Structure−Activity Relation of Human β-Defensin 3:  Influence of Disulfide Bonds and Cysteine Substitution on Antimicrobial Activity and Cytotoxicity

Abstract

Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

β‐Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved

“…This could imply that disulfide bonding is essential to the functional structure of the molecules. However, in the case of HBD-3, disulfide bonding was relevant only for its chemotactic but not its antimicrobial activities [17,33]. In keeping with these studies, we observed that a linear form of HBD-3 had lost its ability to chemoattract macrophages.…”

“…In addition, a linear form of HBD-3 was synthesized by alkylation of reduced HBD-3 with iodoacetamide [33]. In this peptide, all cysteine residues are present as S-carboxamidomethylcysteine to suppress the reactivity of the thiol groups.…”

Studies of the Biological Properties of Human β-Defensin 1

“…The most significant determinant of the anti-E. coli activity of hBD1 mutants is the net charge. The importance of basic residues for antimicrobial activity of defensins has been reported previously for bovine ␤-defensin 2 and 12 (48,51) and hBD3 (46,47). A distribution of the residues important for anti-E. coli properties of hBD1 over the molecule (surface) is illustrated in Fig.…”

“…The charged residues are not the only ones to play a role in the antimicrobial properties of defensins (43,47,54,55). In our anti-E. coli assays, mutants Y03A, N04A, V06A, S08A, and L13A are either inactive or very poorly active.…”

Through the Looking Glass, Mechanistic Insights from Enantiomeric Human Defensins

“…Clearly, native ␣-defensin structure, while dispensable in the killing of E. coli, is required for efficient killing of S. aureus, reminiscent of D HNP1 and D HD5. Due to an unusually high number of cationic charges, the bactericidal activity of HBD3 is generally insensitive to loss of or topological change in disulfide bonding (44,62,63). To extend our previous observation (44), we quantified dose-dependent killing of E. coli and S. aureus by HBD3, .…”