Advan. Enzyme Regul., Voi. 32, pp. 117-129, 1992 Printed in Great Britain. All rights reserved
ANTIMICROBIAL FROG
(1065-2571/92/$15.00 © 1992 Pergamon Press pie.
PEPTIDES SKIN
OF
JOHN H. SPENCER Queen's University, Department of Biochemistry, Kingston, ON, Canada, K7L 3N6 PREFACE
Why do birds and mammals have protection from bacterial and fungal infections of the buccal area, the nasal passages, the lungs and the gastrointestinal tract even though these areas are continuously in contact with infectious micro-organisms? One explanation is that there must be a protective system in operation, separate from, but integrated with, the immune system. Do the nonhemolytic antimicrobial peptides found in the skin and intestine of frogs have counterparts in birds and mammals that can provide such protection?
INTRODUCTION
Frog skin has been shown to be a rich source of peptides, many of which have neural or hormonal properties. Erspamer and colleagues were the pioneers in these studies investigating some 40 different peptides of frog skin (1, 2). They identified a taxonomic and evolutionary relationship between the different types of peptides, the species of frogs in which they occurred, and the location of the frogs on various continents (3). Erspamer predicted that for each of the peptides discovered in frog skin there would be a mammalian counterpart (1). This prediction was supported by the identification of analogs of bombesin in the gastrointestinal tract and brain of mammals, of thyrotropin releasing hormone (TRH), a tripeptide, which is identical in frog skin and in mammals, of xenopsin, an octapeptide with neurotensin-like activities and the caeruleins, decapeptides with cholecystokinin and gastrin-like activities. The association of frog skin with healing powers and witchcraft is part of the folklore of many societies in Europe, Asia and South America and was quoted by Shakespeare (4). One of the first reports associating frog skin peptides and bactericidal properties was in 1969 by Csord~s and Michl (5). They described a slimy skin secretion from Bombina variegata with a sharp smell that protects the animal from desiccation and deters its predators. 117
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From the skin exudate they isolated two nonapeptides which were later shown to be part of a carboxy-amidated tetracosapeptide, bombinin (6). Bombinin, which has the potential hydrophobic moment to adopt an a-helix and be amphipathic, has some similarities to melittin, a cytotoxic, hemolytic amphipathic peptide from bee venom. The rate of identification of skin peptides accelerated when more refined methods of separation such as HPLC were developed. Williams' laboratory capitalized on this technique coupling it to fast atom bombardment (FAB) mass spectroscopy and the sequence analysis of peptides, undertaking an extensive analysis of the skin secretions of Xenopus laevis (7-11). They have reported over 30 components present in HPLC chromatograms, and have presented sequence data showing that many of the peptides are part of the precursor proteins of xenopsin, caerulein and PGLa, a peptide of then unknown biological activity. In 1987, Zasloff (12, 13) isolated two peptides from Xenopus laevis skin, magainins-1 and -2 (MGN), that exhibited broad range anti-microbial properties and speculated that they may be part of a skin protection mechanism against microbial infection. The theory was strengthened with the demonstration that two other peptides present in skin exudates, PGLa and xenopsin precursor fragment (XPF), a peptide that is part of the xenopsin precursor pre-pro-protein, also possess similar antimicrobial properties (14). Thus in Xenopus laevis, a peptide with antimicrobial activity (XPF) and a peptide with neural activity (xenopsin) are synthesized together in a pre-pro-protein and are secreted in skin exudates triggered by epinephrine. This observation has since been extended to other precursor pre-pro-proteins (Fig. 2). The integration of neural, hormonal and antimicrobial properties of peptides in frog skin secretions has led to the development of a plausible hypothesis, that the frogs utilize their own nervous system to produce substances which are targeted at the nervous systems of predators (15). This is supported by the observation of oral dyskinesia, or yawning, in water snakes when in contact with Xenopus laevis, slowing ingestion and allowing the frog to escape when attacked by a snake (16, 17). Frog skin peptides have been the subject of a recent review by Bevins and Zasloff (15), in which they point out that although the identification of a biological property may be certain, it is not necessarily linked to the physiological function of that material.
Occurrence and Organization All of the peptides with antibacterial properties so far identified in
Xenopus laevis frog skin and frog skin exudates are synthesized as pre-pro-proteins (Figs. 1 and 2). They are cationic and form amphipathic
LPF XPF ii'i• ¥Y ~ ~ MGNI&2 ~ F
A.
I
S
I
I ~
P~
I M A S~A S~A A i ~ii~P Q P
pGLa XPF LPF MGN-1 MGN-2 CPF
B.
K I AK K I AK K I AK FGKA FGKA AALK AALK AGLK AGLK AALK AALK AALK AALK AG LK AA LK AALK
ANTIBACTERIAL
G M A S K AGAIAG GWASK IGQTLG GWASK IGQTLG G I U K F LHSAGK G I G K F LHSAKK GFGSF LGKALK GFGSF LGKALK GLASL LGKALK GFGSF LGKALK GFGSF LGKALK GLASL LGKALK GFGSF LGKALK GLASL LGKALK GLASF LGKALK GFASF LGKALK GFASF LGKALK VALKAL'G V G L K Q L I Q P V G L Q G L M Q P F V G E I M K S~K F V G E I M N S~K I G A N A L G G S I G A N A L G G A IGTHFLGGAPQQ~R I G T H F L G G A I G A N A L G G S IGANALGGSPQQ~R I G A N A L G G A I G T H F L G G A I G A H L L G G A I G A N M L G G A IGANALGGAPQQ~R
PE~IDE8
FIG. 1. Comparison of the signal sequences and antimicrobial sequences of frog skin peptides.
F L~i
V
81G]k~kL PEPTIDE8
p P P P
Q Q Q Q
Q~R Q ~R Q~R Q ~R
P Q Q ~R P Q Q~R
p Q Q~R P Q Q ~R
K~R K~R
~D
~
CPF
SP
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XPF
PGLa
CPF
AP
AP
AP
SP
SP
SP
SP
SP
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M1
C
C
L
X
~
C AP
I
CPF
AP
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C
C AP
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CPF
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C
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AP
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( pXC 601 )
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M2
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FIG. 2. Organization of the sequences of the frog skin peptides in the cDNAs. SP, signal peptide; AP, acidic peptide; XPF, xenopsin precursor fragment; X, xenopsin; LPF, levitide precursor fragment; L, levitide; M1, magainin-1; M2, magainin-2; CPF, caerulein precursor fragment; C, caerulein; vertical arrow 1', site of deletion.
Caerulein
Magainin
Levitide
Xenopsin
PGLa
PRECURSOR
Z
FROG SKIN PEP'rIDES
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a-helices in the presence of trifluoroethanol (18) or other lipophilic agents. Each precursor protein contains a signal sequence and these exhibit extensive sequence homology amongst each other (Fig. 1A). The antimicrobial peptides show some sequence similarity within subsets, for example PGLa, XPF and LPF, but there is no overall sequence homology, so the biological properties must be explained by other features. A lot of the data on the organization of the pre-pro-proteins comes from cDNA sequence analyses and comparison with peptide sequence data (Fig. 2) (8-12, 19-21). These data have allowed predictions of processing mechanisms and of thetypes of proteases that would be required. During processing PGLa, xenopsin, levitide, and the caeruleins become amidated at their carboxy-terminii. The reaction occurs by transamidation from glycine since it is the next amino acid residue to their carboxy terminii. XPF, LPF, MGN and CPF are secreted in frog skin exudates with the carboxy terminal amino acid unmodified. However, many studies have used chemically synthesized peptides with the carboxy terminus amidated. All of the processed peptides have a glycine at the amino terminus, due to processing by an Arg-Arg-Lys ~ Gly peptidase. The spacer regions (Fig. 2) are processed by a dipeptidase that cleaves at X-Pro, X-GIy and X-AIa. Based on the dipeptidase predictive processing of the spacer regions it was proposed that acidic peptides would probably be released from the MGN precursor peptide (Fig. 2) (11). These were searched for by Williams' group and the three different types of acidic peptides based on slight differences in sequence of the spacer regions that would be generated from the MGN precursor were found, identified and sequenced (11). Williams' group has also identified and sequenced similar acidic peptides from PGLa, XPF, LPF and CPF precursor proteins (22). The role and biological activity of these acidic peptides is unknown but their occurrence in skin exudates is intriguing. Their structural properties make them likely candidates for neural-peptide activity. The PGLa precursor protein is the simplest, organization-wise, of the antibiotic pre-pro-peptides comprising a leader sequence, a signal sequence, a spacer sequence containing an acid peptide, followed by PGLa (Table 1 and Fig. 2). A second cDNA of PGLa precursor has been reported (23) but Xenopus laevis is tetraploid, thus whether it is an allelic form or a true second gene copy is unknown. PGLa since it is carboxy amidated is less susceptible to proteolysis having the longest half life of all the antimicrobial skin peptides after processing and secretion. The xenopsin precursor protein includes one copy each of a signal peptide, xenopsin precursor fragment (XPF), an acid peptide, and the octapeptide xenopsin. Although only one cDNA sequence has been examined in detail, there is clear evidence that there are at least two xenopsin pre-pro-precursor molecules (8). Again, these may be allelic forms due to the tetraploidy of
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TABLE 1. PRECURSORPRE-PRO-PROTEINCOMPONENTS Antibacterial peptide PGLa XPF LPF MGN CPF
Hormone/neural peptide None Xenopsin Levitide None Caerulein
Data from cDNA sequences and peptide sequences. All pre-proproteins have signal peptides and acid peptides (Fig. 2).
Xenopus laevis. However, the possibility of a second gene or multiple gene copies has not been eliminated. The most recent peptide identified from Xenopus laevis skin exudates is levitide (10). Pre-pro-levitide is very similar to pre-pro-xenopsin, but they are different gene products. Levitide itself is a small tetradecapeptide with no known biological properties but its similarity to xenopsin indicates putative neural-peptide activity. The magainins are not associated with any known neuropeptides (Table 1). They are 23 amino acid peptides, MGN-1 differing from MGN-2 by two amino acids only (12). The organization of the magainins in the pre-pro-protein has the signal peptide followed by a leader peptide and then MGN-1 followed by a spacer region and then five alternating copies of MGN-2 and spacer regions (Fig. 2). A similar organization of frog skin peptides, the dermenkephalin and dermorphin opioid peptides from Phyllomedusa sauvagii, has been reported (24). The MGN spacer regions include the acid peptides found in skin exudates. There are no reports of multiple cDNA copies of MGN. The caeruleins have the most complex precusor organization (8, 19, 21). There are multiple copies of identical caeruleins in different precursors (Fig. 2). The precursors all comprise an identical signal sequence, various copies of identical caeruleins, various copies of caerulein precursor fragments (CPF) with slight sequence variations (Fig. 1) and various copies of identical acid peptides. The complexity of the sequence arrangements can be explained by a series of deletions and transpositions (Fig. 2). At the genetic level the sizes of the various genes differ quite extensively. There is a similar exon pattern in the genes, for the precursors of PGLa, xenopsin, levitide, and caerulein (25, 26). Exon-2 codes for the signal peptide in each case, which is compatible with the similarity in their sequences. Clearly this exon is very significant in this group of peptides, having been preserved through evolutionary time. On the basis of computer analysis of the sequences it has been suggested that all of the peptides and precursors arise from a common ancestral gene and then have been
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subjected to duplications and exon shuffling (27). This is probably somewhat of a simplistic overview at the present time since complete sequence data are not yet available. There are no data available on the magainin gene organization but the similarity of the signal sequence indicates that it probably will have an exon-2 organization similar to the other antimicrobial peptides. Mechanism of Action All of the frog skin antimicrobial peptides, when plotted on the Schiffer-Edmunson a-helical wheel (28), reveal amphipathic structures. Amphipathic structures have been linked with membrane penetration and formation of channel structures. Thus the presence of a signal peptide, the ot-helicity, and nature of the amphipathic helix, led to speculations that the bacteriostatic action was possibly due to channel formation resulting in disruption of permeability of the bacterial membrane (13). Evidence supporting this idea has come from a variety of sources. Two dimensional NMR spectroscopy of MGN-2 in the presence of small amounts of trifluoroethanol revealed the formation of an a-helix (18). The transition involves the entire peptide and the rate of change indicated that association of individual peptides also occurs. Lowering the pH of an aqueous solution of MGN-2 and increasing the salt concentration resulted in the peptide self-polymerizing into long filaments (29). This observation and the NMR studies linked the formation of an oL-helix in a hydrophobic environment with the association of the peptides. Lipid bilayer and patch clamp studies (30, 31) showed that MGN-2 forms anion channels which transport chloride and metasulfate and may involve 3 to 6 monomers of MGN per channel. CD spectroscopy revealed a-helix formation with phosphatidylserine vesicles but not with phosphatidylcholine vesicles (32). Other acidic phospholipids also induce a-helix formation and it was suggested that the positive N-terminal region of MGN interacts with the acidic lipid membranes inducing a-helix formation. The a-helix can bind shallowly to the hydrophobic regions of the membrane with the helix axis parallel to the membrane surface, affecting membrane permeability. In a theoretical analysis Guy and Raghunathan (33) proposed channels with 12 MGN monomers arranged in a rosette with the hydrophilic groups in the centre forming the anion channel and the hydrophobic groups in contact with the lypophilic parts of the membrane. They pointed out that the a-helix must aggregate first on the membrane surface and then cross the membrane. Westerhoff et al. (34, 35) used the concept of mitochondria originating from bacteria as a rationale for studies of the effect of MGNs on
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mitochondrial metabolism. They showed in a series of elegant studies that MGN resulted in dissipation of the mitochondrial membrane potential resulting in uncoupling of respiration and inhibition of the uncoupled respiration, consistent with the MGN's acting as ionophores or ion channels and an OH- efflux causing the uncoupling. In bacteria this would result in the death of the bacteria. The respiratory rate was linked to the concentration of MGN and they predicted that an ion channel would be formed by four to six MGN molecules presumably arranged in a rosette (34). Gram-negative bacteria have a double membrane, an outer lipopolysaccharide membrane and an inner plasma membrane. Salmonella typhimurium with its well-defined lipopolysaccharide (LPS) outer membrane surface and availability of LPS mutants of known structure has been used as a model for studies of the interaction of MGN with the surface LPS. Viability tests of LPS mutants exposed to MGN showed the intact LPS, smooth parent strain, was least sensitive (36, 37). Sensitivity increased as the polysaccharide length decreased in the mutants (degree of roughness increased). VI'-IR spectroscopy, used as a measure of the phase behavior of LPS, revealed that MGN disorders the LPS (38). The disordering was more related to LPS charge than length of the LPS polysaccharide. The results indicate that the MGN effects are associated with facilitation of the insertion of MGN into the bacterial membrane. All of these studies indicate that the or-helical amphipathic properties of the antimicrobial peptides are a very important feature in the mechanism of action, not the primary sequence per se. This view is supported by other data that show that analogs of MGN with more a-helix content are more potent in their antimicrobial action (39, 40); also Raman and FT-IR spectroscopic studies show that the peptides form a-helical structures when mixed with acidic phospholipid liposomes (41), a far more natural type of experimental situation than in the presence of trifluoroethanol. Using FT-IR (M. Jackson et al., submitted) we have confirmed that MGN, XPF, and PGLa all form a-helical structures in the presence of phosphatidylserine liposomes over a range of pD. They also exhibit a small amount of ~-sheet with increasing pD, presumably at the carboxyl terminus of the peptide. This latter finding agrees with data from CD spectra (40). v r - I R spectra of MGN with no amide at the carboxy terminus show no helicity at high pD but good or-helix structure at pD5 again with some [~-sheet structure. Note that natural frog MGN has no amide group and the antibacterial activity is half that of the amidated peptide (42). PGLa is less sensitive to pD and shows evidence of surface aggregation under some conditions. Combining all these data, an explanation of the mechanism of action is emerging. Antibacterial peptides secreted from glands in the frog skin into an aqueous medium contact the surface of bacteria. The
125
FROG SKIN PEPTIDES
positively charged amino terminus of the peptides interact with acidic lipid membrane moieties, inducing a-helix formation and polymerization. The a-helical structure binds shallowly and parallel to the hydrophobic surface and starts to penetrate the membrane. Binding must be transient, otherwise the non-polar face of the helix which is toward the solvent is in an energetically unfavorable position. Penetration of the membrane will continue, the peptide structure finally being held in place by the 13-sheet portion which remains in the aqueous phase, and the channel is formed. If the peptides are polymerized end on end both membranes could be penetrated. The channel allows chloride to enter the cell and there is an efflux of OH- resulting in uncoupling of respiration which kills the bacteria. The non-hemolytic property of the peptides may be linked to cholesterol in cell membranes since zwitterions such as phosphatidyl choline do not induce formation of a-helices in these peptides.
Mammalian Counterparts to the Frog Skin Antibacterial Peptides As noted in the Introduction, Erspamer predicted mammalian counterparts to all frog skin peptides (1). Since the antimicrobial frog skin peptides have little sequence homology as a group, it is unlikely that any nucleic acid hybridization approach would be successful in identifying mammalian counterparts to them. The other approaches used in the search have centered either on isolation of peptides directly from acid tissue extracts or using
TABLE 2. HUMAN TISSUE REACTIVITY TO POLYCLONAL MAGAININ-2 ANTIBODY Non-reactive Brain Choroid plexus Arteries (muscular and elastic) Muscle Peripheral nerve Dorsal root and sympathetic ganglia Cardiac muscle Pleura and alveoli Thymus Placenta Pancreas Spleen Liver
Kidney Ureter Bladder Stomach Small intestine Prostate Ovary Vagina Skin Cartilage Testicle Ductus epididymis (testis)
Data from Ramsay et al. (submitted).
Reactive Microvillus lining of epithelial cells of the crypts of the colon (infrequent) Ciliated Tissues Ependyma Trachea Bronchi Fallopian tube Spermatozoa Rete testis Ductuli efferentes (testis) Uterus - - Proliferative endometrium
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immunohistochemical techniques. The immunohistochemical process has been the method of choice. Since the peptides are small, 21 to 35 amino acids in length, they can be synthesized chemically and polyclonal antibodies raised against them which, when affinity purified, are very specific in reactivity. Using such a polyclonal magainin antibody, reactivity has been demonstrated in the submandibular and labial salivary glands of the human but not in bovine, rat, hamster, and mouse (43). This immunohistochemical reactivity of the magainin antibody with the salivary glands has not been confirmed by isolation of the peptides to date. In a similar series of investigations Ramsey et al. (submitted) used identical polyclonal antibodies to synthetic magainin to investigate reactivity in a wide variety of human tissues (Table 2). Reactivity was found in the bronchii, the ependyma of the brain, the fete testes, fallopian tubes, the proliferative endometrium of the uterus and it was absent in control tissues such as the liver and kidney (Table 2). All of the tissues which showed positive reactivity are ciliated and close examination of the staining revealed that it was the apical portions of the cilia which were stained. In the bronchii, the proliferative endometrium and the ductuli efferentes of the testes some epithelial cells were stained. On western blots of acid extracts of tissues the magainin antibody reacted to magainin when it was included as a control peptide but there was no staining of tubulin or any other identifiable bands. Thus, although the histochemistry is very clearly associated with cilia, the reasons for this association are unknown. The authors have speculated that two types of reaction may be occurring. In one reaction, the polyclonal antibody is reacting with an unknown protein in the cilia which has an epitope to MGN. In the second reaction the cellular staining (reaction) indicates the presence of small amounts of a magainin type peptide in the human bronchus, uterus, and testes. The secretion of a magainin type peptide from the ciliated cells, which accumulates on the surface of the cilia as an aggregate, has not been excluded. SUMMARY
A mechanism of action for frog skin antimicrobial peptides has been proposed, based on the amphipathic nature of the peptides when they contact bacterial surfaces. This results in anion channel formation and penetration of the membrane which allows efflux of OH- and uncoupling of respiration in the bacteria. The question of occurrence of human antimicrobial peptides analogous to those in frogs has not been answered but early studies indicate that Erspamer's prediction is correct.
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ACKNOWLEDGEMENTS
The author thanks L. Hellinga and A. Phelan for preparation of the manuscript and H. Metz for Figure 2. Studies in the author's laboratory were funded by a grant from the School of Graduate Studies and Research, Queen's University, Kingston, Ontario, Canada. REFERENCES 1. V. ERSPAMER and P. MELCHIORRI, Active polypeptides of the amphibian skin and their synthetic analogues, Pure Appl. Chem. 35,463-494 (1973). 2. V. ERSPAMER, G. FALCONIER ERSPAMER, G. MAZZANTI and R. ENDEAN, Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea, Comp. Biochem. Physiol. C. 77, 99-108 (1984). 3. J. M. CEI, Taxonomic and evolutionary significance of peptides in amphibian skin, Peptides 6, suppl. 3, 13-16 (1985). 4. W. SHAKESPEARE, in Macbeth, Act IV, Scene 1, lines 6-9, Cambridge University Press, Cambridge, England (1623). 5. A. CSORD,~S and H. MICHL, Primary structure of two oligopeptides of the toxin of bombina variegata L., Toxicon 7, 103-108 (1969). 6. A. CSORDAS and H. MICHL, Isolierung und strukturaufkl~irung eines h~imolytisch wirkenden polypeptides aus dem abwehrsekret europ~iischer unken, Monat Chemie 101, 182-189 (1970). 7. B . W . GIBSON, L. POULTER and D. H. WILLIAMS, A mass spectrometric assay for novel peptides: application to Xenopus laevis skin secretions, Peptides 6, suppl. 3, 23-27 (1985). 8. B. W GIBSON, L. POULTER, D. H. WILLIAMS and J. E. MAGGIO, Novel peptide fragments originating from PGL a and the caerulein and xenopsin precursors from Xenopus laevis, J. Biol. Chem. 261, 5341-5349 (1986). 9. M. G. GIOVANNINI, L. POULTER, B. W. GIBSON and D. H. WILLIAMS, Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones, Biochem. J. 243, 113-120 (1987). 10. L. POULTER, A. S. TERRY, D. H. WILLIAMS, M. G. GIOVANNINI, C. H. MOORE and B. W. GIBSON, Levitide, a neurohormone-like peptide from the skin of Xenopus laevis, J. Biol. Chem. 263, 3279-3283 (1988). 11. A. S. TERRY, L. POULTER, D. H. WILLIAMS, J. C. NUTKINS, M. G. GIOVANNINI, C. H. MOORE and B. W. GIBSON, The cDNA sequence coding for prepro-PGS (prepro-magainins) and aspects of the processing of this prepro-polypeptide, J. Biol. Chem. 263, 5745-5751 (1988). 12. M. ZASLOFF, 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 (1987). 13. M. ZASLOFF, B. MARTIN and H.-C. CHEN, Antimicrobial activity of synthetic magainin peptides and several analogues, Proc. Natl. Acad. Sci. USA 85, 910-913 (1988). 14. E. SORAVIA, G. MARTINI and M. ZASLOFF, Antimicrobial properties of peptides from Xenopus granular gland secretions, FEB& Lett. 228,337-340 (1988). 15. C. L. BEVINS and M. ZASLOFF, Peptides from frog skin, Annu. Rev. Biochem. 59, 395-414 (1990). 16. G. T. BARTHALMUS and W. J. ZIELINSKI, Xenopus skin mucus induces oral dyskinesias that promote escape from snakes, Pharmacol. Biochem. & Behavior 30, 957-959 (1988). 17. G . T . BARTHALMUS, Neuroleptic modulation of oral dyskinesias induced in snakes by Xenopus skin mucus, Pharmacol. Biochem. & Behavior 34, 95-99 (1989). 18. D. MARION, M. ZASLOFF and A. BAX, A two-dimensional NMR study of the antimicrobial peptide magainin 2, FEBS. Lett. 227, 21-26 (1988).
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38.
J . H . SPENCER K. RICHTER, H. A S C H A U E R and G. KREIL, Biosynthesis of peptides in the skin of Xenopus laevis: isolation of novel peptides predicted from the sequence of cloned cDNAs, Peptides 6, 17-21 (1985). I. SURES and M. CRIPPA, Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor, Proc. Natl. Acad. Sci. USA 81, 380-384 (1984). T. WAKABAYASHI, H. KATO and S. TACHIBANA, Complete nucleotide sequence of mRNA for caerulein precursor from Xenopus skin: the mRNA contains an unusual repetitive structure, Nucl. Acids Res. 13, 1817-1828 (1985). J . C . NUTKINS and D. H. WILLIAMS, unpublished work, quoted in ref. 11. W. HOFFMANN, K. RICHTER and G. KREIL, A novel peptide designated PYL a and its precursor as predicted from cloned mRNA of Xenopus laevis skin, EMBO J. 2, 711-714 (1983). A. MOR, A. D E L F O U R and P. NICOLAS, Identification of a o-alanine-containing polypeptide precursor for the peptide opioid, dermorphin, J. Biol. Chem. 266, 6264-6270 (1991). K. KUCHLER, G. KREIL and I. SURES, The genes for the frog skin peptides GLa, xenopsin, ievitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide, Eur. J. Biochem. 179, 281-285 (1989). R. VLASAK, O. WIBORG, K. RICHTER, S. B U R G S C H W A I G E R , J. VUUST and G. KREIL, Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis, Eur. J. Biochem. 169, 53-58 (1987). L. T. HUNT and W. C. BARKER, Relationship of promagainin to three other prohormones from the skin of Xenopus laevis: a different perspective, FEBS. Lett. 233, 282-288 (1988). M. SCHIFFER and A. EDMUNDSON, Use of helical wheels to represent the structures of proteins and to identify segments with helical potential, Biophys J. 7, 121-135 (1967). R. URRUTIA, R. A. CRUCIANI, J. L. B A R K E R and B. KACHAR, Spontaneous polymerization of the antibiotic peptide magainin 2, FEBS. Lett. 247, 17-21 (1989). R. A. CRUC1ANI, E. F. STANLEY, M. ZASLOFF, D. L. LEWIS and J. L. BARKER, The antibiotic magainin II from the African clawed frog forms an anion permeable ionophore in artificial membranes, Biophys. J. 53, 9a (1988). H. DUCLOHIER, G. MOLLE and G. SPACH, Antimicrobial peptide magainin 1 from Xenopus skin forms anion-permeable channels in planar lipid bilayers, Biophys. J. 56, 1017-1021 (1989). K. MATSUZAKI, M. H A R A D A , T. HANDA, S. FUNAKOSHI, N. FUJII, H. YAJIMA and K. MIYAJIMA, Magainin 1-induced leakage of entrapped calcein out of negatively-charged lipid vesicles, Biochim. Biophys. Acta 981,130-134 (1989). H . R . GUY and G. R A G H U N A T H A N , Structural models for membrane insertion and channel formation by antiparallel alpha helical membrane peptides, Jerusalem Symp. Quantum Chem. Biochem. 369-379 (1988). H. V. WESTERHOFF, D. JURETIC, R. W. HENDLER and M. ZASLOFF, Magainins and the disruption of membrane-linked free-energy transduction, Proc. Natl. Acad. Sci. USA 86, 6597--6601 (1989). H. W. WESTERHOFF, R. W. HENDLER, M. ZASLOFF and D. JURETIC, Interactions between a new class of eukaryotic antimicrobial agents and isolated rat liver mitochondria, Biochim. Biophys. Acta 975, 361-369 (1989). E. A. MACIAS, F. R. RANA, J. BLAZYK and M. C. MODRZAKOWSKI, Bactericidal activity of magainin 2: use of lipopolysaccharide mutants, Can. J. Microbiol. 36, 582-584 (1990). F. R. RANA, E. A. MACIAS, C. M. SULTANY, M. C. MODZRAKOWSKI and J. BLAZYK, Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure, Biochemistry 30, 5858-5866 (1991). F. R. RANA, C. M. SULTANY and J. BLAZYK, Interactions between Salmonella
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typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide, FEBS. Lett. 261,464-467 (1990).
39. D. JURETIC, H.-C. CHEN, J. H. BROWN, J. L. MORELL, R. W. HENDLER and H. V. WESTERHOFF, Magainin 2 amide and analogues, FEBS. Lett. 249, 219-223 (1989). 40. H.-C. CHEN, J. H. BROWN, J. L. MORELL and C. M. HUANG, Synthetic magainin analogues with improved antimicrobial activity, FEBS. Lett. 236, 462-466 (1988). 41. R.W. WILLIAMS, R. STARMAN, K. M. P. TAYLOR, K. GABLE, T. BEELER, M. ZASLOFF and D. COVELL, Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa, Biochemistry 29, 4490-4496 (1990). 42. R. W. WILLIAMS, D. COVEL and H.-C. CHEN, Magainin peptide analogs show structure-activity correlations, J. Cell. Biochem. Suppl. 13A, 96 (1989). 43. A. WOLFE, J. E. MOREIRA, C. L. BEVINS, A. R. HAND and P. C. FOX, Magainin-like immunoreactivity in human submandibular and labial salivary glands, J. H istochem, and Cytochem. 38 (11), 1531-1534 (1990).
ANTIMICROBIAL FROG
(1065-2571/92/$15.00 © 1992 Pergamon Press pie.
PEPTIDES SKIN
OF
JOHN H. SPENCER Queen's University, Department of Biochemistry, Kingston, ON, Canada, K7L 3N6 PREFACE
Why do birds and mammals have protection from bacterial and fungal infections of the buccal area, the nasal passages, the lungs and the gastrointestinal tract even though these areas are continuously in contact with infectious micro-organisms? One explanation is that there must be a protective system in operation, separate from, but integrated with, the immune system. Do the nonhemolytic antimicrobial peptides found in the skin and intestine of frogs have counterparts in birds and mammals that can provide such protection?
INTRODUCTION
Frog skin has been shown to be a rich source of peptides, many of which have neural or hormonal properties. Erspamer and colleagues were the pioneers in these studies investigating some 40 different peptides of frog skin (1, 2). They identified a taxonomic and evolutionary relationship between the different types of peptides, the species of frogs in which they occurred, and the location of the frogs on various continents (3). Erspamer predicted that for each of the peptides discovered in frog skin there would be a mammalian counterpart (1). This prediction was supported by the identification of analogs of bombesin in the gastrointestinal tract and brain of mammals, of thyrotropin releasing hormone (TRH), a tripeptide, which is identical in frog skin and in mammals, of xenopsin, an octapeptide with neurotensin-like activities and the caeruleins, decapeptides with cholecystokinin and gastrin-like activities. The association of frog skin with healing powers and witchcraft is part of the folklore of many societies in Europe, Asia and South America and was quoted by Shakespeare (4). One of the first reports associating frog skin peptides and bactericidal properties was in 1969 by Csord~s and Michl (5). They described a slimy skin secretion from Bombina variegata with a sharp smell that protects the animal from desiccation and deters its predators. 117
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From the skin exudate they isolated two nonapeptides which were later shown to be part of a carboxy-amidated tetracosapeptide, bombinin (6). Bombinin, which has the potential hydrophobic moment to adopt an a-helix and be amphipathic, has some similarities to melittin, a cytotoxic, hemolytic amphipathic peptide from bee venom. The rate of identification of skin peptides accelerated when more refined methods of separation such as HPLC were developed. Williams' laboratory capitalized on this technique coupling it to fast atom bombardment (FAB) mass spectroscopy and the sequence analysis of peptides, undertaking an extensive analysis of the skin secretions of Xenopus laevis (7-11). They have reported over 30 components present in HPLC chromatograms, and have presented sequence data showing that many of the peptides are part of the precursor proteins of xenopsin, caerulein and PGLa, a peptide of then unknown biological activity. In 1987, Zasloff (12, 13) isolated two peptides from Xenopus laevis skin, magainins-1 and -2 (MGN), that exhibited broad range anti-microbial properties and speculated that they may be part of a skin protection mechanism against microbial infection. The theory was strengthened with the demonstration that two other peptides present in skin exudates, PGLa and xenopsin precursor fragment (XPF), a peptide that is part of the xenopsin precursor pre-pro-protein, also possess similar antimicrobial properties (14). Thus in Xenopus laevis, a peptide with antimicrobial activity (XPF) and a peptide with neural activity (xenopsin) are synthesized together in a pre-pro-protein and are secreted in skin exudates triggered by epinephrine. This observation has since been extended to other precursor pre-pro-proteins (Fig. 2). The integration of neural, hormonal and antimicrobial properties of peptides in frog skin secretions has led to the development of a plausible hypothesis, that the frogs utilize their own nervous system to produce substances which are targeted at the nervous systems of predators (15). This is supported by the observation of oral dyskinesia, or yawning, in water snakes when in contact with Xenopus laevis, slowing ingestion and allowing the frog to escape when attacked by a snake (16, 17). Frog skin peptides have been the subject of a recent review by Bevins and Zasloff (15), in which they point out that although the identification of a biological property may be certain, it is not necessarily linked to the physiological function of that material.
Occurrence and Organization All of the peptides with antibacterial properties so far identified in
Xenopus laevis frog skin and frog skin exudates are synthesized as pre-pro-proteins (Figs. 1 and 2). They are cationic and form amphipathic
LPF XPF ii'i• ¥Y ~ ~ MGNI&2 ~ F
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K I AK K I AK K I AK FGKA FGKA AALK AALK AGLK AGLK AALK AALK AALK AALK AG LK AA LK AALK
ANTIBACTERIAL
G M A S K AGAIAG GWASK IGQTLG GWASK IGQTLG G I U K F LHSAGK G I G K F LHSAKK GFGSF LGKALK GFGSF LGKALK GLASL LGKALK GFGSF LGKALK GFGSF LGKALK GLASL LGKALK GFGSF LGKALK GLASL LGKALK GLASF LGKALK GFASF LGKALK GFASF LGKALK VALKAL'G V G L K Q L I Q P V G L Q G L M Q P F V G E I M K S~K F V G E I M N S~K I G A N A L G G S I G A N A L G G A IGTHFLGGAPQQ~R I G T H F L G G A I G A N A L G G S IGANALGGSPQQ~R I G A N A L G G A I G T H F L G G A I G A H L L G G A I G A N M L G G A IGANALGGAPQQ~R
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FIG. 1. Comparison of the signal sequences and antimicrobial sequences of frog skin peptides.
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I
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FIG. 2. Organization of the sequences of the frog skin peptides in the cDNAs. SP, signal peptide; AP, acidic peptide; XPF, xenopsin precursor fragment; X, xenopsin; LPF, levitide precursor fragment; L, levitide; M1, magainin-1; M2, magainin-2; CPF, caerulein precursor fragment; C, caerulein; vertical arrow 1', site of deletion.
Caerulein
Magainin
Levitide
Xenopsin
PGLa
PRECURSOR
Z
FROG SKIN PEP'rIDES
121
a-helices in the presence of trifluoroethanol (18) or other lipophilic agents. Each precursor protein contains a signal sequence and these exhibit extensive sequence homology amongst each other (Fig. 1A). The antimicrobial peptides show some sequence similarity within subsets, for example PGLa, XPF and LPF, but there is no overall sequence homology, so the biological properties must be explained by other features. A lot of the data on the organization of the pre-pro-proteins comes from cDNA sequence analyses and comparison with peptide sequence data (Fig. 2) (8-12, 19-21). These data have allowed predictions of processing mechanisms and of thetypes of proteases that would be required. During processing PGLa, xenopsin, levitide, and the caeruleins become amidated at their carboxy-terminii. The reaction occurs by transamidation from glycine since it is the next amino acid residue to their carboxy terminii. XPF, LPF, MGN and CPF are secreted in frog skin exudates with the carboxy terminal amino acid unmodified. However, many studies have used chemically synthesized peptides with the carboxy terminus amidated. All of the processed peptides have a glycine at the amino terminus, due to processing by an Arg-Arg-Lys ~ Gly peptidase. The spacer regions (Fig. 2) are processed by a dipeptidase that cleaves at X-Pro, X-GIy and X-AIa. Based on the dipeptidase predictive processing of the spacer regions it was proposed that acidic peptides would probably be released from the MGN precursor peptide (Fig. 2) (11). These were searched for by Williams' group and the three different types of acidic peptides based on slight differences in sequence of the spacer regions that would be generated from the MGN precursor were found, identified and sequenced (11). Williams' group has also identified and sequenced similar acidic peptides from PGLa, XPF, LPF and CPF precursor proteins (22). The role and biological activity of these acidic peptides is unknown but their occurrence in skin exudates is intriguing. Their structural properties make them likely candidates for neural-peptide activity. The PGLa precursor protein is the simplest, organization-wise, of the antibiotic pre-pro-peptides comprising a leader sequence, a signal sequence, a spacer sequence containing an acid peptide, followed by PGLa (Table 1 and Fig. 2). A second cDNA of PGLa precursor has been reported (23) but Xenopus laevis is tetraploid, thus whether it is an allelic form or a true second gene copy is unknown. PGLa since it is carboxy amidated is less susceptible to proteolysis having the longest half life of all the antimicrobial skin peptides after processing and secretion. The xenopsin precursor protein includes one copy each of a signal peptide, xenopsin precursor fragment (XPF), an acid peptide, and the octapeptide xenopsin. Although only one cDNA sequence has been examined in detail, there is clear evidence that there are at least two xenopsin pre-pro-precursor molecules (8). Again, these may be allelic forms due to the tetraploidy of
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TABLE 1. PRECURSORPRE-PRO-PROTEINCOMPONENTS Antibacterial peptide PGLa XPF LPF MGN CPF
Hormone/neural peptide None Xenopsin Levitide None Caerulein
Data from cDNA sequences and peptide sequences. All pre-proproteins have signal peptides and acid peptides (Fig. 2).
Xenopus laevis. However, the possibility of a second gene or multiple gene copies has not been eliminated. The most recent peptide identified from Xenopus laevis skin exudates is levitide (10). Pre-pro-levitide is very similar to pre-pro-xenopsin, but they are different gene products. Levitide itself is a small tetradecapeptide with no known biological properties but its similarity to xenopsin indicates putative neural-peptide activity. The magainins are not associated with any known neuropeptides (Table 1). They are 23 amino acid peptides, MGN-1 differing from MGN-2 by two amino acids only (12). The organization of the magainins in the pre-pro-protein has the signal peptide followed by a leader peptide and then MGN-1 followed by a spacer region and then five alternating copies of MGN-2 and spacer regions (Fig. 2). A similar organization of frog skin peptides, the dermenkephalin and dermorphin opioid peptides from Phyllomedusa sauvagii, has been reported (24). The MGN spacer regions include the acid peptides found in skin exudates. There are no reports of multiple cDNA copies of MGN. The caeruleins have the most complex precusor organization (8, 19, 21). There are multiple copies of identical caeruleins in different precursors (Fig. 2). The precursors all comprise an identical signal sequence, various copies of identical caeruleins, various copies of caerulein precursor fragments (CPF) with slight sequence variations (Fig. 1) and various copies of identical acid peptides. The complexity of the sequence arrangements can be explained by a series of deletions and transpositions (Fig. 2). At the genetic level the sizes of the various genes differ quite extensively. There is a similar exon pattern in the genes, for the precursors of PGLa, xenopsin, levitide, and caerulein (25, 26). Exon-2 codes for the signal peptide in each case, which is compatible with the similarity in their sequences. Clearly this exon is very significant in this group of peptides, having been preserved through evolutionary time. On the basis of computer analysis of the sequences it has been suggested that all of the peptides and precursors arise from a common ancestral gene and then have been
FROG SKIN PEPTIDES
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subjected to duplications and exon shuffling (27). This is probably somewhat of a simplistic overview at the present time since complete sequence data are not yet available. There are no data available on the magainin gene organization but the similarity of the signal sequence indicates that it probably will have an exon-2 organization similar to the other antimicrobial peptides. Mechanism of Action All of the frog skin antimicrobial peptides, when plotted on the Schiffer-Edmunson a-helical wheel (28), reveal amphipathic structures. Amphipathic structures have been linked with membrane penetration and formation of channel structures. Thus the presence of a signal peptide, the ot-helicity, and nature of the amphipathic helix, led to speculations that the bacteriostatic action was possibly due to channel formation resulting in disruption of permeability of the bacterial membrane (13). Evidence supporting this idea has come from a variety of sources. Two dimensional NMR spectroscopy of MGN-2 in the presence of small amounts of trifluoroethanol revealed the formation of an a-helix (18). The transition involves the entire peptide and the rate of change indicated that association of individual peptides also occurs. Lowering the pH of an aqueous solution of MGN-2 and increasing the salt concentration resulted in the peptide self-polymerizing into long filaments (29). This observation and the NMR studies linked the formation of an oL-helix in a hydrophobic environment with the association of the peptides. Lipid bilayer and patch clamp studies (30, 31) showed that MGN-2 forms anion channels which transport chloride and metasulfate and may involve 3 to 6 monomers of MGN per channel. CD spectroscopy revealed a-helix formation with phosphatidylserine vesicles but not with phosphatidylcholine vesicles (32). Other acidic phospholipids also induce a-helix formation and it was suggested that the positive N-terminal region of MGN interacts with the acidic lipid membranes inducing a-helix formation. The a-helix can bind shallowly to the hydrophobic regions of the membrane with the helix axis parallel to the membrane surface, affecting membrane permeability. In a theoretical analysis Guy and Raghunathan (33) proposed channels with 12 MGN monomers arranged in a rosette with the hydrophilic groups in the centre forming the anion channel and the hydrophobic groups in contact with the lypophilic parts of the membrane. They pointed out that the a-helix must aggregate first on the membrane surface and then cross the membrane. Westerhoff et al. (34, 35) used the concept of mitochondria originating from bacteria as a rationale for studies of the effect of MGNs on
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mitochondrial metabolism. They showed in a series of elegant studies that MGN resulted in dissipation of the mitochondrial membrane potential resulting in uncoupling of respiration and inhibition of the uncoupled respiration, consistent with the MGN's acting as ionophores or ion channels and an OH- efflux causing the uncoupling. In bacteria this would result in the death of the bacteria. The respiratory rate was linked to the concentration of MGN and they predicted that an ion channel would be formed by four to six MGN molecules presumably arranged in a rosette (34). Gram-negative bacteria have a double membrane, an outer lipopolysaccharide membrane and an inner plasma membrane. Salmonella typhimurium with its well-defined lipopolysaccharide (LPS) outer membrane surface and availability of LPS mutants of known structure has been used as a model for studies of the interaction of MGN with the surface LPS. Viability tests of LPS mutants exposed to MGN showed the intact LPS, smooth parent strain, was least sensitive (36, 37). Sensitivity increased as the polysaccharide length decreased in the mutants (degree of roughness increased). VI'-IR spectroscopy, used as a measure of the phase behavior of LPS, revealed that MGN disorders the LPS (38). The disordering was more related to LPS charge than length of the LPS polysaccharide. The results indicate that the MGN effects are associated with facilitation of the insertion of MGN into the bacterial membrane. All of these studies indicate that the or-helical amphipathic properties of the antimicrobial peptides are a very important feature in the mechanism of action, not the primary sequence per se. This view is supported by other data that show that analogs of MGN with more a-helix content are more potent in their antimicrobial action (39, 40); also Raman and FT-IR spectroscopic studies show that the peptides form a-helical structures when mixed with acidic phospholipid liposomes (41), a far more natural type of experimental situation than in the presence of trifluoroethanol. Using FT-IR (M. Jackson et al., submitted) we have confirmed that MGN, XPF, and PGLa all form a-helical structures in the presence of phosphatidylserine liposomes over a range of pD. They also exhibit a small amount of ~-sheet with increasing pD, presumably at the carboxyl terminus of the peptide. This latter finding agrees with data from CD spectra (40). v r - I R spectra of MGN with no amide at the carboxy terminus show no helicity at high pD but good or-helix structure at pD5 again with some [~-sheet structure. Note that natural frog MGN has no amide group and the antibacterial activity is half that of the amidated peptide (42). PGLa is less sensitive to pD and shows evidence of surface aggregation under some conditions. Combining all these data, an explanation of the mechanism of action is emerging. Antibacterial peptides secreted from glands in the frog skin into an aqueous medium contact the surface of bacteria. The
125
FROG SKIN PEPTIDES
positively charged amino terminus of the peptides interact with acidic lipid membrane moieties, inducing a-helix formation and polymerization. The a-helical structure binds shallowly and parallel to the hydrophobic surface and starts to penetrate the membrane. Binding must be transient, otherwise the non-polar face of the helix which is toward the solvent is in an energetically unfavorable position. Penetration of the membrane will continue, the peptide structure finally being held in place by the 13-sheet portion which remains in the aqueous phase, and the channel is formed. If the peptides are polymerized end on end both membranes could be penetrated. The channel allows chloride to enter the cell and there is an efflux of OH- resulting in uncoupling of respiration which kills the bacteria. The non-hemolytic property of the peptides may be linked to cholesterol in cell membranes since zwitterions such as phosphatidyl choline do not induce formation of a-helices in these peptides.
Mammalian Counterparts to the Frog Skin Antibacterial Peptides As noted in the Introduction, Erspamer predicted mammalian counterparts to all frog skin peptides (1). Since the antimicrobial frog skin peptides have little sequence homology as a group, it is unlikely that any nucleic acid hybridization approach would be successful in identifying mammalian counterparts to them. The other approaches used in the search have centered either on isolation of peptides directly from acid tissue extracts or using
TABLE 2. HUMAN TISSUE REACTIVITY TO POLYCLONAL MAGAININ-2 ANTIBODY Non-reactive Brain Choroid plexus Arteries (muscular and elastic) Muscle Peripheral nerve Dorsal root and sympathetic ganglia Cardiac muscle Pleura and alveoli Thymus Placenta Pancreas Spleen Liver
Kidney Ureter Bladder Stomach Small intestine Prostate Ovary Vagina Skin Cartilage Testicle Ductus epididymis (testis)
Data from Ramsay et al. (submitted).
Reactive Microvillus lining of epithelial cells of the crypts of the colon (infrequent) Ciliated Tissues Ependyma Trachea Bronchi Fallopian tube Spermatozoa Rete testis Ductuli efferentes (testis) Uterus - - Proliferative endometrium
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immunohistochemical techniques. The immunohistochemical process has been the method of choice. Since the peptides are small, 21 to 35 amino acids in length, they can be synthesized chemically and polyclonal antibodies raised against them which, when affinity purified, are very specific in reactivity. Using such a polyclonal magainin antibody, reactivity has been demonstrated in the submandibular and labial salivary glands of the human but not in bovine, rat, hamster, and mouse (43). This immunohistochemical reactivity of the magainin antibody with the salivary glands has not been confirmed by isolation of the peptides to date. In a similar series of investigations Ramsey et al. (submitted) used identical polyclonal antibodies to synthetic magainin to investigate reactivity in a wide variety of human tissues (Table 2). Reactivity was found in the bronchii, the ependyma of the brain, the fete testes, fallopian tubes, the proliferative endometrium of the uterus and it was absent in control tissues such as the liver and kidney (Table 2). All of the tissues which showed positive reactivity are ciliated and close examination of the staining revealed that it was the apical portions of the cilia which were stained. In the bronchii, the proliferative endometrium and the ductuli efferentes of the testes some epithelial cells were stained. On western blots of acid extracts of tissues the magainin antibody reacted to magainin when it was included as a control peptide but there was no staining of tubulin or any other identifiable bands. Thus, although the histochemistry is very clearly associated with cilia, the reasons for this association are unknown. The authors have speculated that two types of reaction may be occurring. In one reaction, the polyclonal antibody is reacting with an unknown protein in the cilia which has an epitope to MGN. In the second reaction the cellular staining (reaction) indicates the presence of small amounts of a magainin type peptide in the human bronchus, uterus, and testes. The secretion of a magainin type peptide from the ciliated cells, which accumulates on the surface of the cilia as an aggregate, has not been excluded. SUMMARY
A mechanism of action for frog skin antimicrobial peptides has been proposed, based on the amphipathic nature of the peptides when they contact bacterial surfaces. This results in anion channel formation and penetration of the membrane which allows efflux of OH- and uncoupling of respiration in the bacteria. The question of occurrence of human antimicrobial peptides analogous to those in frogs has not been answered but early studies indicate that Erspamer's prediction is correct.
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ACKNOWLEDGEMENTS
The author thanks L. Hellinga and A. Phelan for preparation of the manuscript and H. Metz for Figure 2. Studies in the author's laboratory were funded by a grant from the School of Graduate Studies and Research, Queen's University, Kingston, Ontario, Canada. REFERENCES 1. V. ERSPAMER and P. MELCHIORRI, Active polypeptides of the amphibian skin and their synthetic analogues, Pure Appl. Chem. 35,463-494 (1973). 2. V. ERSPAMER, G. FALCONIER ERSPAMER, G. MAZZANTI and R. ENDEAN, Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea, Comp. Biochem. Physiol. C. 77, 99-108 (1984). 3. J. M. CEI, Taxonomic and evolutionary significance of peptides in amphibian skin, Peptides 6, suppl. 3, 13-16 (1985). 4. W. SHAKESPEARE, in Macbeth, Act IV, Scene 1, lines 6-9, Cambridge University Press, Cambridge, England (1623). 5. A. CSORD,~S and H. MICHL, Primary structure of two oligopeptides of the toxin of bombina variegata L., Toxicon 7, 103-108 (1969). 6. A. CSORDAS and H. MICHL, Isolierung und strukturaufkl~irung eines h~imolytisch wirkenden polypeptides aus dem abwehrsekret europ~iischer unken, Monat Chemie 101, 182-189 (1970). 7. B . W . GIBSON, L. POULTER and D. H. WILLIAMS, A mass spectrometric assay for novel peptides: application to Xenopus laevis skin secretions, Peptides 6, suppl. 3, 23-27 (1985). 8. B. W GIBSON, L. POULTER, D. H. WILLIAMS and J. E. MAGGIO, Novel peptide fragments originating from PGL a and the caerulein and xenopsin precursors from Xenopus laevis, J. Biol. Chem. 261, 5341-5349 (1986). 9. M. G. GIOVANNINI, L. POULTER, B. W. GIBSON and D. H. WILLIAMS, Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones, Biochem. J. 243, 113-120 (1987). 10. L. POULTER, A. S. TERRY, D. H. WILLIAMS, M. G. GIOVANNINI, C. H. MOORE and B. W. GIBSON, Levitide, a neurohormone-like peptide from the skin of Xenopus laevis, J. Biol. Chem. 263, 3279-3283 (1988). 11. A. S. TERRY, L. POULTER, D. H. WILLIAMS, J. C. NUTKINS, M. G. GIOVANNINI, C. H. MOORE and B. W. GIBSON, The cDNA sequence coding for prepro-PGS (prepro-magainins) and aspects of the processing of this prepro-polypeptide, J. Biol. Chem. 263, 5745-5751 (1988). 12. M. ZASLOFF, 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 (1987). 13. M. ZASLOFF, B. MARTIN and H.-C. CHEN, Antimicrobial activity of synthetic magainin peptides and several analogues, Proc. Natl. Acad. Sci. USA 85, 910-913 (1988). 14. E. SORAVIA, G. MARTINI and M. ZASLOFF, Antimicrobial properties of peptides from Xenopus granular gland secretions, FEB& Lett. 228,337-340 (1988). 15. C. L. BEVINS and M. ZASLOFF, Peptides from frog skin, Annu. Rev. Biochem. 59, 395-414 (1990). 16. G. T. BARTHALMUS and W. J. ZIELINSKI, Xenopus skin mucus induces oral dyskinesias that promote escape from snakes, Pharmacol. Biochem. & Behavior 30, 957-959 (1988). 17. G . T . BARTHALMUS, Neuroleptic modulation of oral dyskinesias induced in snakes by Xenopus skin mucus, Pharmacol. Biochem. & Behavior 34, 95-99 (1989). 18. D. MARION, M. ZASLOFF and A. BAX, A two-dimensional NMR study of the antimicrobial peptide magainin 2, FEBS. Lett. 227, 21-26 (1988).
128 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
38.
J . H . SPENCER K. RICHTER, H. A S C H A U E R and G. KREIL, Biosynthesis of peptides in the skin of Xenopus laevis: isolation of novel peptides predicted from the sequence of cloned cDNAs, Peptides 6, 17-21 (1985). I. SURES and M. CRIPPA, Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor, Proc. Natl. Acad. Sci. USA 81, 380-384 (1984). T. WAKABAYASHI, H. KATO and S. TACHIBANA, Complete nucleotide sequence of mRNA for caerulein precursor from Xenopus skin: the mRNA contains an unusual repetitive structure, Nucl. Acids Res. 13, 1817-1828 (1985). J . C . NUTKINS and D. H. WILLIAMS, unpublished work, quoted in ref. 11. W. HOFFMANN, K. RICHTER and G. KREIL, A novel peptide designated PYL a and its precursor as predicted from cloned mRNA of Xenopus laevis skin, EMBO J. 2, 711-714 (1983). A. MOR, A. D E L F O U R and P. NICOLAS, Identification of a o-alanine-containing polypeptide precursor for the peptide opioid, dermorphin, J. Biol. Chem. 266, 6264-6270 (1991). K. KUCHLER, G. KREIL and I. SURES, The genes for the frog skin peptides GLa, xenopsin, ievitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide, Eur. J. Biochem. 179, 281-285 (1989). R. VLASAK, O. WIBORG, K. RICHTER, S. B U R G S C H W A I G E R , J. VUUST and G. KREIL, Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis, Eur. J. Biochem. 169, 53-58 (1987). L. T. HUNT and W. C. BARKER, Relationship of promagainin to three other prohormones from the skin of Xenopus laevis: a different perspective, FEBS. Lett. 233, 282-288 (1988). M. SCHIFFER and A. EDMUNDSON, Use of helical wheels to represent the structures of proteins and to identify segments with helical potential, Biophys J. 7, 121-135 (1967). R. URRUTIA, R. A. CRUCIANI, J. L. B A R K E R and B. KACHAR, Spontaneous polymerization of the antibiotic peptide magainin 2, FEBS. Lett. 247, 17-21 (1989). R. A. CRUC1ANI, E. F. STANLEY, M. ZASLOFF, D. L. LEWIS and J. L. BARKER, The antibiotic magainin II from the African clawed frog forms an anion permeable ionophore in artificial membranes, Biophys. J. 53, 9a (1988). H. DUCLOHIER, G. MOLLE and G. SPACH, Antimicrobial peptide magainin 1 from Xenopus skin forms anion-permeable channels in planar lipid bilayers, Biophys. J. 56, 1017-1021 (1989). K. MATSUZAKI, M. H A R A D A , T. HANDA, S. FUNAKOSHI, N. FUJII, H. YAJIMA and K. MIYAJIMA, Magainin 1-induced leakage of entrapped calcein out of negatively-charged lipid vesicles, Biochim. Biophys. Acta 981,130-134 (1989). H . R . GUY and G. R A G H U N A T H A N , Structural models for membrane insertion and channel formation by antiparallel alpha helical membrane peptides, Jerusalem Symp. Quantum Chem. Biochem. 369-379 (1988). H. V. WESTERHOFF, D. JURETIC, R. W. HENDLER and M. ZASLOFF, Magainins and the disruption of membrane-linked free-energy transduction, Proc. Natl. Acad. Sci. USA 86, 6597--6601 (1989). H. W. WESTERHOFF, R. W. HENDLER, M. ZASLOFF and D. JURETIC, Interactions between a new class of eukaryotic antimicrobial agents and isolated rat liver mitochondria, Biochim. Biophys. Acta 975, 361-369 (1989). E. A. MACIAS, F. R. RANA, J. BLAZYK and M. C. MODRZAKOWSKI, Bactericidal activity of magainin 2: use of lipopolysaccharide mutants, Can. J. Microbiol. 36, 582-584 (1990). F. R. RANA, E. A. MACIAS, C. M. SULTANY, M. C. MODZRAKOWSKI and J. BLAZYK, Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure, Biochemistry 30, 5858-5866 (1991). F. R. RANA, C. M. SULTANY and J. BLAZYK, Interactions between Salmonella
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