Gallinacin-3, an Inducible Epithelial Defensin in the Chicken

  10.1128/IAI.69.4.2684-2691.2001.

2001, 69(4):2684. DOI:Infect. Immun. Kim A. Brogden and Robert I. LehrerChengquan Zhao, Tung Nguyen, Lide Liu, Randy E. Sacco, -Defensin in the Chicken

βGallinacin-3, an Inducible Epithelial

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INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.4.2684–2691.2001

Apr. 2001, p. 2684–2691 Vol. 69, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Gallinacin-3, an Inducible Epithelial b-Defensin in the ChickenCHENGQUAN 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

Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial b-defensins of the chicken (Gallusgallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 wasespecially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, includingthe skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased sig-nificantly after experimental infection of chickens with Haemophilus paragallinarum, whereas its expression inthe tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a longC-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 andGPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that readthrough the original stop codon and allowed the chicken propeptide to lengthen. The striking structural re-semblance of the precursors of b-defensins to those of crotamines (highly toxic peptides found in rattlesnakevenom) supports their homology, even though defensins are specialized to kill microorganisms and crotaminesare specialized to kill much larger prey.

Defensins are endogenous b-sheet peptides that contributeto the antimicrobial properties inherent in mammalian granu-locytes, epithelial cells, and certain secretions. Three defensinsubfamilies exist in vertebrates. Two of these, a- and b-defen-sins, occur in humans (11, 17) and the third, theta (u)-defen-sins, has been identified to date only in leukocytes of the rhesusmonkey (36). Compelling evidence indicates that a-, b-, and u-defensins originated from a common ancestral defensin gene(22). Because only b-defensins have been found in birds, theymay constitute the oldest of these three defensin subfamilies(15).

Human tissues express at least six a-defensins and threeb-defensins. Four of the a-defensins (HNP-1, -2, -3, and -4) arestored within the primary (azurophil) granules of the neutro-phil, and two others (HD-5 and -6) occur in cytoplasmic gran-ules of small intestinal Paneth cells. HD-5 was also found inthe vaginas and ectocervixes of healthy females and was ex-pressed by inflamed fallopian tubes (26).

The b-defensins of humans (3, 14, 25, 32, 40, 43), mice (1,24), and cattle (28, 31, 33, 35, 37, 42) have received consider-able attention. Human b-defensin-1 (HBD-1) is expressed con-stitutively by epithelial cells throughout the body (43) and isespecially prominent in genitourinary tract organs, includingthe vagina and kidneys (3, 40). HBD-2 is inducible and occursin the skin, respiratory passages, and intestine (14, 25, 32).Impaired defensin function, which may or may not (18) beattributable to local airway hypersalinity, has been implicatedin the pathogenesis of bronchopulmonary infections in cysticfibrosis patients (2, 12).

In cattle, b-defensin expression occurs in epithelial cells of

the trachea, tongue, and intestine and in alveolar macro-phages. In these sites, peptide expression is induced by lipo-polysaccharide, injury, and/or cytokines (28, 31, 37). In con-trast, bovine granulocytes express b-defensins in a constitutivemanner (33, 35, 42).

a-Defensins are remarkably abundant in human, rabbit, andrat neutrophils (20), but only b-defensins (at least 13 differentisoforms) appear in the neutrophils of cattle (33). b-Defensinsalso occur in the polymorphonucleated granulocytes (het-erophils) of chickens and turkeys (4, 7, 15, 16). While defensinsare often very prominent in granulocytes, they are not ubiqui-tous, since the neutrophils of mice (6), pigs (19), and horses (5)lack any defensins at all. Although avian b-defensins have beenobserved in bone marrow cells (4, 7, 15, 16), neither theirinduction during infection nor their expression in epithelialcells has been reported until now.

MATERIALS AND METHODS

cDNA cloning. Total cellular RNA was purified from the tracheal tissues ofchickens and turkeys using the Tri-Reagent RNA isolation procedure with re-agents and procedures recommended by the manufacturer (Molecular ResearchCenter, Cincinnati, Ohio). First-strand cDNA synthesis was done with an Ad-vantage reverse transcription-PCR kit (Clontech, Palo Alto, Calif.) using primersdesigned according to the cDNA sequences of gallinacin-1 (Gal-1) and turkeyheterophil peptide-1 (THP-1) (4). The sense primer P1 (59-AAACCATGCGGATCGTGTACCTGC-39) corresponded to the 59 regions of both peptides. Theantisense primer P2 (59-GCAATGCCTAAACTGCACGACCAAAT-39) wascomplementary to the 39 cDNA preceding the poly(A) tails of both peptides.Chicken and turkey tracheal cDNAs were PCR amplified, inserted intoTOPO-TA vectors (Invitrogen, Carlsbad, Calif.), and sequenced by the fluores-cein-labeled dideoxynucleotide terminator method on an Applied Biosystems373A DNA sequencer (Perkin-Elmer, Palo Alto, Calif.).

Tissue expression in the chicken. A healthy 3-month-old chicken was sacri-ficed, and 21 tissue samples were obtained at necropsy. These were rinsed incold, sterile saline, frozen immediately, and stored at 280°C until used. TotalRNA purification and cDNA synthesis were done as described above. PCRprimers were designed according to the cDNA sequences of Gal-1, Gal-2, andGal-3. P3 (59-CCCTTACCTCACTCTCATC-39), corresponding to bp 222 to 240of the Gal-1 cDNA sequence, and P2 (described above) were used to amplify

* 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: [email protected].

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Gal-1 and Gal-1a. P4 (59-GTTCTGTAAAGGAGGGTCCTGCCAC-39), corre-sponding to bp 114 to 138 of the Gal-2 cDNA sequence, and P5 (59-ACTCTATAACACAAAACATATTGC-39), complementary to bp 327 to 350 of the Gal-2cDNA sequence, were used to amplify Gal-2. P2 and P6 (59-CTGCCGCTTCCCACACATAG-39), corresponding to bp 113 to 132 of the Gal-3 cDNA se-quence, were used to amplify Gal-3. Two b-actin primers, P7 (59-GAGCACCCTGTGCTGCTCACAGAGG-39) and P8 (59-CATTGCCAATGGTGATGACCTGACC-39), corresponded to the sequence of chicken b-actin cDNA and wereused to assess the quality and quantity of the chicken mRNA samples.

Thirty-five PCR cycles were performed with an automated thermal cycler, asfollows: 94°C for 20 s, 55°C for 20 s, and 72°C for 40 s. We used a master reagentmixture to ensure tube-to-tube consistency in cDNA synthesis and PCR ampli-fication. Reaction products were visualized after electrophoresis in 1.4% agarosegels.

Experimental infections. Using a protocol that had been approved by theNational Animal Disease Center (Ames, Iowa) Animal Care and Use Commit-tee, mature female chickens were challenged via intranasal inoculation with 0.1

ml of an egg yolk inoculum that contained approximately 5 3 106 organisms ofthe Modesto strain of Haemophilus paragallinarum. Age-matched, noninfectedcontrol birds were housed under similar environmental conditions. Chickenswere euthanized on the fifth day after infection, following the onset of clinicalsigns. Tissue samples were collected, snap frozen in liquid nitrogen, and storedat 280°C.

In situ hybridization. Paraffin-embedded chicken tongue biopsy specimenswere sectioned to a thickness of about 5 mm. The sections were baked at 60°C for1 h, deparaffinized in two changes of xylene for 5 min, immersed in two changesof absolute ethanol for 1 min, and air dried for 10 min. Sections were treated at37°C for 10 min with 40 mg 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 dehy-drated through a graded ethanol series and air dried.

The specific antisense probe (59-CACAGAATTCAGGGCATCAACCTCATATGCTCTTCCACAGCAGG-39) 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

FIG. 1. cDNA and peptide sequences of Gal-1a. The deduced chicken prepropeptide contains 65 residues with a mass of 7,286 Da and a pIof 10.21. The stop codon (TGA) is double underlined.

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 of9.42. The stop codon (TGA) is double underlined, and the peptide’s C-terminal extension is in boldface.

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labeled using the BioPrime DNA labeling system (Life Technologies, Rockville,Md.). Briefly, 500 ng of oligonucleotides was mixed with 20 ml of 2.53 randomprimers solution, denatured by boiling for 5 min, and ice cooled for 5 min. To thiswas added 5 ml of 103 deoxynucleoside triphosphates, 1.3 ml of Klenow frag-ment, and distilled water to bring the total volume up to 50 ml. Reaction mixtureswere incubated at 37°C for 60 min, ethanol precipitated twice with 5 ml of 3 Msodium acetate and 100 ml of cold 95% ethanol, frozen at 280°C for 2 h, andcentrifuged at 15,000 3 g for 10 min.

Hybridization and detection were carried out using an in situ hybridization anddetection system (Life Technologies). DNA probes were dissolved at 0.5 ml/ml inhybridization buffer and 20% dextran sulfate solution. Probes were denatured byboiling for 10 min and chilled on ice for 5 min. Ten microliters of probe was usedper 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. Afterhybridization, the coverslips were removed, and the slides were immersed inthree changes of 0.23 SSC (13 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 ml of blocking solution at roomtemperature for 15 min in a humidified chamber. The blocking solution was thenremoved, and the slides were next incubated with 10 ml of streptavidin-alkalinephosphatase conjugate plus 90 ml of buffer for another 15 min. The slides werewashed twice in Tris-buffered saline (100 mM Tris base, 150 mM sodium chlo-ride, pH 7.5) for 15 min and once in alkaline-substrate buffer (100 mM Tris base,150 mM sodium chloride, 50 mM MgCl2 z 6H2O, pH 9.5) for 5 min at roomtemperature. Color development was carried out in nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate solution at 37°C and terminated by rinsingthe slides several times in deionized water. Slides were counterstained withsafranin, dehydrated through a graded ethanol series (50, 70, 90, and 100%) for1 min in each concentration, air dried, and permanently mounted with Gel/Mount (Biomeda Corp., Foster City, Calif.). Photographs were taken at a mag-nification of 3100 with a Nikon Optiphot microscope.

Nucleotide sequence accession numbers. The GenBank accession numbers forthe sequences described in this paper are as follows: Gal-1a, AF181951; Gal-3,AF181952; and GPV-1, AF18195.

RESULTS

Gallinacins. We sequenced 12 clones from the chicken tra-chea PCR product. One showed 99% identity with the Gal-1cDNA sequence (Fig. 1), and its deduced mature amino acidsequence was identical to that of the Gal-1a peptide we pre-viously purified from chicken leukocytes (15). The carboxyl-terminal glycine shown in the cDNA sequence was absent fromthe mature Gal-1a peptide, indicating that the native peptideundergoes C-terminal processing. The Gal-1a prepropeptidecomprised 65 residues, including a signal sequence of 20 resi-dues, a propiece with 5 residues, a mature peptide with 39residues, and the C-terminal glycine discussed above. Threesingle-nucleotide substitutions distinguished Gal-1a fromGal-1, and each caused an amino acid change. In Gal-1a andGal-1, the amino acids and codons were as follows, respec-tively: Ser9 and Asn9 (AGT and AAT), Ser20 and Tyr20 (TCCand TAC), and His32 and Tyr32 (TAC and CAC). We hadoriginally purified Gal-1a from chicken leukocytes (15, 16),and while the present results suggest that it may also be ex-pressed by chicken tracheal cells, its origin from tissue leuko-

FIG. 3. cDNA and peptide sequences of Gallopavin 1. The deduced turkey prepropeptide contains 59 residues with a mass of 6,598 Da anda pI of 9.49. The stop codon (TGA) is double underlined.

FIG. 4. Partial cDNA sequences of Gal-3 and Gallopavin 1. The inserted nucleotide bases of Gal-3 (i.e., those without counterparts inGallopavin 1) are shown in boldface. Both in-frame TGA stop codons are double underlined.

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cytes cannot be excluded, as some macrophages can expressb-defensins (29).

All of the other 11 cDNA clones obtained from the chickentracheal PCR product encoded a novel b-defensin, Gal-3,whose sequence is shown in Fig. 2. The deduced Gal-3 pre-propeptide contained 80 amino acids and had a mass of 8,723.3Da and a pI of 9.42. The propeptide included a 20-residuesignal sequence, followed by a short propiece and a typicalcationic b-defensin domain. The latter contained 38 residues,and its mass and pI were 4,234 Da and 9.49, respectively. Thedefensin domain was followed by a distinctly unusual 22- to24-residue anionic extension, (AY)EVD . . . NPH.

GPV-1. We identified a homologous epithelial b-defensin inturkey tracheal tissue. Named GPV-1, its cDNA and deducedamino acid sequences are shown in Fig. 3. Unlike Gal-3, GPV-1 did not contain a C-terminal extension. Instead, its 240-bpreading frame encoded a 59-amino-acid residue and a cationicprepropeptide, with a calculated mass of 6,598 Da (oxidizedcysteines) and pI of 9.49. The signal sequence and 39 untrans-lated portion of GPV-1 cDNA were each 83% identical toTHP-1 cDNA at the nucleotide level, but the sequences en-coding the mature peptides were considerably more divergent.

The Gal-3 and Gal-1 and -1a cDNA sequences showed;75% overall identity, which was most marked in their signalsequences and 39 untranslated regions. The correspondingcDNA sequences of chicken Gal-3 and turkey GPV-1 were91% identical. The C-terminal extension of Gal-3 evidentlyarose from the insertion of two bases just before its originalTGA stop codon (retained in GPV-1), introducing a frameshiftand read through of the old stop codon (Fig. 4). These changes,plus insertion of a 15-bp fragment not found in GPV-1, gener-ated the “postpiece” of Gal-3 (Fig. 2). To confirm that the in-sert, frameshift, and extension did not result from a PCR artifact,we used another PCR primer set to prepare and amplify cDNAfrom the chicken tongue and bursa of Fabricius. Cloning andsequencing of both PCR products confirmed the above-de-scribed findings (data not shown).

Expression in healthy tissues. We examined the expressionof Gal-1, -2, and -3 in 21 different healthy chicken tissues,including (i) skin, (ii) the gastrointestinal tract (tongue, esoph-agus, proventriculus, gizzard, liver, small intestine, large intes-

tine, cloaca, bursa of Fabricius, and gall bladder), (iii) therespiratory tract (trachea, lung, and air sacs), (iv) the genito-urinary tract (kidney, ovary, oviduct, and egg yolk sacs), and (v)miscellaneous tissues (spleen, pancreas, and bone marrow).Figure 5 shows that Gal-1 and -1a (our primers did not dis-tinguish between them) and Gal-2 were expressed stronglyonly in healthy bone marrow and, to a lesser extent, in lung. Incontrast, Gal-3 was weakly expressed in the bone marrow andwas strongly expressed in the tongue, bursa of Fabricius, andtrachea. Moderate Gal-3 expression was noted in the skin,esophagus and air sacs. Weaker expression of Gal-3 was seenin the large intestine, kidney, and ovary. Thus, whereas expres-sion of Gal-1 (and -1a) and -2 was restricted to bone marrowcells, Gal-3 showed widespread expression in nonmyeloid cells.

FIG. 5. Expression of b-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, andcaudal); 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.

FIG. 6. Gal-3 expression in the chicken tongue (in situ hybridiza-tion). (Left panel) Section of tongue that was probed with antisensemessage. Sites of Gal-3 expression (arrows) are stained brown. (Rightpanel) Control that was processed with the corresponding sense probe.

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The expression of Gal-3 by epithelial cells of the tongue wasconfirmed by in situ hybridization (Fig. 6).

Inducibility. To determine if tracheal Gal-3 production wasconstitutive or increased in response to infection, we chal-lenged six chickens with H. paragallinarum, reserving six non-infected, age-matched animals of the same lines as controls(Fig. 7). The results were analyzed quantitatively by phosphor-imaging the PCR products and normalizing Gal-3 expressionto that of b-actin in the same sample. One sample from aninfected chicken (sample I-1) lacked both Gal-3 and b-actinand was therefore not analyzed further. The other 11 trachealsamples (6 control and 5 infected) were analyzed by a Mann-Whitney rank sum test. The median values were 1.140 (con-trol) and 6.800 (infected). These differences were significant(T 5 43.000; P [exact] 5 0.017), indicating that tracheal ex-pression of Gal-3 rose in response to H. paragallinarum infec-tion. 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 itwas constitutive in nature (data not shown).

DISCUSSION

A database search (BLAST) performed on the nucleotidesequences of Gal-3 and GPV-1 identified only chicken Gal-1and turkey THP-1 as their close relatives. When this search wasperformed on the peptide sequences of Gal-3 and GPV-1,many additional b-defensin homologues were recognized. Therelationship of Gal-3 and GPV-1 to other b-defensins is shownin a dendrogram, based on amino acid sequences (Fig. 8).

An unusual structural feature of the Gal-3 precursor was the22- to 24-residue peptide domain that extended beyond itsexpected C terminus. The mechanism that established this wasdiscussed above. The functional significance (if any) of theextension is unknown. Since we did not purify mature Gal-3peptide, we cannot exclude its posttranslational removal bylimited proteolysis. Very similar peptide architecture has beennoted in certain defensin-like peptides from invertebrates.MGD-1, a 39-residue, defensin-like antibacterial peptide fromhemocytes of the Mediterranean mussel, Mytilus galloprovin-cialis, provides the best example (23). It has a signal peptide of21 residues (versus 20 residues in Gal-3), an active peptide of39 amino acids (versus 38 residues in Gal-3), and an acidic,21-residue carboxyl-terminal extension (versus 22 to 24 resi-dues in Gal-3). The insect defensins of bees (Apis mellifera andBombus pascuorum) have C-terminal extensions that make themabout 12 residues longer than other insect defensins (10, 27).What, if anything, the extensions contribute to function re-mains to be determined. “Big defensin,” an antimicrobial pep-tide of Limulus, the horseshoe crab, has a 35-residue N-terminal hydrophobic domain and a 37-residue, C-terminalb-defensin-like domain (30). Here, both domains contributesignificantly 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 forcattle (31) and pigs (34). Immunohistochemical studies showedthat b-defensin was concentrated in a 0.1-mm-thick layer at the

FIG. 7. Induction of Gal-3 in the chicken trachea after infection.Tracheal tissues were obtained from six control chickens (lanes C) andsix infected chickens (lanes I) on the fifth day after the experimentalgroup had been infected with H. paragallinarum. Reverse transcrip-tion-PCR was performed as described in the text, using primers spe-cific for Gal-3 and chicken b-actin. The bar graph shows the Gal-3/b-actin ratio.

FIG. 8. Dendrogram. Avian and mammalian b-defensins are shown.The relationship is based on amino acid sequence homology. TAP,LAP, and EBD, b-defensins from epithelial cells of the bovine trachea,tongue, and intestine, respectively.

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cornified tips of filiform papillae on the dorsal tongue of thepig and in superficial squamous cell layers of its buccal mucosa,presumably constituting an antimicrobial barrier against or-ganisms that enter the mouth (34).

The prominent Gal-3 expression in the bursa of Fabricius(Fig. 5) was fascinating, given recent reports that describeinteractions between defensins and B or T lymphocytes. For

example, human b-defensins are chemotactic for immaturedendritic cells and memory T cells and bind CCR6, a chemo-kine receptor preferentially expressed by these cells (41).There is also evidence that defensins can enhance systemicimmunoglobulin G antibody responses in vitro and in vivo,acting through help provided by CD41 Th1- and Th2-type cyto-kines (21). Finally, a-defensins and prodefensins have been

FIG. 9. Primary sequences of chicken and turkey b-defensins. Heterophils are equivalent to polymorphonuclear neutrophils in humans.Identical residues in each set are connected by vertical lines. Boldface indicates conserved residues.

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 arattlesnake crotamine are shown, with gaps (–) placed to maximize alignment. Cysteine connectivity, shown at the top, is identical for all of thesepeptides. Boldface indicates residues identical to those found in both Gal-3 and GPV-1. Underlining shows conservative substitutions of residuesfound in both Gal-3 and GPV-1.

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recovered as HLA-DR-associated ligands from the peripheralblood mononuclear cells of two patients with plasmacytoma (13).

Figure 9 compares the primary sequences of Gal-3 andGPV-1 to each other and to those of the myeloid b-defensinsof these fowl. Except for its C-terminal extension, Gal-3 re-sembles GPV-1 more closely than it resembles myeloid galli-nacins. Although other factors may have contributed to main-taining the stability of epithelial b-defensins, a need to retainstructural features involved in receptor-ligand interactions(e.g., with lymphocytes or cytokine receptors) may have con-strained their ability to diverge.

It is noteworthy that b-defensins show striking similarities tocertain toxins. Figure 10 aligns chicken Gal-3 and turkey GPV-1 with three other peptides: (i) crotamine, a myotoxic peptidefound in rattlesnake (Crotalus viridis viridis) venom; (ii) a b-de-fensin-like peptide from the venom of the male duck-billedplatypus (Ornithorhynchus anatiformis) (8, 38); and (iii) HBD-3(the HBD-3 sequence is entry gi:8163794 on the National Cen-ter for Biotechnology server of the National Institutes ofHealth [http://www.ncbi.nlm.nih.gov/]).

Even though homology at the nucleotide level is no longerevident, HBD-3 and chicken Gal-3 are clearly the descendantsof common ancestral genes. The platypus peptide has not beencloned, but its solution structure and cysteine disulfide pairingare identical to those of the bovine b-defensin BNBD-12 (39).The crotalid myotoxins, several of which have been cloned,share the identical disulfide pairing motif (9). Because thesignal peptide of defensins typically shows greater conservationthan does the defensin domain, we performed a BLAST searchon the amino acid sequence of HBD-3 and obtained 62 hitsfrom among the 509,459 sequences analyzed. Remarkably, 19of the first 20 hits were peptides that contained six cysteineswith an identical disulfide pairing pattern. Of these, 11 weremammalian b-defensins (including BNBD-1), 2 were avianb-defensins (gallinacin and THP), and 6 were rattlesnake pep-tides of the crotamine type (Table 1). Since the signal sequencesearch did not use any sequence or conformational informa-tion derived from the cysteine-rich b-defensin or toxin do-mains, it seems highly improbable that rattlesnake crotamines

and b-defensins are not homologous. While this dual nature ofpeptides derived from a common gene is new, perhaps Lucre-tius (who also anticipated atomic structure) had somethingsimilar in mind when he wrote in De Rerum Natura, “Quod alicibus est aliis fuat acre venenum.” (Lucretius [98 to 55 BCE] inDe Rerum Natura. Translation: “What is food to one may be afierce poison to others.”)

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TABLE 1. Signal sequence conservationa

Species Accessionno. Peptide

No. of residues/total (%)

Identical Conserved

Human AF217245 HBD-3 22/22 (100) 22/22 (100)Chimpanzee AF209855 b-Defensin-2 13/20 (65) 18/20 (90)Human AF040153 HBD-2 13/20 (65) 18/20 (90)Mouse AF092929 b-Defensin-3 12/17 (70) 13/17 (75)Pig AF031666 b-Defensin-1 11/20 (55) 14/20 (70)Turkey AF033338 THP-2 14/20 (70) 15/20 (75)Chicken AF033336 Gallinacin 14/20 (70) 15/20 (75)Rat AF068861 b-Defensin-2 11/18 (61) 14/18 (77)Mouse AF068861 b-Defensin-4 11/18 (61) 13/18 (72)Tropical rattlesnake AF044674 Crotamine 12/21 (57) 15/21 (71)Prairie rattlesnake JC5324 Myotoxin A 12/21 (57) 15/21 (71)Bovine AAD43032.1 BNBD-12 12/20 (60) 14/20 (70)Bovine S76279 LAP (tongue) 11/15 (73) 13/15 (86)Bovine M63023 TAP (trachea) 11/15 (73) 13/15 (86)Bovine AF000362 EAP (enteric) 10/15 (66) 12/15 (79)

a Four additional rattlesnake peptides (accession numbers P24333, P24332,P24331, and AAC06241.1) that were among the top 20 hits are not shown hereto conserve space.

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Editor: R. N. Moore

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