Antibacterial activities and conformations of bovine β-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity

Mandal, Mahitosh; Jagannadham, Medicharla V.; Nagaraj, Ramakrishnan

doi:10.1016/s0196-9781(01)00628-3

Cited by 56 publications

(45 citation statements)

References 30 publications

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“…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.…”

Section: Discussionsupporting

confidence: 54%

Studies of the Biological Properties of Human β-Defensin 1

Pazgier

Prahl²,

Hoover³

et al. 2007

Journal of Biological Chemistry

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Defensins are small (30 -45 amino acid residues) cationic proteins with broad antimicrobial activity against many bacteria and fungi, some enveloped viruses, and other activities such as chemoattraction of a range of different cell types to the sites of inflammation. These proteins represent attractive targets for developing novel antimicrobial agents and modulators of immune responses with therapeutic applicability. In this report, we present the results of functional and structural studies of 26 single-site mutants of human ␤-defensin 1 (hBD1). All mutants were assayed for antimicrobial activity against Escherichia coli (ATCC strain 25922) and for chemotactic activity with CCR6-transfected HEK293 cells. To analyze the structural implications of mutagenesis and to verify the correctness of the disulfide connectivity, we used x-ray crystallography to conduct complete structural studies for 10 mutants in which the topology of disulfides was the same as in the native hBD1. Mutations did not induce significant changes of the tertiary structure, suggesting that the observed alterations of biological properties of the mutants were solely associated with changes in the respective side chains. We found that cationic residues located near the C terminus (Arg Defensins are recognized as an important element of the human innate immune system (1-4). The defensin family is composed of small (3-5 kDa), cationic, and cysteine-rich proteins. In human defensins, the connectivity of three disulfide bridges forms the basis for assigning them into one of two classes, the ␣-and ␤-defensins (5-7). In ␤-defensins, the topology of the three disulfide bridges is 1-5, 2-4, and 3-6, meaning that the first cysteine in the sequence is covalently linked to the fifth, and so on. Three human ␤-defensins, hBD1-3, 4 have been isolated from natural sources and characterized in detail (8 -11). Additionally, the recombinant or synthetic preparations of hBD4, hBD27, and hBD28 have also been described and characterized functionally (12, 13). Analysis of the human genome indicates the existence of over 40 potential coding regions for these peptides (14,15).In addition to broad antimicrobial activity, hBDs have also been recognized as modulators of cell-mediated adaptive immunity, due to their chemotactic and immunoenhancing activity (16 -19). Recent studies revealed that ␤-defensins also play a role in cell differentiation, tissue remodeling, and sperm maturation (20 -22).The first human ␤-defensin to be discovered, hBD1, is constitutively expressed in the epithelial cells of the urinary and respiratory tracts and in keratinocytes of skin (9,23,24). A naturally occurring 36-amino acid hBD1 peptide shows anti-bacterial activity at micromolar concentrations against some Gramnegative bacteria (i.e. Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae), as well as yeast Candida albicans. When tested in vitro, hBD1 is relatively less potent against the Gram-positive Streptococcus aureus (9, 10, 23).HBD1 and hBD2 selectively chemoattract hum...

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Section: Discussionsupporting

confidence: 54%

Studies of the Biological Properties of Human β-Defensin 1

Pazgier

Prahl²,

Hoover³

et al. 2007

Journal of Biological Chemistry

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“…While it has been demonstrated that all three disulfides or the order of connectivity is not an essential feature for activity in defensins (27,29,49), our results indicate that linear peptides without cysteines can still possess antibacterial activity, although it is 10-to 20-fold lower than that of HNP-1. The antibacterial activity is only marginally inhibited by high concentrations of NaCl, especially against E. coli and S. aureus.…”

contrasting

confidence: 71%

Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Varkey

Nagaraj

2005

Antimicrob Agents Chemother

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The antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1⌬C) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1⌬C18) or the G was replaced with A (HNP-1⌬C18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1⌬C and HNP-1⌬C18 in the presence of 100 mM NaCl, except in the case of Pseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active against P. aeruginosa even in the presence of 100 mM NaCl. The antibacterial activity of HNP-1⌬C, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.Mammalian defensins are a family of cationic antimicrobial peptides characterized by three disulfide bridges (13,22,35). They are expressed in granulocytes, monocytes, macrophages, and epithelial cells of the skin, intestines, and other tissues (2, 13, 39). Based on the pattern of disulfide bonding, mammalian defensins have been classified into ␣ and ␤ families. In another class named the -defensins, in addition to three disulfide bridges, the amino-and carboxyl-terminal ends are linked via an amide bond (13). In humans, ␣-defensins are produced in neutrophils and Paneth cells of the small intestine, while ␤-defensins are expressed by epithelial cells at various sites (13,22). The -defensins have been identified only in primates (13,19). The expression of defensins is constitutive as well as induced by infection and inflammation (13,22,23). Defensin biosynthesis and release have been observed to be regulated by microbial signals, developmental signals, and cytokines (13,14). Apart from their role as microbicidal agents, defensins have also been implicated as effective immune modulators in adaptive immunity (11, 54). They have potency to enhance phagocytosis, induce the activation and degranulation of mast cells, and indirectly regulate innate antimicrobial immunity by affecting the production of chemokines such as interleukin-8, interleukin-10, and tumor necrosis factor from different cells (3,24,53). Studies have also revealed the role of defensins in adaptive immunity, as reflected by their chemotactic effect on monocytes, T cells, and immature dendritic cells (5,32,35,43,48,54).Human neutrophil defensins (HNP-1 to HNP-4) are stored in azurophilic granules of the neutrophil. They are transferred to phagolysosomes upon phagocytosis to kill the ingested microbes (14, 15). The crystal structure of HNP-3 rev...

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“…Of the six hBD-3 variants with mispaired Cys connectivities analyzed (32), the microbicidal activities of native hBD-3, mispaired variants, and disulfide-null hBD-3 were the same (24,32). Similarly, the bactericidal activity of bovine ␤-defensin BNBD-12 against E. coli was also independent of the disulfide array (33). The possible role of ␤-defensin disulfide connectivities in conferring resistance to proteolysis is unknown to our knowledge, perhaps because the mechanisms of ␤-defensin posttranslational processing remain obscure.…”

Section: Discussionmentioning

confidence: 97%

Functional Analysis of the α-Defensin Disulfide Array in Mouse Cryptdin-4

Maemoto¹,

Qu²,

Rosengren

et al. 2004

Journal of Biological Chemistry

123

166

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The ␣-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines ␣-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell ␣-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the ␣-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining ␣-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

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Antibacterial activities and conformations of bovine β-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity

Cited by 56 publications

References 30 publications

Studies of the Biological Properties of Human β-Defensin 1

Studies of the Biological Properties of Human β-Defensin 1

Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Functional Analysis of the α-Defensin Disulfide Array in Mouse Cryptdin-4

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