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β Defensins |
cationic peptides
with a broad spectrum of anti microbial activity.
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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 doesnӮt 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.
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.
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
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.
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
Nishi Y., et al. World J Gastroenterol 2005;11(1):99-103
β-Defensin 1 (H-072-53)
 
 
 
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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)
 
 
β-Defensin 2 (G-072-48)
 
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|
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)
 
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|
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 |
 
 
|
|
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 |
Synthetic human β-Defensin-2
 
Mouse β-Defensin-8 is also available for your research
 
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
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
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.
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.
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