Previous Article | Next Article 
Infection and Immunity, April 2001, p. 2684-2691, Vol. 69, No. 4
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.2684-2691.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Gallinacin-3, an Inducible Epithelial
-Defensin in the Chicken
Chengquan
Zhao,1
Tung
Nguyen,1
Lide
Liu,1
Randy E.
Sacco,2
Kim A.
Brogden,2 and
Robert I.
Lehrer1,3,*
Department of
Medicine1 and Molecular Biology
Institute,3 University of California, Los
Angeles, California 90095, and Respiratory Diseases of
Livestock Research Unit, National Animal Disease Center, USDA
Agricultural Research Service, Ames, Iowa 500102
Received 16 October 2000/Returned for modification 19 December
2000/Accepted 8 January 2001
 |
ABSTRACT |
Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized,
epithelial 
-defensins of the chicken (Gallus gallus)
and turkey (Meleagris gallopavo), respectively. In
normal chickens, the expression of gallinacin-3 was especially
prominent in the tongue, bursa of Fabricius, and trachea. It also
occurred in other organs, including the skin, esophagus, air sacs,
large intestine, and kidney. Tracheal expression of gallinacin-3
increased significantly after experimental infection of chickens
with Haemophilus paragallinarum, whereas its expression
in the tongue, esophagus, and bursa of Fabricius was unaffected. The
precursor of gallinacin-3 contained a long C-terminal extension not
present in the prepropeptide. By comparing the cDNA sequences of
gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into
the gallinacin-3 gene had induced a frameshift that read through the
original stop codon and allowed the chicken propeptide to lengthen. The
striking structural resemblance of the precursors of -defensins
to those of crotamines (highly toxic peptides found in rattlesnake
venom) supports their homology, even though defensins are specialized
to kill microorganisms and crotamines are specialized to kill much
larger prey.
| |
INTRODUCTION |
Defensins are endogenous -sheet
peptides that contribute to the antimicrobial properties inherent in
mammalian granulocytes, epithelial cells, and certain secretions. Three
defensin subfamilies exist in vertebrates. Two of these, 
- and
-defensins, occur in humans (11, 17) and the third,
theta (
)-defensins, has been identified to date only in
leukocytes of the rhesus monkey (36). Compelling evidence
indicates that -, -, and -defensins originated from a
common ancestral defensin gene (22). Because only
-defensins have been found in birds, they may constitute the oldest
of these three defensin subfamilies (15).
Human tissues express at least six -defensins and three
-defensins. Four of the -defensins (HNP-1, -2, -3, and -4) are stored within the primary (azurophil) granules of the neutrophil, and
two others (HD-5 and -6) occur in cytoplasmic granules of small
intestinal Paneth cells. HD-5 was also found in the vaginas and
ectocervixes of healthy females and was expressed by inflamed fallopian
tubes (26).
The -defensins of humans (3, 14, 25, 32, 40, 43), mice
(1, 24), and cattle (28, 31, 33, 35, 37, 42)
have received considerable attention. Human -defensin-1 (HBD-1) is
expressed constitutively by epithelial cells throughout the body
(43) and is especially prominent in genitourinary tract organs, including the vagina and kidneys (3, 40). HBD-2 is inducible and occurs in the skin, respiratory passages, and intestine (14, 25, 32). Impaired defensin function, which may or may not (18) be attributable to local airway hypersalinity,
has been implicated in the pathogenesis of bronchopulmonary infections in cystic fibrosis patients (2, 12).
In cattle, -defensin expression occurs in epithelial cells of the
trachea, tongue, and intestine and in alveolar macrophages. In
these sites, peptide expression is induced by lipopolysaccharide, injury, and/or cytokines (28, 31, 37). In contrast, bovine granulocytes express -defensins in a constitutive manner (33, 35, 42).
-Defensins are remarkably abundant in human, rabbit, and rat
neutrophils (20), but only -defensins (at least 13 different isoforms) appear in the neutrophils of cattle
(33). -Defensins also occur in the polymorphonucleated
granulocytes (heterophils) of chickens and turkeys (4, 7, 15,
16). While defensins are often very prominent in granulocytes,
they are not ubiquitous, since the neutrophils of mice
(6), pigs (19), and horses (5)
lack any defensins at all. Although avian -defensins have been
observed in bone marrow cells (4, 7, 15, 16), neither their induction during infection nor their expression in epithelial cells has been reported until now.
| |
MATERIALS AND METHODS |
cDNA cloning.
Total cellular RNA was purified from the
tracheal tissues of chickens and turkeys using the Tri-Reagent RNA
isolation procedure with reagents and procedures recommended by the
manufacturer (Molecular Research Center, Cincinnati, Ohio).
First-strand cDNA synthesis was done with an Advantage reverse
transcription-PCR kit (Clontech, Palo Alto, Calif.) using primers
designed according to the cDNA sequences of gallinacin-1 (Gal-1) and
turkey heterophil peptide-1 (THP-1) (4). The sense primer
P1 (5'-AAACCATGCGGATCGTGTACCTGC-3') corresponded to the 5'
regions of both peptides. The antisense primer P2
(5'-GCAATGCCTAAACTGCACGACCAAAT-3') was complementary to the
3' cDNA preceding the poly(A) tails of both peptides. Chicken and
turkey tracheal cDNAs were PCR amplified, inserted into TOPO-TA vectors
(Invitrogen, Carlsbad, Calif.), and sequenced by the
fluorescein-labeled dideoxynucleotide terminator method on an Applied
Biosystems 373A DNA sequencer (Perkin-Elmer, Palo Alto, Calif.).
Tissue expression in the chicken.
A healthy 3-month-old
chicken was sacrificed, and 21 tissue samples were obtained at
necropsy. These were rinsed in cold, sterile saline, frozen
immediately, and stored at 
80°C until used. Total RNA purification
and cDNA synthesis were done as described above. PCR primers were
designed according to the cDNA sequences of Gal-1, Gal-2, and Gal-3. P3
(5'-CCCTTACCTCACTCTCATC-3'), corresponding to bp 222 to 240 of the Gal-1 cDNA sequence, and P2 (described above) were used to
amplify Gal-1 and Gal-1. P4
(5'-GTTCTGTAAAGGAGGGTCCTGCCAC-3'), corresponding to bp 114 to 138 of the Gal-2 cDNA sequence, and P5
(5'-ACTCTATAACACAAAACATATTGC-3'), complementary to bp 327 to 350 of the Gal-2 cDNA sequence, were used to amplify Gal-2. P2 and P6
(5'-CTGCCGCTTCCCACACATAG-3'), corresponding to bp 113 to 132 of the Gal-3 cDNA sequence, were used to amplify Gal-3. Two -actin
primers, P7 (5'-GAGCACCCTGTGCTGCTCACAGAGG-3') and P8
(5'-CATTGCCAATGGTGATGACCTGACC-3'), corresponded to the
sequence of chicken -actin cDNA and were used to assess the quality
and quantity of the chicken mRNA samples.
Thirty-five PCR cycles were performed with an automated thermal cycler,
as follows: 94°C for 20 s, 55°C for 20 s, and 72°C for
40 s. We used a master reagent mixture to ensure tube-to-tube consistency in cDNA synthesis and PCR amplification. Reaction products
were visualized after electrophoresis in 1.4% agarose gels.
Experimental infections.
Using a protocol that had been
approved by the National Animal Disease Center (Ames, Iowa) Animal Care
and Use Committee, mature female chickens were challenged via
intranasal inoculation with 0.1 ml of an egg yolk inoculum that
contained approximately 5 × 106 organisms
of the Modesto strain of Haemophilus paragallinarum. Age-matched, noninfected control birds were housed under similar environmental conditions. Chickens were euthanized on the fifth day
after infection, following the onset of clinical signs. Tissue samples
were collected, snap frozen in liquid nitrogen, and stored at 80°C.
In situ hybridization.
Paraffin-embedded chicken tongue
biopsy specimens were sectioned to a thickness of about 5 µm. The
sections were baked at 60°C for 1 h, deparaffinized in two
changes of xylene for 5 min, immersed in two changes of absolute
ethanol for 1 min, and air dried for 10 min. Sections were treated at
37°C for 10 min with 40 µg of proteinase K per ml in
phosphate-buffered saline (PBS), rinsed in PBS, and fixed at room
temperature for 1 min with fresh 4% paraformaldehyde in PBS. After
being rinsed with PBS, the sections were dehydrated through a graded
ethanol series and air dried.
The specific antisense probe
(5'-CACAGAATTCAGGGCATCAACCTCATATGCTCTTCCACAGCAGG-3') was
complementary to bp 160 to 203 of Gal-3.
A sense probe of the same
sequence was used as the control. Both
probes were labeled using the
BioPrime DNA labeling system (Life
Technologies, Rockville, Md.).
Briefly, 500 ng of oligonucleotides
was mixed with 20 µl of 2.5×
random primers solution, denatured
by boiling for 5 min, and ice cooled
for 5 min. To this was added
5 µl of 10× deoxynucleoside
triphosphates, 1.3 µl of Klenow fragment,
and distilled water to
bring the total volume up to 50 µl. Reaction
mixtures were incubated
at 37°C for 60 min, ethanol precipitated
twice with 5 µl of 3 M
sodium acetate and 100 µl of cold 95% ethanol,
frozen at
80°C
for 2 h, and centrifuged at 15,000 ×
g for 10
min.
Hybridization and detection were carried out using an in situ
hybridization and detection system (Life Technologies). DNA
probes were
dissolved at 0.5 µl/ml in hybridization buffer and
20% dextran
sulfate solution. Probes were denatured by boiling
for 10 min and
chilled on ice for 5 min. Ten microliters of probe
was used per slide,
and slides were covered with 22- by 22-mm
coverslips (Fisher
Scientific, Hanover Park, Ill.) and hybridized
in a humid chamber at
42°C overnight. After hybridization, the
coverslips were removed, and
the slides were immersed in three
changes of 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) at room temperature for 15
min.
Next, the slides were incubated with 200 µl of blocking solution at
room temperature for 15 min in a humidified chamber. The
blocking
solution was then removed, and the slides were next incubated
with 10 µl of streptavidin-alkaline phosphatase conjugate plus
90 µl of
buffer for another 15 min. The slides were washed twice
in
Tris-buffered saline (100 mM Tris base, 150 mM sodium chloride,
pH 7.5)
for 15 min and once in alkaline-substrate buffer (100
mM Tris base, 150 mM sodium chloride, 50 mM MgCl
2 · 6H
2O, pH 9.5)
for 5 min at room temperature.
Color development was carried out
in nitroblue
tetrazolium-5-bromo-4-chloro-3-indolylphosphate solution
at 37°C and
terminated by rinsing the slides several times in
deionized water.
Slides were counterstained with safranin, dehydrated
through a graded
ethanol series (50, 70, 90, and 100%) for 1 min
in each concentration,
air dried, and permanently mounted with
Gel/Mount (Biomeda Corp.,
Foster City, Calif.). Photographs were
taken at a magnification of
×100 with a Nikon Optiphot
microscope.
Nucleotide sequence accession numbers.
The GenBank accession
numbers for the sequences described in this paper are as follows:
Gal-1, AF181951; Gal-3, AF181952; and GPV-1, AF18195.
| |
RESULTS |
Gallinacins.
We sequenced 12 clones from the chicken trachea
PCR product. One showed 99% identity with the Gal-1 cDNA sequence
(Fig. 1), and its deduced mature amino
acid sequence was identical to that of the Gal-1 peptide we
previously purified from chicken leukocytes (15). The
carboxyl-terminal glycine shown in the cDNA sequence was absent from
the mature Gal-1 peptide, indicating that the native peptide
undergoes C-terminal processing. The Gal-1 prepropeptide comprised
65 residues, including a signal sequence of 20 residues, a
propiece with 5 residues, a mature peptide with 39 residues, and the
C-terminal glycine discussed above. Three single-nucleotide substitutions distinguished Gal-1 from Gal-1, and each caused an amino acid change. In Gal-1 and Gal-1, the amino acids and codons
were as follows, respectively: Ser9 and
Asn9 (AGT and AAT), Ser20
and Tyr20 (TCC and TAC), and
His32 and Tyr32 (TAC and
CAC). We had originally purified Gal-1 from chicken leukocytes
(15, 16), and while the present results suggest that it
may also be expressed by chicken tracheal cells, its origin from tissue leukocytes cannot be excluded, as some macrophages can express -defensins (29).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 1.
cDNA and peptide sequences of Gal-1. The deduced
chicken prepropeptide contains 65 residues with a mass of 7,286 Da and
a pI of 10.21. The stop codon (TGA) is double underlined.
|
|
All of the other 11 cDNA clones obtained from the chicken tracheal PCR
product encoded a novel
-defensin, Gal-3, whose sequence
is shown in
Fig.
2. The deduced Gal-3 prepropeptide
contained
80 amino acids and had a mass of 8,723.3 Da and a pI of 9.42.
The propeptide included a 20-residue signal sequence, followed
by a
short propiece and a typical cationic
-defensin domain.
The latter
contained 38 residues, and its mass and pI were 4,234
Da and 9.49, respectively. The defensin domain was followed by
a distinctly unusual
22- to 24-residue anionic extension, (AY)EVD
... NPH.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 2.
cDNA and peptide sequences of Gal-3. The deduced chicken
prepropeptide contains 78 residues with a mass of 8,746 Da and a pI of
9.42. The stop codon (TGA) is double underlined, and the
peptide's C-terminal extension is in boldface.
|
|
GPV-1.
We identified a homologous epithelial -defensin in
turkey tracheal tissue. Named GPV-1, its cDNA and deduced amino acid
sequences are shown in Fig. 3. Unlike
Gal-3, GPV-1 did not contain a C-terminal extension. Instead, its
240-bp reading frame encoded a 59-amino-acid residue and a cationic
prepropeptide, with a calculated mass of 6,598 Da (oxidized cysteines)
and pI of 9.49. The signal sequence and 3' untranslated portion of
GPV-1 cDNA were each 83% identical to THP-1 cDNA at the nucleotide
level, but the sequences encoding the mature peptides were considerably
more divergent.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 3.
cDNA and peptide sequences of Gallopavin 1. The deduced
turkey prepropeptide contains 59 residues with a mass of 6,598 Da and a
pI of 9.49. The stop codon (TGA) is double underlined.
|
|
The Gal-3 and Gal-1 and -1
cDNA sequences showed ~75% overall
identity, which was most marked in their signal sequences and
3'
untranslated regions. The corresponding cDNA sequences of chicken
Gal-3
and turkey GPV-1 were 91% identical. The C-terminal extension
of Gal-3
evidently arose from the insertion of two bases just
before its
original TGA stop codon (retained in GPV-1), introducing
a
frameshift and read through of the old stop codon (Fig.
4).
These changes, plus insertion of a
15-bp fragment not found in
GPV-1, generated the "postpiece"
of Gal-3 (Fig.
2). To confirm
that the insert, frameshift,
and extension did not result from
a PCR artifact, we used another
PCR primer set to prepare and
amplify cDNA from the chicken tongue and
bursa of Fabricius. Cloning
and sequencing of both PCR products
confirmed the above-described
findings (data not shown).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 4.
Partial cDNA sequences of Gal-3 and Gallopavin 1. The
inserted nucleotide bases of Gal-3 (i.e., those without counterparts in
Gallopavin 1) are shown in boldface. Both in-frame TGA stop codons
are double underlined.
|
|
Expression in healthy tissues.
We examined the expression of
Gal-1, -2, and -3 in 21 different healthy chicken tissues, including
(i) skin, (ii) the gastrointestinal tract (tongue, esophagus,
proventriculus, gizzard, liver, small intestine, large intestine,
cloaca, bursa of Fabricius, and gall bladder), (iii) the respiratory
tract (trachea, lung, and air sacs), (iv) the genitourinary tract
(kidney, ovary, oviduct, and egg yolk sacs), and (v) miscellaneous
tissues (spleen, pancreas, and bone marrow). Figure
5 shows that Gal-1 and -1 (our primers did not distinguish between them) and Gal-2 were expressed strongly only in healthy bone marrow and, to a lesser extent, in lung. In
contrast, Gal-3 was weakly expressed in the bone marrow and was
strongly expressed in the tongue, bursa of Fabricius, and trachea.
Moderate Gal-3 expression was noted in the skin, esophagus and air
sacs. Weaker expression of Gal-3 was seen in the large intestine,
kidney, and ovary. Thus, whereas expression of Gal-1 (and -1) and -2 was restricted to bone marrow cells, Gal-3 showed widespread expression
in nonmyeloid cells. The expression of Gal-3 by epithelial cells of the
tongue was confirmed by in situ hybridization (Fig.
6).
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 5.
Expression of -defensins in the tissues of normal
chickens. Lanes: 1, skin; 2, tongue; 3, esophagus and crop; 4, proventriculus; 5, gizzard; 6, liver; 7, small intestine; 8, large
intestine; 9, cloaca (coprodaeum, urodaeum, and proctodaeum); 10, bursa
of Fabricius; 11, liver; 12, trachea; 13, lung; 14, air sacs
(interclavicular, cervical, anterior thoracic, posterior thoracic, and
abdominal); 15, kidney (cranial, middle, and caudal); 16, ovary; 17, oviduct (pooled ostium, magnum, isthmus, uterine shell gland, and
vagina); 18, egg yolk sacs; 19, spleen; 20, pancreas; 21, bone marrow;
M, ladder standards. Each tissue sample was obtained from a single
individual chicken.
|
|
View larger version (153K):
[in this window]
[in a new window]
|
FIG. 6.
Gal-3 expression in the chicken tongue (in situ
hybridization). (Left panel) Section of tongue that was probed with
antisense message. Sites of Gal-3 expression (arrows) are stained
brown. (Right panel) Control that was processed with the corresponding
sense probe.
|
|
Inducibility.
To determine if tracheal Gal-3 production was
constitutive or increased in response to infection, we challenged six
chickens with H. paragallinarum, reserving six noninfected,
age-matched animals of the same lines as controls (Fig.
7). The results were analyzed
quantitatively by phosphorimaging the PCR products and normalizing
Gal-3 expression to that of -actin in the same sample. One sample
from an infected chicken (sample I-1) lacked both Gal-3 and -actin
and was therefore not analyzed further. The other 11 tracheal samples
(6 control and 5 infected) were analyzed by a Mann-Whitney rank sum
test. The median values were 1.140 (control) and 6.800 (infected).
These differences were significant (T = 43.000;
P [exact] = 0.017), indicating that tracheal expression of
Gal-3 rose in response to H. paragallinarum infection. In
contrast, the expression of Gal-3 in the skin, tongue, esophagus, and
bursa of Fabricius did not differ significantly in tissues from control and infected chickens, suggesting that it was constitutive in nature
(data not shown).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 7.
Induction of Gal-3 in the chicken trachea after
infection. Tracheal tissues were obtained from six control chickens
(lanes C) and six infected chickens (lanes I) on the fifth day after
the experimental group had been infected with H.
paragallinarum. Reverse transcription-PCR was performed as
described in the text, using primers specific for Gal-3 and chicken
-actin. The bar graph shows the Gal-3/-actin ratio.
|
|
| |
DISCUSSION |
A database search (BLAST) performed on the nucleotide sequences of
Gal-3 and GPV-1 identified only chicken Gal-1 and turkey THP-1 as their
close relatives. When this search was performed on the peptide
sequences of Gal-3 and GPV-1, many additional -defensin homologues
were recognized. The relationship of Gal-3 and GPV-1 to other
-defensins is shown in a dendrogram, based on amino acid sequences
(Fig. 8).
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 8.
Dendrogram. Avian and mammalian -defensins are
shown. The relationship is based on amino acid sequence homology.
TAP, LAP, and EBD, -defensins from epithelial cells of the
bovine trachea, tongue, and intestine, respectively.
|
|
An unusual structural feature of the Gal-3 precursor was the 22- to
24-residue peptide domain that extended beyond its expected C terminus.
The mechanism that established this was discussed above. The functional
significance (if any) of the extension is unknown. Since we did not
purify mature Gal-3 peptide, we cannot exclude its posttranslational
removal by limited proteolysis. Very similar peptide architecture has
been noted in certain defensin-like peptides from invertebrates. MGD-1,
a 39-residue, defensin-like antibacterial peptide from hemocytes of the
Mediterranean mussel, Mytilus galloprovincialis, provides
the best example (23). It has a signal peptide of 21 residues (versus 20 residues in Gal-3), an active peptide of 39 amino
acids (versus 38 residues in Gal-3), and an acidic, 21-residue
carboxyl-terminal extension (versus 22 to 24 residues in Gal-3). The
insect defensins of bees (Apis mellifera and
Bombus pascuorum) have C-terminal extensions that make
them about 12 residues longer than other insect defensins (10, 27). What, if anything, the extensions
contribute to function remains to be determined. "Big defensin," an
antimicrobial peptide of Limulus, the horseshoe
crab, has a 35-residue N-terminal hydrophobic domain and a 37-residue,
C-terminal -defensin-like domain (30). Here, both
domains contribute significantly to the peptide's antimicrobial
properties (30).
The extensive expression of Gal-3 in the chicken tongue, evident
in Fig. 5 and 6, is reminiscent of findings reported for cattle
(31) and pigs (34). Immunohistochemical
studies showed that -defensin was concentrated in a 0.1-mm-thick
layer at the cornified tips of filiform papillae on the dorsal tongue
of the pig and in superficial squamous cell layers of its buccal
mucosa, presumably constituting an antimicrobial barrier against
organisms that enter the mouth (34).
The prominent Gal-3 expression in the bursa of Fabricius (Fig. 5) was
fascinating, given recent reports that describe interactions between
defensins and B or T lymphocytes. For example, human -defensins are
chemotactic for immature dendritic cells and memory T cells and bind
CCR6, a chemokine receptor preferentially expressed by these cells
(41). There is also evidence that defensins can enhance
systemic immunoglobulin G antibody responses in vitro and in vivo,
acting through help provided by CD4+ Th1- and
Th2-type cytokines (21). Finally, -defensins
and prodefensins have been recovered as HLA-DR-associated ligands from
the peripheral blood mononuclear cells of two patients with plasmacytoma (13).
Figure 9 compares the primary sequences
of Gal-3 and GPV-1 to each other and to those of the myeloid
-defensins of these fowl. Except for its C-terminal extension, Gal-3
resembles GPV-1 more closely than it resembles myeloid gallinacins.
Although other factors may have contributed to maintaining the
stability of epithelial -defensins, a need to retain structural
features involved in receptor-ligand interactions (e.g., with
lymphocytes or cytokine receptors) may have constrained their ability
to diverge.
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 9.
Primary sequences of chicken and turkey -defensins.
Heterophils are equivalent to polymorphonuclear neutrophils in
humans. Identical residues in each set are connected by vertical lines.
Boldface indicates conserved residues.
|
|
It is noteworthy that -defensins show striking similarities to
certain toxins. Figure 10 aligns
chicken Gal-3 and turkey GPV-1 with three other peptides: (i)
crotamine, a myotoxic peptide found in rattlesnake (Crotalus
viridis viridis) venom; (ii) a -defensin-like peptide from the
venom of the male duck-billed platypus (Ornithorhynchus anatiformis) (8, 38); and (iii) HBD-3
(the HBD-3 sequence is entry gi:8163794 on the
National Center for Biotechnology server of the National Institutes of
Health [http://www.ncbi.nlm.nih.gov/]).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 10.
A little further from the tree? The amino acid
sequences of Gal-3, Gal-1, HBD-3, platypus defensin-like peptide (DLP),
and a rattlesnake crotamine are shown, with gaps (-) placed to
maximize alignment. Cysteine connectivity, shown at the top, is
identical for all of these peptides. Boldface indicates residues
identical to those found in both Gal-3 and GPV-1. Underlining shows
conservative substitutions of residues found in both Gal-3 and
GPV-1.
|
|
Even though homology at the nucleotide level is no longer evident,
HBD-3 and chicken Gal-3 are clearly the descendants of common ancestral
genes. The platypus peptide has not been cloned, but its solution
structure and cysteine disulfide pairing are identical to those of the
bovine -defensin BNBD-12 (39). The crotalid myotoxins,
several of which have been cloned, share the identical disulfide
pairing motif (9). Because the signal peptide of defensins
typically shows greater conservation than does the defensin domain, we
performed a BLAST search on the amino acid sequence of HBD-3 and
obtained 62 hits from among the 509,459 sequences analyzed. Remarkably,
19 of the first 20 hits were peptides that contained six cysteines with
an identical disulfide pairing pattern. Of these, 11 were mammalian
-defensins (including BNBD-1), 2 were avian -defensins
(gallinacin and THP), and 6 were rattlesnake peptides of the crotamine
type (Table 1). Since the signal sequence search did not use any sequence or conformational information derived
from the cysteine-rich -defensin or toxin domains, it seems
highly improbable that rattlesnake crotamines and -defensins are not
homologous. While this dual nature of peptides derived from a common
gene is new, perhaps Lucretius (who also anticipated atomic structure)
had something similar in mind when he wrote in De Rerum
Natura, "Quod ali cibus est aliis fuat acre
venenum." (Lucretius [98 to 55 BCE] in
De Rerum Natura. Translation: "What is food to one may be
a fierce poison to others.")
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Room CHS 37-062, UCLA School of Medicine, 10833 LeConte
Ave., Los Angeles, CA 90095-1690. Phone: (310) 825-5340. Fax: (310) 206-8766. E-mail: rlehrer{at}mednet.ucla.edu.
Editor:
R. N. Moore
| |
REFERENCES |
| 1.
|
Bals, R.,
M. J. Goldman, and J. M. Wilson.
1998.
Mouse beta-defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract.
Infect. Immun.
66:1225-1232[Abstract/Free Full Text].
|
| 2.
|
Bals, R.,
X. Wang,
Z. Wu,
T. Freeman,
V. Bafna,
M. Zasloff, and J. M. Wilson.
1998.
Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung.
J. Clin. Investig.
102:874-880[Medline].
|
| 3.
|
Bensch, K. W.,
M. Raida,
H. J. Magert,
P. Schulz-Knappe, and W. G. Forssmann.
1995.
hBD-1: a novel beta-defensin from human plasma.
FEBS Lett.
368:331-335[CrossRef][Medline].
|
| 4.
|
Brockus, C. W.,
M. W. Jackwood, and B. G. Harmon.
1998.
Characterization of beta-defensin prepropeptide mRNA from chicken and turkey bone marrow.
Anim. Genet.
29:283-289[CrossRef][Medline].
|
| 5.
|
Couto, M. A.,
S. S. Harwig,
J. S. Cullor,
J. P. Hughes, and R. I. Lehrer.
1992.
Identification of eNAP-1, an antimicrobial peptide from equine neutrophils.
Infect. Immun.
60:3065-3071[Abstract/Free Full Text].
|
| 6.
|
Eisenhauer, P. B., and R. I. Lehrer.
1992.
Mouse neutrophils lack defensins.
Infect. Immun.
60:3446-3447[Abstract/Free Full Text].
|
| 7.
|
Evans, E. W.,
G. G. Beach,
J. Wunderlich, and B. G. Harmon.
1994.
Isolation of antimicrobial peptides from avian heterophils.
J. Leukoc. Biol.
56:661-665[Abstract].
|
| 8.
|
Fenner, P. J.,
J. A. Williamson, and D. Myers.
1992.
Platypus envenomation a painful learning experience.
Med. J. Aust.
157:829-832[Medline].
|
| 9.
|
Fox, J. W.,
M. Elzinga, and A. T. Tu.
1979.
Amino acid sequence and disulfide bond assignment of myotoxin a isolated from the venom of Prairie rattlesnake (Crotalus viridis viridis).
Biochemistry
18:678-684[CrossRef][Medline].
|
| 10.
|
Fujiwara, S.,
J. Imai,
M. Fujiwara,
T. Yaeshima,
T. Kawashima, and K. Kobayashi.
1990.
A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin.
J. Biol. Chem.
265:11333-11337[Abstract/Free Full Text].
|
| 11.
|
Ganz, T., and J. Weiss.
1997.
Antimicrobial peptides of phagocytes and epithelia.
Semin. Hematol.
34:343-354[Medline].
|
| 12.
|
Goldman, M. J.,
G. M. Anderson,
E. D. Stolzenberg,
U. P. Kari,
M. Zasloff, and J. M. Wilson.
1997.
Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.
Cell
88:553-560[CrossRef][Medline].
|
| 13.
|
Halder, T. M.,
M. Bluggel,
S. Heinzel,
G. Pawelec,
H. E. Meyer, and H. Kalbacher.
2000.
Defensins are dominant HLA-DR-associated self-peptides from CD34() peripheral blood mononuclear cells of different tumor patients (plasmacytoma, chronic myeloid leukemia).
Blood
95:2890-2896[Abstract/Free Full Text].
|
| 14.
|
Harder, J.,
U. Meyer-Hoffert,
L. M. Teran,
L. Schwichtenberg,
J. Bartels,
S. Maune, and J. M. Schroder.
2000.
Mucoid Pseudomonas aeruginosa, TNF-, and IL-1beta, but not IL-6, induce human beta-defensin-2 in respiratory epithelia.
Am. J. Respir. Cell Mol. Biol.
22:714-721[Abstract/Free Full Text].
|
| 15.
|
Harwig, S. S.,
K. M. Swiderek,
V. N. Kokryakov,
L. Tan,
T. D. Lee,
E. A. Panyutich,
G. M. Aleshina,
O. V. Shamova, and R. I. Lehrer.
1994.
Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes.
FEBS Lett.
342:281-285[CrossRef][Medline].
|
| 16.
|
Harwig, S. S.,
K. M. Swiderek,
V. Kokryakov,
T. D. Lee, and R. I. Lehrer.
1994.
Primary structure of Gallinacin-1, an antimicrobial -defensin from chicken leukocytes, p. 81-88.
In
J. Crabbs (ed.), Techniques in protein chemistry, vol. IV. Academic Press, San Diego, Calif.
|
| 17.
|
Huttner, K. M., and C. L. Bevins.
1999.
Antimicrobial peptides as mediators of epithelial host defense.
Pediatr. Res.
45:785-794[Medline].
|
| 18.
|
Knowles, M. R.,
J. M. Robinson,
R. E. Wood,
C. A. Pue,
W. M. Mentz,
G. C. Wager,
J. T. Gatzy, and R. C. Boucher.
1997.
Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.
J. Clin. Investig.
100:2588-2595[Medline].
|
| 19.
|
Kokryakov, V. N.,
S. S. Harwig,
E. A. Panyutich,
A. A. Shevchenko,
G. M. Aleshina,
O. V. Shamova,
H. A. Korneva, and R. I. Lehrer.
1993.
Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins.
FEBS Lett.
327:231-236[CrossRef][Medline].
|
| 20.
|
Lehrer, R. I.,
A. K. Lichtenstein, and T. Ganz.
1993.
Defensins: antimicrobial and cytotoxic peptides of mammalian cells.
Annu. Rev. Immunol.
11:105-128[CrossRef][Medline].
|
| 21.
|
Lillard, J. W., Jr.,
P. N. Boyaka,
O. Chertov,
J. J. Oppenheim, and J. R. McGhee.
1999.
Mechanisms for induction of acquired host immunity by neutrophil peptide defensins.
Proc. Natl. Acad. Sci. USA
96:651-656[Abstract/Free Full Text].
|
| 22.
|
Liu, L.,
C. Zhao,
H. H. Heng, and T. Ganz.
1997.
The human beta-defensin-1 and alpha-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry.
Genomics
43:316-320[CrossRef][Medline].
|
| 23.
|
Mitta, G.,
F. Vandenbulcke,
F. Hubert, and P. Roch.
1999.
Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge J.
Cell Sci.
112:4233-4242[Abstract].
|
| 24.
|
Morrison, G. M.,
D. J. Davidson, and J. R. Dorin.
1999.
A novel mouse beta defensin, Defb2, which is upregulated in the airways by lipopolysaccharide.
FEBS Lett.
442:112-116[CrossRef][Medline].
|
| 25.
|
O'Neil, D. A.,
E. M. Porter,
D. Elewaut,
G. M. Anderson,
L. Eckmann,
T. Ganz, and M. F. Kagnoff.
1999.
Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium.
J. Immunol.
163:6718-6724[Abstract/Free Full Text].
|
| 26.
|
Quayle, A. J.,
E. M. Porter,
A. A. Nussbaum,
Y. M. Wang,
C. Brabec,
K. P. Yip, and S. C. Mok.
1998.
Gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract.
Am. J. Pathol.
152:1247-1258[Abstract].
|
| 27.
|
Rees, J. A.,
M. Moniatte, and P. Bulet.
1997.
Novel antibacterial peptides isolated from a European bumblebee, Bombus pascuorum (Hymenoptera, Apoidea).
Insect Biochem. Mol. Biol.
27:413-422[CrossRef][Medline].
|
| 28.
|
Russell, J. P.,
G. Diamond,
A. P. Tarver,
T. F. Scanlin, and C. L. Bevins.
1996.
Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha.
Infect. Immun.
64:1565-1568[Abstract].
|
| 29.
|
Ryan, L. K.,
J. Rhodes,
M. Bhat, and G. Diamond.
1998.
Expression of beta-defensin genes in bovine alveolar macrophages.
Infect. Immun.
66:878-881[Abstract/Free Full Text].
|
| 30.
|
Saito, T.,
S. Kawabata,
T. Shigenaga,
Y. Takayenoki,
J. Cho,
H. Nakajima,
M. Hirata, and S. Iwanaga.
1995.
A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity.
J. Biochem. (Tokyo)
117:1131-1137[Abstract/Free Full Text].
|
| 31.
|
Schonwetter, B. S.,
E. D. Stolzenberg, and M. A. Zasloff.
1995.
Epithelial antibiotics induced at sites of inflammation.
Science
267:1645-1648[Abstract/Free Full Text].
|
| 32.
|
Schroder, J. M., and J. Harder.
1999.
Human beta-defensin-2.
Int. J. Biochem. Cell Biol.
31:645-651[CrossRef][Medline].
|
| 33.
|
Selsted, M. E.,
Y. Q. Tang,
W. L. Morris,
P. A. McGuire,
M. J. Novotny,
W. Smith,
A. H. Henschen, and J. S. Cullor.
1993.
Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils.
J. Biol. Chem.
268:6641-6648[Abstract/Free Full Text].
|
| 34.
|
Shi, J.,
G. Zhang,
H. Wu,
C. Ross,
F. Blecha, and T. Ganz.
1999.
Porcine epithelial beta-defensin 1 is expressed in the dorsal tongue at antimicrobial concentrations.
Infect. Immun.
67:3121-3127[Abstract/Free Full Text].
|
| 35.
|
Tang, Y. Q., and M. E. Selsted.
1993.
Characterization of the disulfide motif in BNBD-12, an antimicrobial beta-defensin peptide from bovine neutrophils.
J. Biol. Chem.
268:6649-6653[Abstract/Free Full Text].
|
| 36.
|
Tang, Y. Q.,
J. Yuan,
G. Osapay,
K. Osapay,
D. Tran,
C. J. Miller,
A. J. Ouellette, and M. E. Selsted.
1999.
A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins.
Science
286:498-502[Abstract/Free Full Text].
|
| 37.
|
Tarver, A. P.,
D. P. Clark,
G. Diamond,
J. P. Russell,
H. Erdjument-Bromage,
P. Tempst,
K. S. Cohen,
D. E. Jones,
R. W. Sweeney,
M. Wines,
S. Hwang, and C. L. Bevins.
1998.
Enteric beta-defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with Cryptosporidium parvum infection.
Infect. Immun.
66:1045-1056[Abstract/Free Full Text].
|
| 38.
|
Tonkin, M. A., and J. Negrine.
1994.
Wild platypus attack in the antipodes. A case report.
J. Hand Surg.
19:162-164[Medline].
|
| 39.
|
Torres, A. M.,
G. M. de Plater,
M. Doverskog,
L. C. Birinyi-Strachan,
G. M. Nicholson,
C. H. Gallagher, and P. W. Kuchel.
2000.
Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold.
Biochem. J.
348:649-656.
|
| 40.
|
Valore, E. V.,
C. H. Park,
A. J. Quayle,
K. R. Wiles,
P. B. McCray, Jr., and T. Ganz.
1998.
Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.
J. Clin. Investig.
101:1633-1642[Medline].
|
| 41.
|
Yang, D.,
O. Chertov,
S. N. Bykovskaia,
Q. Chen,
M. J. Buffo,
J. Shogan,
M. Anderson,
J. M. Schroder,
J. M. Wang,
O. M. Howard, and J. J. Oppenheim.
1999.
Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.
Science
286:525-528[Abstract/Free Full Text].
|
| 42.
|
Yount, N. Y.,
J. Yuan,
A. Tarver,
T. Castro,
G. Diamond,
P. A. Tran,
J. N. Levy,
C. McCullough,
J. S. Cullor,
C. L. Bevins, and M. E. Selsted.
1999.
Cloning and expression of bovine neutrophil beta-defensins. Biosynthetic profile during neutrophilic maturation and localization of mature peptide to novel cytoplasmic dense granules.
J. Biol. Chem.
274:26249-26258[Abstract/Free Full Text].
|
| 43.
|
Zhao, C.,
I. Wang, and R. I. Lehrer.
1996.
Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells.
FEBS Lett.
396:319-322[CrossRef][Medline].
|
Infection and Immunity, April 2001, p. 2684-2691, Vol. 69, No. 4
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.2684-2691.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Gao, B., Sherman, P., Luo, L., Bowie, J., Zhu, S.
(2009). Structural and functional characterization of two genetically related meucin peptides highlights evolutionary divergence and convergence in antimicrobial peptides. FASEB J.
23: 1230-1245
[Abstract]
[Full Text]
-
Shimizu, M., Watanabe, Y., Isobe, N., Yoshimura, Y.
(2008). Expression of Avian {beta}-Defensin 3, an Antimicrobial Peptide, by Sperm in the Male Reproductive Organs and Oviduct in Chickens: An Immunohistochemical Study. Poult. Sci.
87: 2653-2659
[Abstract]
[Full Text]
-
Akbari, M. R., Haghighi, H. R., Chambers, J. R., Brisbin, J., Read, L. R., Sharif, S.
(2008). Expression of Antimicrobial Peptides in Cecal Tonsils of Chickens Treated with Probiotics and Infected with Salmonella enterica Serovar Typhimurium. CVI
15: 1689-1693
[Abstract]
[Full Text]
-
Whittington, C. M., Papenfuss, A. T., Bansal, P., Torres, A. M., Wong, E. S.W., Deakin, J. E., Graves, T., Alsop, A., Schatzkamer, K., Kremitzki, C., Ponting, C. P., Temple-Smith, P., Warren, W. C., Kuchel, P. W., Belov, K.
(2008). Defensins and the convergent evolution of platypus and reptile venom genes. Genome Res
18: 986-994
[Abstract]
[Full Text]
-
Mageed, A. M. A., Isobe, N., Yoshimura, Y.
(2008). Expression of Avian {beta}-Defensins in the Oviduct and Effects of Lipopolysaccharide on Their Expression in the Vagina of Hens. Poult. Sci.
87: 979-984
[Abstract]
[Full Text]
-
Subedi, K., Isobe, N., Nishibori, M., Yoshimura, Y.
(2007). Changes in the expression of gallinacins, antimicrobial peptides, in ovarian follicles during follicular growth and in response to lipopolysaccharide in laying hens (Gallus domesticus). Reproduction
133: 127-133
[Abstract]
[Full Text]
-
Hasenstein, J. R., Zhang, G., Lamont, S. J.
(2006). Analyses of Five Gallinacin Genes and the Salmonella enterica Serovar Enteritidis Response in Poultry.. Infect. Immun.
74: 3375-3380
[Abstract]
[Full Text]
-
Townes, C. L., Michailidis, G., Nile, C. J., Hall, J.
(2004). Induction of Cationic Chicken Liver-Expressed Antimicrobial Peptide 2 in Response to Salmonella enterica Infection. Infect. Immun.
72: 6987-6993
[Abstract]
[Full Text]
-
Landon, C., Thouzeau, C., Labbe, H., Bulet, P., Vovelle, F.
(2004). Solution Structure of Spheniscin, a {beta}-Defensin from the Penguin Stomach. J. Biol. Chem.
279: 30433-30439
[Abstract]
[Full Text]
-
Wang, W., Owen, S. M., Rudolph, D. L., Cole, A. M., Hong, T., Waring, A. J., Lal, R. B., Lehrer, R. I.
(2004). Activity of {alpha}- and {theta}-Defensins against Primary Isolates of HIV-1. J. Immunol.
173: 515-520
[Abstract]
[Full Text]
-
Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., Bulet, P.
(2003). Spheniscins, Avian {beta}-Defensins in Preserved Stomach Contents of the King Penguin, Aptenodytes patagonicus. J. Biol. Chem.
278: 51053-51058
[Abstract]
[Full Text]
-
Lehrer, R. I., Andrew Tincu, J., Taylor, S. W., Menzel, L. P., Waring, A. J.
(2003). Natural Peptide Antibiotics from Tunicates: Structures, Functions and Potential Uses. Integr. Comp. Biol.
43: 313-322
[Abstract]
[Full Text]
-
Cole, A. M., Hong, T., Boo, L. M., Nguyen, T., Zhao, C., Bristol, G., Zack, J. A., Waring, A. J., Yang, O. O., Lehrer, R. I.
(2002). Retrocyclin: A primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1. Proc. Natl. Acad. Sci. USA
99: 1813-1818
[Abstract]
[Full Text]
-
Leonova, L., Kokryakov, V. N., Aleshina, G., Hong, T., Nguyen, T., Zhao, C., Waring, A. J., Lehrer, R. I.
(2001). Circular minidefensins and posttranslational generation of molecular diversity. J. Leukoc. Biol.
70: 461-464
[Abstract]
[Full Text]
Top
Abstract
Introduction
