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β Defensins

cationic peptides with a broad spectrum of anti microbial activity.

β-defensin 2 (BD-2) also known as skin-antimicrobial peptide 1 (SAP1) is a peptide that in humans is encoded by the DEFB4 (defensin, β4) gene. Human β-defensin-2 (hBD-2) is a cysteine-rich cationic low molecular weight antimicrobial peptide recently discovered in lesional skin.

hBD-2 is a protein whose primary structure is made by 64 aminoacids. At concentrations ӆ2.4 mM, hBD-2 is monomeric. The structure is amphiphilic with a nonuniform surface distribution of positive charge and contains several key structural elements, including a triple-stranded, antiparallel beta sheet with strands 2 and 3 in a beta hairpin conformation. The determination of other structural elements depends on the technique used. When X-ray crystallography is used an alpha helix can be observed at the C-terminal end of the protein. When using NMR this alpha-helix does not appear.

Human β-defensin 2 is produced by a number of epithelial cells and exhibits potent antimicrobial activity against Gram-negative bacteria and Candida, but not Gram-positive S. aureus. It has been speculated that β-defensin 2 may contribute to the infrequency of Gram-negative infections on skin and lung tissue.

hBD-2 represents the first human defensin that is produced following stimulation of epithelial cells by contact with microorganisms such as P. aeruginosa or cytokines such as TNF-α and IL-1 β. The HBD-2 gene and protein are locally expressed in keratinocytes associated with inflammatory skin lesions. It is intriguing to speculate that HBD-2 is a dynamic component of the local epithelial defense system of the skin and respiratory tract having a role to protect surfaces from infection, and providing a possible reason why skin and lung infections with Gram-negative bacteria are rather rare. Although this protein doesn”Æt have any antibacterial activity against Gram-positive bacteria, there is a study showing that there is a synergy between hBD-2 and other proteins. One example of this synergistic effect is with epiP, a protein segregated by some strains of S. epidermidis. hBD2, holding hands with epiP, is capable of killing S. aureus, a Gram-positive bacteria responsible of human diseases.

defensin 2

β-Defensin 2

Other Defensins

See more defensin like Canine β-Defensin at CBD section

 

Schematic representation of the solution structures of mBD-7 (A), mBD-8 (B) and hBD-1(C). The elements of secondary structure and the disulfide bonds are indicated. Same orientation as in Fig. 3a. The figure was drawn with Molscript (Kraulis 1991) and Raster 3D (Merritt and Murphy 1994). (D) Overlay of the structures of the two human defensins hBD-1and hBD-2 (dark gray) with the structures of the two murine defensins mBD-7 and mBD-8 (light gray); figure drawn with SYBYL 6.5.

BAUER F., et al. Protein Science (2001), 10:2470?479

Electrostatic surface plots of the most representative NMR structures of the three human β-defensins. HBD1, HBD2, and HBD3 are diagramed from left to right in the figure. In the top half of the figure the C terminus of each of the structures is positioned on the right, and the N-terminal helix is at the top, whereas in the bottom half of the figure the structures are rotated 180?and the C termini are now on the left, whereas the N-terminal helical regions remain at the top. The basic regions of the protein are colored blue, whereas the red regions are acidic. This figure was generated with GRASP (62).

Schibli D. J., et al. J. Biol. Chem., Vol. 277, Issue 10, 8279-8289, March 8, 2002

Possible orientations of two HBD3 monomers to form a dimer. Intermonomer hydrogen bonds between strand 2 of each of the monomers would establish a 6-stranded sheet. The dimer interface could be stabilized by the electrostatic interaction of Lys-32 and Glu-28 in addition to inter-monomer hydrogen bonds between the side chains of Gln-29. It should be noted that the positioning of the two monomers is only a model to illustrate the potential interactions if strand 2 were the dimer interface. Atoms of the two monomers may be overlapping, and correct bond distances have not been accounted for. This figure was created using the program MOLMOL.

Schibli D. J., et al. J. Biol. Chem., Vol. 277, Issue 10, 8279-8289, March 8, 2002

Sequence homology of HBD1-3 and bovine tracheal antimicrobial protein (TAP). Homologous residues among the defensins are highlighted. It should be noted that whereas the 42-residue sequence of HBD1 has been used for the alignment, the alignment does not alter for the 36-residue fragment used in this study. In addition to the sequence homology between the β-defensins, the net positive charge is also indicated. a, the net positive charge for both the 42-residue and the 36-residue HBD1 peptides is indicated. Alignment was performed with the ClustalX program .

Schibli D. J., et al. J. Biol. Chem., Vol. 277, Issue 10, 8279-8289, March 8, 2002

Ribbon diagrams of the three human -defensins. A, HBD1; B, HBD2; C, HBD3. All three structures are in approximately the same orientation, with the three-disulfide bonds shown in gold. This figure was generated with the program MOLMOL.

Schibli D. J., et al. J. Biol. Chem., Vol. 277, Issue 10, 8279-8289, March 8, 2002

 

Stereo-diagrams of the backbone trace of the 20 lowest energy structures of HBD3 with residues 6-44 overlaid (A) and HBD1 with residues 2-35 overlaid (B). The backbone and heavy atom r.m.s. distances of HBD3 are 0.616 and 1.337 ?for residues 6-45. The backbone and heavy atom r.m.s. distances of HBD1 are 0.451 and 0.992 ?for residues 2-35. This figure was generated with the program MOLMOL.

Defensins to the rescue.

Human neutrophils secrete the antimicrobial peptides α-defensin-1, -2, and -3 in response to bacterial infection. These peptides form dimeric structures that contain a total of six β-strands (purple arrows). The positively charged side chains of α-defensin-3 are shown in blue, negative charges in red, and disulfide bonds in orange.

Tomas Gan. Science 1 November 2002: Vol. 298. no. 5595, pp. 977 - 979

beta-defensin 2

Figure 2. Although murine beta-defensin 2 is attracted to iDCs via CCR6 (8), TLR-4 is the receptor for DC activation. Both mDF2-β and LPS, but not MCP-3 fusion protein (MCP3), induce maturation of iDCs from CCR6 KO mice (A). The CCR6 KO phenotype was verified by PCR analysis. Black bar, CD11c+ / B7.2+ / CD40+; hatched bars, CD11c+/B7.2+/I-Ahigh (MHC class II). Data are representative of two independent experiments. (B) iDCs from the mice with TLR-4 mutation or TLR-4 locus deletion failed to mature by treatment with mDF2-beta or LPS (C3H/HeJ and C5710ScNr, respectively), compared with DCs from wild-type mice (C3H/HeN). DCs were treated with LPS (10 ng/ml) or recombinant proteins (5 µg/ml). Open bar, C3H/HeN; hatched bar, C3H/HeJ; and cross-hatched bar, C5710ScNr. Experiment was repeated three times. (C) Activation of the luciferase reporter gene with mDF2-β. Data are representative of two independent experiments. Cells were transiently cotransfected with murine TLR-4 and MD2 and treated with mDF2-β (5 or 25 µg, mDF2-β 5 or mDF2-β 25) or control recombinant protein sFv315 at 5 or 25 µg/ml. All samples were in culture medium (CM) containing 10 µg/ml polymixin B. Control group was treated with 10 ng/ml LPS in CM without polymixin B (11). A representative recombinant protein N24mDF2-beta (fig. S1) was used as a source of mDF2-β.

Biragyn A, et al. Science. 2002 Nov 1;298(5595):1025-9.

 

Tissue expression of hBD-3 mRNA.

BD-3


Low hBD-3 mRNA expression (analyzed by real-time RT-PCR) was detected in many tissues (A). Normal skin and tonsils showed the highest hBD-3 transcript level. (n.d., not detected.) hBD-3 mRNA is expressed in cultivated human primary keratinocytes (B) or primary tracheal epithelial cells (C) and is up-regulated by treatment of the cells with heat-inactivated bacteria (108 cells/ml) or TNF-alpha (10 ng/ml) for 6 h. The mucoid clinical isolate of P. aeruginosa proved to be the strongest inducer of hBD-3. Bars represent the relative hBD-3 transcript levels normalized to β-actin transcript levels.

Jürgen Harder et al. J. Biol. Chem., Vol. 276, Issue 8, 5707-5713, February 23, 2001

Coomassie-stained 16% Tricine gel of the human β-defensins before (lanes 1, 3, and 5) and after reduction of the disulfide bonds with dithiothreitol (lanes 2, 4, and 6). Lanes 1 and 2, HBD1; lanes 3 and 4, HBD2; lanes 5 and 6, HBD3; lane 7, protein molecular mass markers.

Schibli D. J., et al. J. Biol. Chem., Vol. 277, Issue 10, 8279-8289, March 8, 2002

NOEs observed for HBD1 after the final ARIA run. A, short and medium range NOEs. The strong, medium, and weak NOEs are signified by a decrease in the relative intensity of each of the lines. Those NOEs that were ambiguous by either chemical shift overlap with other atoms or were overlapped by the H2O resonance are signified as dotted lines. The CSI of the H backbone atoms is also represented in addition to the location of the secondary structures in the sequence. B, NOEs between the separate strands of the sheets. Long dashed lines represent unambiguous NOEs, whereas short dashed lines represent ambiguous NOEs.

Isomoto H., et al. World J Gastroenterol  2005 August 21;11(31):4782-4787

defensin


defensin

defensin

Nishi Y., et al. World J Gastroenterol 2005;11(1):99-103

β-Defensin 1 (H-072-53)

  

BD-1  BD-1

BD-1  BD-1

Tissue Sample

Human lung cancer tissues , mouse and rat lung tissues

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

N/A

Blocking

3% H2O2, 2% Normal Goat Serum

Primary Antibody

Rabbit Anti-Beta-Defensin I (H) Antiserum (Catalog No.: H-072-53)

Optimal Dilution

1:500, 1 hour at RT

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

Streptavidin-HRP (Vector), 1:400, 30 min

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

 

β-Defensin 2 (H-072-48)

BD-2  BD-2

BD-2  BD-2

 

β-Defensin 2 (G-072-48)

  

Tissue Sample

Rat and mouse lung tissues

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

N/A

Blocking

3% H2O2, 2% Normal Goat Serum

Primary Antibody

Rabbit Anti-Beta-Defensin 2 (H) Purified IgG (Catalog No.: G-072-48)

Optimal Dilution

1:500 (2 ug/ml), 1 hour at RT

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

Streptavidin-HRP (Vector), 1:400, 30 min

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

 

β-Defensin 3 (H-072-42)

BD-3  BD-3

Tissue Sample

Rat and mouse lung tissues

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

N/A

Blocking

3% H2O2, 2% Normal Goat Serum

Primary Antibody

Rabbit Anti-Beta-Defensin 3 (H) Antiserum (Catalog No.: H-072-42)

Optimal Dilution

1:500, 1 hour at RT

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

Streptavidin-HRP (Vector), 1:400, 30 min

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

 

β-Defensin 3 (H-072-44)

BD-8  BD-8

BD-8  BD-8

Tissue Sample

Rat and Mouse heart tissues

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

N/A

Blocking

3% H2O2, 2% Normal Goat Serum

Primary Antibody

Rabbit Anti-Beta-Defensin 8(H) Antiserum (Catalog No.: H-072-44)

Optimal Dilution

1:500, 1 hour at RT

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

Streptavidin-HRP (Vector), 1:400, 30 min

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

β-Defensin 1 & 2 RIA Kits (RK-072-53) & (RK-072-48)

  BD-2

β-Defensin 2 ELISA Kits (EK-072-37)

  BD-2

 

Synthetic human β-Defensin-2

 

Mouse β-Defensin-8 is also available for your research

defensin-8

Sequence alignment of mammalian beta-defensins (BD)

The alignment includes the sequences of four human defensins (hBD-1, hBD-2, hBD-3, hBD-4), six murine defensins (mBD-1 to mBD-4, mBD-7, mBD-8), three bovine defensins (bBD-1, bBD-2, bBD-12), and the bovine tracheal anitmicrobial peptide (bTAP). Strictly conserved amino acid residues are highlighted by a black box and residues occurring with a frequency of 50% are marked by gray boxes. The alignment was generated using the programs ClustalW (Higgins et al. 1992) and Alscript (Barton 1993). Because of the low sequence similarity, DALI analysis (Holm and Sander 1996) of the three-dimensional structures was used to allow a correct placement of the gaps in the sequences of mBD-7 and mBD-8. Elements of secondary structure found in hBD-1, hBD-2, mBD-7, and mBD-8 are schematically indicated below the alignment. The numbering scheme refers to the full-length hBD-2 including the amino-terminal signal sequence.

BAUER F., et al. Protein Science (2001), 10:2470?479

Peptide sequence of hBD-3

The deduced amino acid sequence (single-letter code) of the native hBD-3 peptide based on the complementary DNA sequence obtained from human keratinocytes and tracheal epithelia cells is shown. For comparison, amino acid sequences of the human β-defensins hBD-1 and hBD-2, bovine epithelial β-defensins TAP, LAP, and EBD bovine neutrophil β-defensin BNBD-12, as well as the beta-defensin consensus sequence (including the putative disulfide bridges) are aligned. (The dashes in the β-defensin sequences represent gaps due to the alignment).

Jürgen Harder et al. J. Biol. Chem., Vol. 276, Issue 8, 5707-5713, February 23, 2001

Proposed salt-bridge between Lys-17 and Glu-27 of HBD3. This figure was generated with the program MOLMOL.

Schibli D. J., et al. J. Biol. Chem., Vol. 277, Issue 10, 8279-8289, March 8, 2002

New Peptide Ligands for Melanocortin Receptors

Canine Beta-Defensin CBD103, Human BD-1 and BD-3

New ligands for melanocortin receptors

Named originally for their effects on peripheral end organs, the melanocortin system controls a diverse set of physiological processes through a series of five G-protein-coupled receptors and several sets of small peptide ligands. The central melanocortin system plays an essential role in homeostatic regulation of body weight, in which two alternative ligands, alpha-melanocyte-stimulating hormone and agouti-related protein, stimulate and inhibit receptor signaling in several key brain regions that ultimately affect food intake and energy expenditure. Much of what we know about the relationship between central melanocortin signaling and body weight regulation stems from genetic studies. Comparative genomic studies indicate that melanocortin receptors used for controlling pigmentation and body weight regulation existed more than 500 million years ago in primitive vertebrates, but that fine-grained control of melanocortin receptors through neuropeptides and endogenous antagonists developed more recently. Recent studies based on dog coat-color genetics revealed a new class of melanocortin ligands, the beta-defensins, which reveal the potential for cross talk between the melanocortin and the immune systems.

Kaelin et al. Int J Obes (Lond). 2008 Dec;32 Suppl 7:S19-27.

Structural and molecular evolutionary analysis of Agouti and Agouti-related proteins.

Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.

Jackson et al. Chem Biol. 2006 Dec;13(12):1297-305.  

 
MC1R
Reference
human Beta-Defensin 1
30 nM
human Beta-Defensin 3
14 nM

%Defensin%


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