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Pseudin-2: An Antimicrobial Peptide with Low Hemolytic Activity from the Skin of the Paradoxical Frog
Biochemical and Biophysical Research Communications 288, 1001–1005 (2001) doi:10.1006/bbrc.2001.5884, available online at http://www.idealibrary.com on
Pseudin-2: An Antimicrobial Peptide with Low Hemolytic Activity from the Skin of the Paradoxical Frog Loyd Olson III,* Ana Maria Soto,† Floyd C. Knoop,‡ and J. Michael Conlon* ,1 *Department of Biomedical Sciences and ‡Department of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, Nebraska 68178-0405; and †Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198
Received September 28, 2001
Four structurally related peptides (pseudins 1– 4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC ⴝ 2.5 M against Escherichia coli, 80 M against Staphylococcus aureus, and 130 M against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 M. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic ␣-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis. © 2001 Academic Press Key Words: pseudidae; antimicrobial peptide; amphibian skin; amphipathic ␣-helix.
The emergence of strains of pathogenic bacteria and fungi with resistance to commonly used antibiotics has necessitated a search for new types of antimicrobial agents to which these microorganisms will not have developed resistance. Antimicrobial peptides synthesized in granular glands in the skins of certain frogs represent a promising source of such potential therapeutic agents (1, 2). Amphibian antimicrobial peptides, which comprise between 12 and 48 amino acid residues are usually synthesized as members of a structurally related family and are released into skin secretions in a holocrine manner in response to stress or injury (3). The peptides that have been characterized to date are, 1
To whom correspondence and reprint requests should be addressed. Fax: 402-280-2690. E-mail: [email protected].
almost without exception, hydrophobic, cationic and adopt amphipathic ␣-helical conformations on interaction with the bacterial cell membrane (4, 5). Although often displaying broad spectrum antimicrobial activity, the peptides are, to varying degrees, hemolytic against human erythrocytes which severely limits their therapeutic potential (6). The synthesis of cationic ␣-helical antimicrobial peptides in the skin is by no means a universal feature of Anurans. The extant Anurans (at least 3500 species) have been divided into 21 families (7). Antimicrobial peptides of this type have been isolated from skin secretions and/or skin extracts of frogs belonging to genera that are classified in the families Pipidae [Xenopus (8)], Discoglossidae [Bombina (9, 10)], Hyperoliidae [Kassina (11)], Ranidae [Rana (12, 13)], Hylidae [Phyllomedusa (14), Agalychnis (15), and Litoria (16)], and Myobatrachidae [Uperoleia (17)]. In contrast, up to this time, the author’s laboratory has failed to detect the production of cationic ␣-helical antimicrobial peptides in the skins of several species of frogs belonging to the families Pelobatidae, Bufonidae, Microhylidae, and Rhacophoridae (unpublished data). The family Pseudidae comprises four species of semiaquatic frogs, organized in two genera Pseudis and Lysapus, that are found in tropical lowland areas in the eastern part of South America. The paradoxical frog Pseudis paradoxa is so called because the tadpoles of the species attain a length of 220 mm whereas the largest adults rarely exceed a size of 70 mm. This study describes the purification and characterization of four previously undescribed peptides with antimicrobial activity from an extract of the skin of P. paradoxa. MATERIALS AND METHODS Skin secretions. Adult specimens of P. paradoxa (n ⫽ 2) were injected with 0.1 mM norepinephrine (0.5 ml) at a dorsal site and each placed in a buffer solution (100 ml) of composition 50 mM sodium chloride, 25 mM ammonium acetate, pH 7.0, for 5 min (18). The animals were returned to the aquarium and appeared to suffer
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no ill-effects. After centrifugation (4000g for 30 min at 4°C), the supernatants were pooled and pumped onto three Sep-Pak C-18 cartridges (Waters Associates, Milford, MA) connected in series at a flow rate of 2 ml min ⫺1. Bound material was eluted with acetonitrile/ water/trifluoroacetic acid (700:299:1, v/v/v) and freeze-dried. Tissue extraction. Skin (10.1 g) was removed from pithed adult specimens of P. paradoxa (n ⫽ 2) and immediately frozen on dry ice. The frozen tissue was cut into small pieces and extracted by homogenization in ethanol/0.7 M HCl (3:1, v/v; 100 ml) at 0°C using a PowerGen rotor/stator-type homogenizer (Fisher Scientific, Pittsburgh, PA). The homogenate was stirred for 1.5 h at 0°C and centrifuged (4000g for 30 min at 4°C). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000g for 30 min at 4°C), the extract was pumped onto 6 Sep-Pak C-18 cartridges connected in series at a flow rate of 2 ml min ⫺1. Bound material was eluted with acetonitrile/water/trifluoroacetic acid (700:299:1, v/v/v) and freeze-dried. Antimicrobial assays. Activity of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent (50 l) in Mueller–Hinton broth (50 l) with an inoculum (50 l of 10 3 CFU/ml) from an overnight culture of either Escherichia coli (ATCC 25922) or Staphylococcus aureus (NCTC 8325) in 96-well microtiter cellculture plates for 18 h at 37°C in a humidified atmosphere of 5% CO 2 in air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35°C. After incubation, the absorbance at 550 nm of each well was determined using a M.A. Bioproducts Model MA308 microtiter plate reader. Minimal inhibitory concentrations (MICs) were measured by a standard microdilution method (19) and were taken as the lowest concentration where no visible growth was observed. To monitor the validity of the bacterial assays, incubations were carried out in parallel with increasing concentrations of the broad-spectrum antibiotic, bacitracin. Incubations with Candida albicans were carried in parallel with amphotercin B. Purification of the peptides. The frog skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 1% (v/v) trifluoroacetic acid/water (4 ml) and injected onto a (25 ⫻ 1 cm) Vydac 218TP510 (C-18) reversed-phase HPLC column (Separations Group, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml min ⫺1. The concentration of acetonitrile in the eluting solvent was raised to 21% over 10 min and to 63% over 60 min using linear gradients. Absorbance was measured at 214 and 280 nm and fractions (1 min) were collected. The fractions containing antimicrobial activity were successively rechromatographed on (250 ⫻ 4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) columns. The concentration of acetonitrile in the eluting solvent was raised from 28 to 56% over 40 min and the flow rate was 1.5 ml min ⫺1. Structural analysis. Amino acid compositions were determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using a Waters AccQ Tag system with fluorescence detection and separation of the amino acid derivatives by reversed-phase HPLC. Hydrolysis in 5.7 M HCl (24 h at 110°C) of approximately 1 nmol of peptide was carried out. The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems Procise Model 491 sequenator. Electrospray mass spectrometry was carried out using a Perkin– Elmer Sciex API 150EX single quadrupole instrument. The accuracy of mass determinations was ⫾0.02%. Peptide synthesis. Pseudin-2 was synthesized by solid-phase methodology on a 0.025-mmol scale on an Applied Biosystems Model 432A peptide synthesizer using a 4-(2⬘,4⬘-dimethoxy-phenyl-Fmocaminomethyl)phenoxyacetamido-ethyl resin (Applied Biosystems, Foster City, CA). Fmoc amino acid derivatives were activated with O-benzotriazol-1-yl-N,N,N⬘,N⬘-tetramethyluronium hexafluorophosphate (0.075 mmol), 1-hydroxy-benzotriazole hydrate (0.075 mmol)
FIG. 1. Reversed-phase HPLC on a semipreparative Vydac C-18 column of an extract of skin secretions of the paradoxical frog Pseudis paradoxa, after partial purification on Sep-Pak cartridges. The dashed line shows the concentration of acetonitrile in the eluting solvent. The fractions denoted by the bars contained material that inhibited the growth of E. coli.
and diisopropylethylamine (0.150 mmol). The peptide was cleaved from the resin with trifluoroacetic acid/water/thioanisole/1,2-ethanedithiol (99.0/0.50/0.25/0.25) at 25°C for 3 h. The synthetic material was purified to near homogeneity by chromatography on a 1 ⫻ 25-cm Vydac 218TP510 C-18 reversed-phase HPLC column using the elution conditions shown in Fig. 2A. Hemolytic assay. Peptides in the concentration range 100 –1000 M were incubated with washed human erythrocytes (2 ⫻ 10 7 cells) from healthy donors (n ⫽ 6) in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 l) for 1 h at 37°C. After centrifugation (12,000g for 15 s), the absorbance at 541 nm of the supernatant was measured. A parallel incubation in the presence of 3% v/v Tween 20 was carried out to determine the absorbance associated with 100% hemolysis. The HC 50 value was taken as the concentration of peptide producing 50% hemolysis. Circular dichroism. The circular dichroism spectrum of pseudin-2 was recorded at 25°C using a Model 202SF stopped flow circular dichroism spectrophotometer (Aviv Instruments, Lakewood, NJ). Spectra were recorded in a 1 mm path length cell at a peptide concentration of 100 M in two solvents: (a) 10 mM sodium phosphate buffer, pH 7.0, and (b) 10 mM sodium phosphate buffer, pH 7.0, containing 50% (v/v) trifluoroethanol. In each case, the circular dichroism spectrum of the solvent was subtracted from the spectrum of the peptide.
RESULTS Purification of the Pseudins The antimicrobial activity, measured against E. coli, in the extract of P. paradoxa skin after partial purification on Sep-Pak cartridges was eluted from a semipreparative Vydac C-18 reverse-phase HPLC column in the three fractions denoted by the bars in Fig. 1. Fraction 1 was subsequently shown to contain pseudin-1, fraction 2 contained pseudin-2, and fraction 3 ⫹ 4 contained both pseudin-3 and pseudin-4. The
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Structural and Biological Characterization The amino acid sequences of the pseudins were determined without ambiguity by automated Edman degradation and the primary structures are shown in Fig. 3. The amino acid compositions of the peptides were consistent with their proposed structures. Electrospray mass spectrometry demonstrated that the peptides did not contain a C-terminally ␣-amidated amino acid residue (pseudin-1: observed M r 2715.4, calculated M r 2715.2; pseudin-2: observed M r 2685.4, calculated M r 2685.2; pseudin-3: observed M r 2569.6, calculated M r 2570.0; pseudin-4: observed M r 2512.0, calculated M r 2511.9). The primary structure of pseudin-2 was confirmed by chemical synthesis. A mixture of endogenous and synthetic pseudin-2 was eluted from an analytical Vydac C-18 column under the conditions of chromatography shown in Fig. 2A as a single symmetric peak. The minimal inhibitory concentrations of the endogenous peptides against the gram-negative bacterium E. coli were pseudin-1 4.5 M, pseudin-2 2.5 M, pseudin-3 12 M, and pseudin-4 6.5 M. The minimal inhibitory concentration of pseudin-2 against the gram-positive bacterium Staphylocococcus aureus was 80 and 130 M against the yeast, Candida albicans. In six independent experiments using human erythrocytes from different donors, the HC 50 value of synthetic pseudin-2 ranged from 320 to 550 M. Circular Dichroism
FIG. 2. Purification of pseudin-2 on analytical (A) Vydac C-4, and (B) Vydac phenyl columns. The peaks containing material that inhibited the growth of E. coli are denoted by (⫹) and the arrows show where peak collection began and ended.
same procedure was used to purify all the peptides and so only the purification of pseudin-2 is described. Rechromatography of fraction 2 (Fig. 1) on an analytical Vydac C-4 column led to the elution of two prominent and well-resolved peaks (Fig. 2A). The later eluting peak inhibited the growth of E. coli and contained pseudin-2. The peptide was purified to near homogeneity, as assessed by peak symmetry and mass spectrometry by a final chromatography on analytical Vydac phenyl column (Fig. 2B). The approximate yields of the pure peptides were pseudin-1, 43 nmol; pseudin-2, 221 nmol; pseudin-3, 39 nmol; and pseudin-4, 33 nmol. Norepinephrine-stimulated skin secretions from P. paradoxa, after concentration on Sep-Pak cartridges, did not inhibit the growth of either E. coli or S. aureus and pseudins were not detected by mass spectrometry following chromatography of the secretions under the conditions shown in Fig. 1.
As shown in Fig. 4, the circular dichroism spectrum of pseudin-2 was markedly solvent-dependent. In aqueous sodium phosphate buffer, pH 7.0, the presence of a broad negative band near 199 nm in the spectrum indicates that the peptide exists predominantly in either an unordered conformation or there is a rapid interconversion of multiple conformations. In the more hydrophobic solvent (50% trifluoroethanol), the negative bands centered at 208 and 222 nm and the positive band centered at 193 nm are indicative of significant ␣-helical character. DISCUSSION The article has described the purification and structural characterization of four antimicrobial peptides (pseudins) from an extract of the skin of the paradoxical frog, Pseudis paradoxa. This is the first report of the presence of such peptides in frogs of the family Pseudidae. The pseudins were not released into norepinephrine-stimulated skin secretions under the conditions that led to release of antimicrobial activity in Xenopus leavis (18), Xenopus tropicalis (20) and Dyscophus guineti (21). As shown in Fig. 5, the pseudins are structurally related to each other but show virtually no amino acid sequence similarity to previously characterized antimicrobial peptides isolated
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FIG. 3. A comparison of the primary structures of the pseudins with antimicrobial peptides isolated from the skins of frogs belonging to different anuran species. Magainin-1, magainin-2 and PGLa are from Xenopus laevis, dermaseptin-B is from Phyllomedusa bicolor, bombinin is from Bombina variegata, caerin 1.1 is from Litoria splendida, and uperin 3.6 is from Uperolia mjobergii.
from the skins of frogs belonging to other families. However, circular dichroism studies have shown that pseudin-2 adopts an ␣-helical conformation in an hydrophobic solvent such as 50% trifluoroethanol that is believed to mimic the environment of the phospholipid bilayer of the bacterial cell membrane. A helical wheel diagram demonstrates the amphipathic nature of the helix with the Lys 6, Lys 7, Lys 17, and Glu 14 residues segregating on the same face (data not shown). Thus, pseudins may be classified along with other cationic, amphipathic ␣-helical antimicrobial peptides previously isolated from frog skin (5). These peptides are believed to adopt an ␣-helical conformation on binding to the bacterial cell membrane and form oligomers that
FIG. 4. Circular dichroism spectra of synthetic pseudin-2 in (A) 10 mM sodium phosphate buffer, pH 7.0, and (B) 10 mM sodium phosphate buffer, pH 7.0 containing 50% trifluoroethanol. Molar ellipticity is expressed as deg cm 2 dmol ⫺1.
insert into the hydrophobic interior leading ultimately to cell lysis (22, 23). A striking feature of the properties of pseudin-2 is the low hemolytic activity of the peptide. Using erythrocytes from six human donors, the concentration of pseudin-2 producing 50% hemolysis (HC 50 value) was always greater than 300 M and no significant hemolysis was detected at a concentration less than 50 M. In contrast, previous studies in the laboratory that have used the same assay methodology report HC 50 values of between 4 and 16 M for temporins from Rana grylio (24), 30 M for kassinatuerin-1 from Kassina senegalensis (11) and 130 M for ranatuerin-1 from Rana catesbeiana (25). The MIC value of pseudin-2 measured against E. coli is 25-fold less than the concentration at which hemolysis is detected suggesting that the amino acid sequence of the peptide may form the basis for the design of analogs with therapeutic potential. The biological function of the pseudins is a matter for speculation. No evidence was obtained for the release of pseudins into skin secretions so that it is unclear whether the peptides are playing a physiologically significant role in protecting the organism against invasion by pathogenic microorganisms. During metamor-
FIG. 5. A comparison of the primary structure of the pseudin-2 with a region at the C-terminus of death effector domain-containing testicular molecule (DEFT), a protein predominantly expressed in mammalian testicular germ cells. Amino acids in common are shaded. A gap has been introduced in pseudin-2 to maximize sequence identity.
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phosis, the large tail of the tadpole of the paradoxical frog disappears by a process that involves massive apoptosis. As shown in Fig. 5, the amino acid sequence of pseudin-2 shows two regions of structural similarity with DEFT, a death effector domain-containing molecule that is predominantly expressed in testicular germ cells of a range of mammals (26). The death effector domain is a structural motif of about 80 amino acids that is found in proteins such as procaspase-8 and Fas-associating death domain-containing protein (FADD) and is involved in the formation of a deathinducing signaling complex (DISC) that leads to apoptosis (27). The region of structural similarity with the pseudins in rat and human DEFT lies at the C-terminus of the protein (between residues 272 and 295) rather than in the death effector domain (residues 25–103). Clearly, further studies are warranted to obtain the amino acid sequence of the biosynthetic precursor of pseudin-2 in order to determine whether DEFT and the pseudins are evolutionarily related. In the meantime, it is tempting to speculate that the pseudins are involved in the regulation of the apoptosis that occurs at metamorphosis when the large tadpole becomes a relatively small frog. ACKNOWLEDGMENTS We thank Dr. Donald Babin, Creighton University, for amino acid analyses; Ms. Eva Lovas, Creighton University, for mass spectrometry measurements; and Dr. Luis Marky, University of Nebraska Medical Center, Omaha, for providing facilities for CD spectroscopy. This work was supported by Restoragen Inc. (Lincoln, NE).
REFERENCES 1. Nicolas, P., and Mor, A. (1995) Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu. Rev. Microbiol. 49, 277–304. 2. Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 61–92. 3. Simmaco, M., Mignogna, G., and Barra, D. (1998) Antimicrobial peptides from amphibian skin: What do they tell us? Biopolymers 47, 435– 450. 4. Hancock, R. E. W., Falla, T., and Brown, M. H. (1995) Cationic bactericidal peptides. Adv. Microb. Physiol. 37, 135–175. 5. Tossi, A., Sandri, L., and Giangaspero, A. (2000) Amphipathic, ␣-helical antimicrobial peptides. Biopolymers 55, 4 –30. 6. Helmerhorst, E. J., Reijnders, I. M., van’t Hof, W., Veerman, E. C. I., and Nieuw Amerongen, A. V. (1999) A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides. FEBS Lett. 449, 105–110. 7. Duellman, W. E., and Trueb, L. (1994) Biology of Amphibians, Johns Hopkins Univ. Press, Baltimore and London. 8. Zasloff, M. (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci USA 84, 5449 –5453. 9. Gibson, B. W., Tang, D., Mandrell, R., Kelly, M., and Spindel, E. R. (1991) Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis. J. Biol. Chem. 266, 23103–23111.
10. Simmaco, M., Barra, D., Chiarini, F., Noviello, L., Melchiorri, P., Kreil, G., and Richter, K. (1991) A family of bombinin-related peptides from the skin of Bombina variegata. Eur. J. Biochem. 199, 217–222. 11. Matutte, B., Knoop, F. C., and Conlon, J. M. (2000) Kassinatuerin-I: A peptide with broad-spectrum antimicrobial activity isolated from the skin of the Hyperoliid frog, Kassina senegalensis. Biochem. Biophys. Res. Commun. 268, 433– 436. 12. Simmaco, M., Mignogna, G., Barra, D., and Bossa, F. (1994) Antimicrobial peptides from skin secretions of Rana esculenta. J. Biol. Chem. 269, 11956 –11961. 13. Goraya, J., Wang, Y., Li, Z., O’Flaherty, M., Knoop, F. C., Platz, J. E., and Conlon, J. M. (2000) Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs, Rana luteiventris, Rana berlandieri and Rana pipiens. Eur. J. Biochem. 267, 894 –900. 14. Mor, A., Hani, K., and Nicolas, P. (1994) The vertebrate peptide antibiotics dermaseptins have overlapping structural features but target specific microorganisms. J. Biol. Chem. 269, 31635– 31641. 15. Wechselberger, C. (1998) Cloning of cDNAs encoding new peptides of the dermaseptin family. Biochim. Biophys. Acta 1388, 279 –283. 16. Steinborner, S. T., Currie, G. J., Bowie, J. H., Wallace, J. C., and Tyler, M. J. (1998) New antibiotic caerin 1 peptides from the skin secretion of the Australian tree frog Litoria chloris. Comparison of the activities of the caerin 1 peptides from the genus Litoria. J. Pept. Res. 51, 121–126. 17. Bradford, A. M., Bowie, J. H., Tyler, M. J., and Wallace, J. C. (1996) New antibiotic uperin peptides from the dorsal gland of the Australian toadlet, Uperoleia mjobergi. Aust. J. Chem. 49, 1325–1333. 18. Nutkins, J. C., and Williams, D. H. (1989) Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis. Eur. J. Biochem. 181, 97–102. 19. Barchiesi, F., Colombo, A. L., McGough, D. A., and Rinaldi, M. G. (1994) Comparative study of broth microdilution and macrodilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards’ proposed standards. J. Clin. Microbiol. 32, 2494 –2500. 20. Ali, M. F., Soto, A., Knoop, F. C., and Conlon, J. M. (2001) Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). Biochim. Biophys. Acta, in press. 21. Conlon, J. M., and Kim, J. B. (2000) A protease inhibitor of the Kunitz family from skin secretions of the tomato frog, Dyscophus guineti (Microhylidae). Biochem. Biophys. Res. Commun. 279, 961–964. 22. Oren, Z., and Shai, Y. (1998) Mode of action of linear amphipathic ␣-helical antimicrobial peptides. Biopolymers 47, 451– 463. 23. Huang, H. W. (2000) Action of antimicrobial peptides: Two-state model. Biochemistry 39, 8347– 8352. 24. Kim, J. B., Halverson, T., Basir, Y. J., Dulka, J., Knoop, F. C., and Conlon, J. M. (2000) Purification and characterization of antimicrobial peptides from skin extracts and skin secretions of the North American pig frog Rana grylio. Regul. Pept. 90, 53– 60. 25. Goraya, J., Knoop, F. C., and Conlon, J. M. (1998) Ranatuerins: Antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana. Biochem. Biophys. Res. Commun. 250, 589 –592. 26. Leo, C. P., Hsu, S. Y., McGee, E. A., Salanova, M., and Hsueh, A. J. W. (1998) DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells. Endocrinology 139, 4839 – 4848. 27. Nagata, S. (1997) Apoptosis by death factor. Cell 88, 355–365.
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Pseudin-2: An Antimicrobial Peptide with Low Hemolytic Activity from the Skin of the Paradoxical Frog Loyd Olson III,* Ana Maria Soto,† Floyd C. Knoop,‡ and J. Michael Conlon* ,1 *Department of Biomedical Sciences and ‡Department of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, Nebraska 68178-0405; and †Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198
Received September 28, 2001
Four structurally related peptides (pseudins 1– 4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC ⴝ 2.5 M against Escherichia coli, 80 M against Staphylococcus aureus, and 130 M against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 M. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic ␣-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis. © 2001 Academic Press Key Words: pseudidae; antimicrobial peptide; amphibian skin; amphipathic ␣-helix.
The emergence of strains of pathogenic bacteria and fungi with resistance to commonly used antibiotics has necessitated a search for new types of antimicrobial agents to which these microorganisms will not have developed resistance. Antimicrobial peptides synthesized in granular glands in the skins of certain frogs represent a promising source of such potential therapeutic agents (1, 2). Amphibian antimicrobial peptides, which comprise between 12 and 48 amino acid residues are usually synthesized as members of a structurally related family and are released into skin secretions in a holocrine manner in response to stress or injury (3). The peptides that have been characterized to date are, 1
To whom correspondence and reprint requests should be addressed. Fax: 402-280-2690. E-mail: [email protected].
almost without exception, hydrophobic, cationic and adopt amphipathic ␣-helical conformations on interaction with the bacterial cell membrane (4, 5). Although often displaying broad spectrum antimicrobial activity, the peptides are, to varying degrees, hemolytic against human erythrocytes which severely limits their therapeutic potential (6). The synthesis of cationic ␣-helical antimicrobial peptides in the skin is by no means a universal feature of Anurans. The extant Anurans (at least 3500 species) have been divided into 21 families (7). Antimicrobial peptides of this type have been isolated from skin secretions and/or skin extracts of frogs belonging to genera that are classified in the families Pipidae [Xenopus (8)], Discoglossidae [Bombina (9, 10)], Hyperoliidae [Kassina (11)], Ranidae [Rana (12, 13)], Hylidae [Phyllomedusa (14), Agalychnis (15), and Litoria (16)], and Myobatrachidae [Uperoleia (17)]. In contrast, up to this time, the author’s laboratory has failed to detect the production of cationic ␣-helical antimicrobial peptides in the skins of several species of frogs belonging to the families Pelobatidae, Bufonidae, Microhylidae, and Rhacophoridae (unpublished data). The family Pseudidae comprises four species of semiaquatic frogs, organized in two genera Pseudis and Lysapus, that are found in tropical lowland areas in the eastern part of South America. The paradoxical frog Pseudis paradoxa is so called because the tadpoles of the species attain a length of 220 mm whereas the largest adults rarely exceed a size of 70 mm. This study describes the purification and characterization of four previously undescribed peptides with antimicrobial activity from an extract of the skin of P. paradoxa. MATERIALS AND METHODS Skin secretions. Adult specimens of P. paradoxa (n ⫽ 2) were injected with 0.1 mM norepinephrine (0.5 ml) at a dorsal site and each placed in a buffer solution (100 ml) of composition 50 mM sodium chloride, 25 mM ammonium acetate, pH 7.0, for 5 min (18). The animals were returned to the aquarium and appeared to suffer
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0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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no ill-effects. After centrifugation (4000g for 30 min at 4°C), the supernatants were pooled and pumped onto three Sep-Pak C-18 cartridges (Waters Associates, Milford, MA) connected in series at a flow rate of 2 ml min ⫺1. Bound material was eluted with acetonitrile/ water/trifluoroacetic acid (700:299:1, v/v/v) and freeze-dried. Tissue extraction. Skin (10.1 g) was removed from pithed adult specimens of P. paradoxa (n ⫽ 2) and immediately frozen on dry ice. The frozen tissue was cut into small pieces and extracted by homogenization in ethanol/0.7 M HCl (3:1, v/v; 100 ml) at 0°C using a PowerGen rotor/stator-type homogenizer (Fisher Scientific, Pittsburgh, PA). The homogenate was stirred for 1.5 h at 0°C and centrifuged (4000g for 30 min at 4°C). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000g for 30 min at 4°C), the extract was pumped onto 6 Sep-Pak C-18 cartridges connected in series at a flow rate of 2 ml min ⫺1. Bound material was eluted with acetonitrile/water/trifluoroacetic acid (700:299:1, v/v/v) and freeze-dried. Antimicrobial assays. Activity of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent (50 l) in Mueller–Hinton broth (50 l) with an inoculum (50 l of 10 3 CFU/ml) from an overnight culture of either Escherichia coli (ATCC 25922) or Staphylococcus aureus (NCTC 8325) in 96-well microtiter cellculture plates for 18 h at 37°C in a humidified atmosphere of 5% CO 2 in air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35°C. After incubation, the absorbance at 550 nm of each well was determined using a M.A. Bioproducts Model MA308 microtiter plate reader. Minimal inhibitory concentrations (MICs) were measured by a standard microdilution method (19) and were taken as the lowest concentration where no visible growth was observed. To monitor the validity of the bacterial assays, incubations were carried out in parallel with increasing concentrations of the broad-spectrum antibiotic, bacitracin. Incubations with Candida albicans were carried in parallel with amphotercin B. Purification of the peptides. The frog skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 1% (v/v) trifluoroacetic acid/water (4 ml) and injected onto a (25 ⫻ 1 cm) Vydac 218TP510 (C-18) reversed-phase HPLC column (Separations Group, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml min ⫺1. The concentration of acetonitrile in the eluting solvent was raised to 21% over 10 min and to 63% over 60 min using linear gradients. Absorbance was measured at 214 and 280 nm and fractions (1 min) were collected. The fractions containing antimicrobial activity were successively rechromatographed on (250 ⫻ 4.6 mm) Vydac 214TP54 (C-4) and Vydac 219TP54 (phenyl) columns. The concentration of acetonitrile in the eluting solvent was raised from 28 to 56% over 40 min and the flow rate was 1.5 ml min ⫺1. Structural analysis. Amino acid compositions were determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using a Waters AccQ Tag system with fluorescence detection and separation of the amino acid derivatives by reversed-phase HPLC. Hydrolysis in 5.7 M HCl (24 h at 110°C) of approximately 1 nmol of peptide was carried out. The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems Procise Model 491 sequenator. Electrospray mass spectrometry was carried out using a Perkin– Elmer Sciex API 150EX single quadrupole instrument. The accuracy of mass determinations was ⫾0.02%. Peptide synthesis. Pseudin-2 was synthesized by solid-phase methodology on a 0.025-mmol scale on an Applied Biosystems Model 432A peptide synthesizer using a 4-(2⬘,4⬘-dimethoxy-phenyl-Fmocaminomethyl)phenoxyacetamido-ethyl resin (Applied Biosystems, Foster City, CA). Fmoc amino acid derivatives were activated with O-benzotriazol-1-yl-N,N,N⬘,N⬘-tetramethyluronium hexafluorophosphate (0.075 mmol), 1-hydroxy-benzotriazole hydrate (0.075 mmol)
FIG. 1. Reversed-phase HPLC on a semipreparative Vydac C-18 column of an extract of skin secretions of the paradoxical frog Pseudis paradoxa, after partial purification on Sep-Pak cartridges. The dashed line shows the concentration of acetonitrile in the eluting solvent. The fractions denoted by the bars contained material that inhibited the growth of E. coli.
and diisopropylethylamine (0.150 mmol). The peptide was cleaved from the resin with trifluoroacetic acid/water/thioanisole/1,2-ethanedithiol (99.0/0.50/0.25/0.25) at 25°C for 3 h. The synthetic material was purified to near homogeneity by chromatography on a 1 ⫻ 25-cm Vydac 218TP510 C-18 reversed-phase HPLC column using the elution conditions shown in Fig. 2A. Hemolytic assay. Peptides in the concentration range 100 –1000 M were incubated with washed human erythrocytes (2 ⫻ 10 7 cells) from healthy donors (n ⫽ 6) in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 l) for 1 h at 37°C. After centrifugation (12,000g for 15 s), the absorbance at 541 nm of the supernatant was measured. A parallel incubation in the presence of 3% v/v Tween 20 was carried out to determine the absorbance associated with 100% hemolysis. The HC 50 value was taken as the concentration of peptide producing 50% hemolysis. Circular dichroism. The circular dichroism spectrum of pseudin-2 was recorded at 25°C using a Model 202SF stopped flow circular dichroism spectrophotometer (Aviv Instruments, Lakewood, NJ). Spectra were recorded in a 1 mm path length cell at a peptide concentration of 100 M in two solvents: (a) 10 mM sodium phosphate buffer, pH 7.0, and (b) 10 mM sodium phosphate buffer, pH 7.0, containing 50% (v/v) trifluoroethanol. In each case, the circular dichroism spectrum of the solvent was subtracted from the spectrum of the peptide.
RESULTS Purification of the Pseudins The antimicrobial activity, measured against E. coli, in the extract of P. paradoxa skin after partial purification on Sep-Pak cartridges was eluted from a semipreparative Vydac C-18 reverse-phase HPLC column in the three fractions denoted by the bars in Fig. 1. Fraction 1 was subsequently shown to contain pseudin-1, fraction 2 contained pseudin-2, and fraction 3 ⫹ 4 contained both pseudin-3 and pseudin-4. The
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Structural and Biological Characterization The amino acid sequences of the pseudins were determined without ambiguity by automated Edman degradation and the primary structures are shown in Fig. 3. The amino acid compositions of the peptides were consistent with their proposed structures. Electrospray mass spectrometry demonstrated that the peptides did not contain a C-terminally ␣-amidated amino acid residue (pseudin-1: observed M r 2715.4, calculated M r 2715.2; pseudin-2: observed M r 2685.4, calculated M r 2685.2; pseudin-3: observed M r 2569.6, calculated M r 2570.0; pseudin-4: observed M r 2512.0, calculated M r 2511.9). The primary structure of pseudin-2 was confirmed by chemical synthesis. A mixture of endogenous and synthetic pseudin-2 was eluted from an analytical Vydac C-18 column under the conditions of chromatography shown in Fig. 2A as a single symmetric peak. The minimal inhibitory concentrations of the endogenous peptides against the gram-negative bacterium E. coli were pseudin-1 4.5 M, pseudin-2 2.5 M, pseudin-3 12 M, and pseudin-4 6.5 M. The minimal inhibitory concentration of pseudin-2 against the gram-positive bacterium Staphylocococcus aureus was 80 and 130 M against the yeast, Candida albicans. In six independent experiments using human erythrocytes from different donors, the HC 50 value of synthetic pseudin-2 ranged from 320 to 550 M. Circular Dichroism
FIG. 2. Purification of pseudin-2 on analytical (A) Vydac C-4, and (B) Vydac phenyl columns. The peaks containing material that inhibited the growth of E. coli are denoted by (⫹) and the arrows show where peak collection began and ended.
same procedure was used to purify all the peptides and so only the purification of pseudin-2 is described. Rechromatography of fraction 2 (Fig. 1) on an analytical Vydac C-4 column led to the elution of two prominent and well-resolved peaks (Fig. 2A). The later eluting peak inhibited the growth of E. coli and contained pseudin-2. The peptide was purified to near homogeneity, as assessed by peak symmetry and mass spectrometry by a final chromatography on analytical Vydac phenyl column (Fig. 2B). The approximate yields of the pure peptides were pseudin-1, 43 nmol; pseudin-2, 221 nmol; pseudin-3, 39 nmol; and pseudin-4, 33 nmol. Norepinephrine-stimulated skin secretions from P. paradoxa, after concentration on Sep-Pak cartridges, did not inhibit the growth of either E. coli or S. aureus and pseudins were not detected by mass spectrometry following chromatography of the secretions under the conditions shown in Fig. 1.
As shown in Fig. 4, the circular dichroism spectrum of pseudin-2 was markedly solvent-dependent. In aqueous sodium phosphate buffer, pH 7.0, the presence of a broad negative band near 199 nm in the spectrum indicates that the peptide exists predominantly in either an unordered conformation or there is a rapid interconversion of multiple conformations. In the more hydrophobic solvent (50% trifluoroethanol), the negative bands centered at 208 and 222 nm and the positive band centered at 193 nm are indicative of significant ␣-helical character. DISCUSSION The article has described the purification and structural characterization of four antimicrobial peptides (pseudins) from an extract of the skin of the paradoxical frog, Pseudis paradoxa. This is the first report of the presence of such peptides in frogs of the family Pseudidae. The pseudins were not released into norepinephrine-stimulated skin secretions under the conditions that led to release of antimicrobial activity in Xenopus leavis (18), Xenopus tropicalis (20) and Dyscophus guineti (21). As shown in Fig. 5, the pseudins are structurally related to each other but show virtually no amino acid sequence similarity to previously characterized antimicrobial peptides isolated
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FIG. 3. A comparison of the primary structures of the pseudins with antimicrobial peptides isolated from the skins of frogs belonging to different anuran species. Magainin-1, magainin-2 and PGLa are from Xenopus laevis, dermaseptin-B is from Phyllomedusa bicolor, bombinin is from Bombina variegata, caerin 1.1 is from Litoria splendida, and uperin 3.6 is from Uperolia mjobergii.
from the skins of frogs belonging to other families. However, circular dichroism studies have shown that pseudin-2 adopts an ␣-helical conformation in an hydrophobic solvent such as 50% trifluoroethanol that is believed to mimic the environment of the phospholipid bilayer of the bacterial cell membrane. A helical wheel diagram demonstrates the amphipathic nature of the helix with the Lys 6, Lys 7, Lys 17, and Glu 14 residues segregating on the same face (data not shown). Thus, pseudins may be classified along with other cationic, amphipathic ␣-helical antimicrobial peptides previously isolated from frog skin (5). These peptides are believed to adopt an ␣-helical conformation on binding to the bacterial cell membrane and form oligomers that
FIG. 4. Circular dichroism spectra of synthetic pseudin-2 in (A) 10 mM sodium phosphate buffer, pH 7.0, and (B) 10 mM sodium phosphate buffer, pH 7.0 containing 50% trifluoroethanol. Molar ellipticity is expressed as deg cm 2 dmol ⫺1.
insert into the hydrophobic interior leading ultimately to cell lysis (22, 23). A striking feature of the properties of pseudin-2 is the low hemolytic activity of the peptide. Using erythrocytes from six human donors, the concentration of pseudin-2 producing 50% hemolysis (HC 50 value) was always greater than 300 M and no significant hemolysis was detected at a concentration less than 50 M. In contrast, previous studies in the laboratory that have used the same assay methodology report HC 50 values of between 4 and 16 M for temporins from Rana grylio (24), 30 M for kassinatuerin-1 from Kassina senegalensis (11) and 130 M for ranatuerin-1 from Rana catesbeiana (25). The MIC value of pseudin-2 measured against E. coli is 25-fold less than the concentration at which hemolysis is detected suggesting that the amino acid sequence of the peptide may form the basis for the design of analogs with therapeutic potential. The biological function of the pseudins is a matter for speculation. No evidence was obtained for the release of pseudins into skin secretions so that it is unclear whether the peptides are playing a physiologically significant role in protecting the organism against invasion by pathogenic microorganisms. During metamor-
FIG. 5. A comparison of the primary structure of the pseudin-2 with a region at the C-terminus of death effector domain-containing testicular molecule (DEFT), a protein predominantly expressed in mammalian testicular germ cells. Amino acids in common are shaded. A gap has been introduced in pseudin-2 to maximize sequence identity.
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phosis, the large tail of the tadpole of the paradoxical frog disappears by a process that involves massive apoptosis. As shown in Fig. 5, the amino acid sequence of pseudin-2 shows two regions of structural similarity with DEFT, a death effector domain-containing molecule that is predominantly expressed in testicular germ cells of a range of mammals (26). The death effector domain is a structural motif of about 80 amino acids that is found in proteins such as procaspase-8 and Fas-associating death domain-containing protein (FADD) and is involved in the formation of a deathinducing signaling complex (DISC) that leads to apoptosis (27). The region of structural similarity with the pseudins in rat and human DEFT lies at the C-terminus of the protein (between residues 272 and 295) rather than in the death effector domain (residues 25–103). Clearly, further studies are warranted to obtain the amino acid sequence of the biosynthetic precursor of pseudin-2 in order to determine whether DEFT and the pseudins are evolutionarily related. In the meantime, it is tempting to speculate that the pseudins are involved in the regulation of the apoptosis that occurs at metamorphosis when the large tadpole becomes a relatively small frog. ACKNOWLEDGMENTS We thank Dr. Donald Babin, Creighton University, for amino acid analyses; Ms. Eva Lovas, Creighton University, for mass spectrometry measurements; and Dr. Luis Marky, University of Nebraska Medical Center, Omaha, for providing facilities for CD spectroscopy. This work was supported by Restoragen Inc. (Lincoln, NE).
REFERENCES 1. Nicolas, P., and Mor, A. (1995) Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu. Rev. Microbiol. 49, 277–304. 2. Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 61–92. 3. Simmaco, M., Mignogna, G., and Barra, D. (1998) Antimicrobial peptides from amphibian skin: What do they tell us? Biopolymers 47, 435– 450. 4. Hancock, R. E. W., Falla, T., and Brown, M. H. (1995) Cationic bactericidal peptides. Adv. Microb. Physiol. 37, 135–175. 5. Tossi, A., Sandri, L., and Giangaspero, A. (2000) Amphipathic, ␣-helical antimicrobial peptides. Biopolymers 55, 4 –30. 6. Helmerhorst, E. J., Reijnders, I. M., van’t Hof, W., Veerman, E. C. I., and Nieuw Amerongen, A. V. (1999) A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides. FEBS Lett. 449, 105–110. 7. Duellman, W. E., and Trueb, L. (1994) Biology of Amphibians, Johns Hopkins Univ. Press, Baltimore and London. 8. Zasloff, M. (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci USA 84, 5449 –5453. 9. Gibson, B. W., Tang, D., Mandrell, R., Kelly, M., and Spindel, E. R. (1991) Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis. J. Biol. Chem. 266, 23103–23111.
10. Simmaco, M., Barra, D., Chiarini, F., Noviello, L., Melchiorri, P., Kreil, G., and Richter, K. (1991) A family of bombinin-related peptides from the skin of Bombina variegata. Eur. J. Biochem. 199, 217–222. 11. Matutte, B., Knoop, F. C., and Conlon, J. M. (2000) Kassinatuerin-I: A peptide with broad-spectrum antimicrobial activity isolated from the skin of the Hyperoliid frog, Kassina senegalensis. Biochem. Biophys. Res. Commun. 268, 433– 436. 12. Simmaco, M., Mignogna, G., Barra, D., and Bossa, F. (1994) Antimicrobial peptides from skin secretions of Rana esculenta. J. Biol. Chem. 269, 11956 –11961. 13. Goraya, J., Wang, Y., Li, Z., O’Flaherty, M., Knoop, F. C., Platz, J. E., and Conlon, J. M. (2000) Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs, Rana luteiventris, Rana berlandieri and Rana pipiens. Eur. J. Biochem. 267, 894 –900. 14. Mor, A., Hani, K., and Nicolas, P. (1994) The vertebrate peptide antibiotics dermaseptins have overlapping structural features but target specific microorganisms. J. Biol. Chem. 269, 31635– 31641. 15. Wechselberger, C. (1998) Cloning of cDNAs encoding new peptides of the dermaseptin family. Biochim. Biophys. Acta 1388, 279 –283. 16. Steinborner, S. T., Currie, G. J., Bowie, J. H., Wallace, J. C., and Tyler, M. J. (1998) New antibiotic caerin 1 peptides from the skin secretion of the Australian tree frog Litoria chloris. Comparison of the activities of the caerin 1 peptides from the genus Litoria. J. Pept. Res. 51, 121–126. 17. Bradford, A. M., Bowie, J. H., Tyler, M. J., and Wallace, J. C. (1996) New antibiotic uperin peptides from the dorsal gland of the Australian toadlet, Uperoleia mjobergi. Aust. J. Chem. 49, 1325–1333. 18. Nutkins, J. C., and Williams, D. H. (1989) Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis. Eur. J. Biochem. 181, 97–102. 19. Barchiesi, F., Colombo, A. L., McGough, D. A., and Rinaldi, M. G. (1994) Comparative study of broth microdilution and macrodilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards’ proposed standards. J. Clin. Microbiol. 32, 2494 –2500. 20. Ali, M. F., Soto, A., Knoop, F. C., and Conlon, J. M. (2001) Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). Biochim. Biophys. Acta, in press. 21. Conlon, J. M., and Kim, J. B. (2000) A protease inhibitor of the Kunitz family from skin secretions of the tomato frog, Dyscophus guineti (Microhylidae). Biochem. Biophys. Res. Commun. 279, 961–964. 22. Oren, Z., and Shai, Y. (1998) Mode of action of linear amphipathic ␣-helical antimicrobial peptides. Biopolymers 47, 451– 463. 23. Huang, H. W. (2000) Action of antimicrobial peptides: Two-state model. Biochemistry 39, 8347– 8352. 24. Kim, J. B., Halverson, T., Basir, Y. J., Dulka, J., Knoop, F. C., and Conlon, J. M. (2000) Purification and characterization of antimicrobial peptides from skin extracts and skin secretions of the North American pig frog Rana grylio. Regul. Pept. 90, 53– 60. 25. Goraya, J., Knoop, F. C., and Conlon, J. M. (1998) Ranatuerins: Antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana. Biochem. Biophys. Res. Commun. 250, 589 –592. 26. Leo, C. P., Hsu, S. Y., McGee, E. A., Salanova, M., and Hsueh, A. J. W. (1998) DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells. Endocrinology 139, 4839 – 4848. 27. Nagata, S. (1997) Apoptosis by death factor. Cell 88, 355–365.
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