Dataset Information

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.

ABSTRACT: Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.

SUBMITTER: Wu Z

PROVIDER: S-EPMC166407 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.

Wu Zhibin Z Hoover David M DM Yang De D Boulègue Cyril C Santamaria Fanny F Oppenheim Joost J JJ Lubkowski Jacek J Lu Wuyuan W

Proceedings of the National Academy of Sciences of the United States of America 20030702 15

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recentl ...[more]

PMID: 12840147

Similar Datasets

Project description:BackgroundAvian beta-defensins (AvBD) are small, cationic, antimicrobial peptides. The potential application of AvBDs as alternatives to antibiotics has been the subject of interest. However, the mechanisms of action remain to be fully understood. The present study characterized the structure-function relationship of AvBD-6 and AvBD-12, two peptides with different net positive charges, similar hydrophobicity and distinct tissue expression profiles.ResultsAvBD-6 was more potent than AvBD-12 against E. coli, S. Typhimurium, and S. aureus as well as clinical isolates of extended spectrum beta lactamase (ESBL)-positive E. coli and K. pneumoniae. AvBD-6 was more effective than AvBD-12 in neutralizing LPS and interacting with bacterial genomic DNA. Increasing bacterial concentration from 10(5) CFU/ml to 10(9) CFU/ml abolished AvBDs' antimicrobial activity. Increasing NaCl concentration significantly inhibited AvBDs' antimicrobial activity, but not the LPS-neutralizing function. Both AvBDs were mildly chemotactic for chicken macrophages and strongly chemotactic for CHO-K1 cells expressing chicken chemokine receptor 2 (CCR2). AvBD-12 at higher concentrations also induced chemotactic migration of murine immature dendritic cells (DCs). Disruption of disulfide bridges abolished AvBDs' chemotactic activity. Neither AvBDs was toxic to CHO-K1, macrophages, or DCs.ConclusionsAvBDs are potent antimicrobial peptides under low-salt conditions, effective LPS-neutralizing agents, and broad-spectrum chemoattractant peptides. Their antimicrobial activity is positively correlated with the peptides' net positive charges, inversely correlated with NaCl concentration and bacterial concentration, and minimally dependent on intramolecular disulfide bridges. In contrast, their chemotactic property requires the presence of intramolecular disulfide bridges. Data from the present study provide a theoretical basis for the design of AvBD-based therapeutic and immunomodulatory agents.

Project description:Human-β-defensins (HBD1-3) are antibacterial peptides containing three disulphide bonds. In the present study, the effect of Escherichia coli lipopolysaccharide (LPS) on the antibacterial activities of HBD2-3, C-terminal analogues having a single disulphide bond, Phd1-3, and their corresponding myristoylated analogues MPhd1-3 were investigated. The effect of LPS on the activities of linear amphipathic peptides melittin, LL37 and non-ribosomally synthesized peptides, polymyxin B, alamethicin, gramicidin A, and gramicidin S was also examined. The antibacterial activity of HBD 2-3, Phd1-3, and MPhd1-3 in the presence of LPS against E. coli and Staphylococcus aureus was inhibited. While LPS inhibited the antibacterial activity of LL37, the inhibition of melittin activity was partial. The hemolytic activity exhibited by MPhd1, MPhd3, melittin, and LL37 was inhibited in the presence of LPS. HBD2-3, Phd1-3, and MPhd1-3 also showed endotoxin neutralizing activity. The antibacterial and hemolytic activities of polymyxin B, alamethicin, gramicidin A, and gramicidin S were not inhibited in the presence of LPS. Fluorescence assays employing dansyl cadaverine showed that HBD2-3 and defensin analogues bind to LPS more strongly as compared to alamethicin, gramicidin A, and gramicidin S. Electron microscopy images indicated that peptides disintegrate the structure of LPS. The inhibition of the antibacterial activity of native defensins and analogues in the presence of LPS indicates that the initial interaction with the bacterial surface is similar. The native defensin sequence or structure is also not essential, although cationic charges are necessary for binding to LPS. Hydrophobic interaction is the main driving force for association of non-ribosomally synthesized polymyxin B, alamethicin, gramicidin A, and gramicidin S with LPS. It is likely that these peptides rapidly insert into membranes and do not interact with the bacterial cell surface, whereas cationic peptides such as β-defensin and their analogues, melittin and LL37, first interact with the bacterial cell surface and then the membrane. Our results suggest that evaluating interaction of antibacterial and hemolytic peptides with LPS is a compelling way of elucidating the mechanism of bacterial killing or hemolysis.

Dataset Information

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.

Publications

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets