane permeabilities, fusogenic effect, and antimicrobial activities. The MIC test the two truncated form of CB both loss its antibacterial activity, suggesting the adequate peptide length of the CB is required in its antimicrobial function. CB1 was constructed by replacing the C-terminal segment with N-terminal sequence of CB, the polycationic property of CB1 make it restored the antimicrobial activity. The N-terminal polycationic and amphipathic sequence may contribute to antimicrobial activity. Furthermore CB-N27 and CB-C17 both inhibited membrane lysis, lipid mixing, and antibacterial activity. Suggesting that the optimal length of N-terminal positively charged residues and C-terminal hydrophobic segment are critical factors for its functions. The helix-hinge-helix structure of CB provides the flexibility for its two □-helices and facilitates the interactions to lipid membrane. Several natural antimicrobial peptides including cecropins, magainins and melittins have been found to kill cancer cells. However, their efficacy may not be adequate for their development as anticancer agents. In this study, we used a natural antimicrobial peptide, cecropin B (CB), as a template to generate a novel anticancer peptide. The consensus pattern of cecropins is W-x(0,2)-[KDN]-x-{L}-K-[KRE]-[LI]-E-[RKN] (PROSITE: PS00268), and this signature sequence is located at N-terminus of CB. CB1a was constructed by repeating the signature sequence of CB three times and including a hinge near C-terminus. The circular dichroism spectra show that CB1a is unstructured in aqueous solution, but adopt a helical conformation in membrane-like environment. The solution structure of CB1a in polar solvent was also studied by NMR. CB1a formed a helix-hinge-helix in 20 % HFIP solution, and the bent angle between two helical segments was ranging from 60° to 110°. A heparin-binding motif is located in the central part of helix 1. Isothermal titration calorimetry reveals the association constant of CB1a bound to low molecular weight heparin is 1.66×105 M-1 at physiological ionic strength at 25°C. Binding of CB1a to heparin produces a large conformational change toward a more structural state. CB1a demonstrated promising activity against several cancer cells with low toxicity to the non-cancer cells. The IC50 of CB1a on leukemia and stomach carcinoma cells were in the range of 2- to 8-fold lower than those of CB. Besides, CB1a displayed low hemolytic property on human red blood cell. These properties might make CB1a a good candidate for use as an anticancer agent.