bombinin-like peptides with antimicrobial activity from ... · three peptides have been isolated...

T H E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 34, Issue of December 5, pp. 23103-23111,1991 Printed in U. S. A.

Bombinin-like Peptides with Antimicrobial Activity from Skin Secretions of the Asian Toad, Bombina orientalis*

(Received for publication, May 23, 1991)

Bradford W. Gibson$.$, Dazhi Tang$., Robert Mandrellll, Michele Kelly((, and Eliot R. Spindell1 From the $Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, the 1Veterans Administration Medical Center, San Francisco, California 94121, and the IIOregon Regional Primute Research Center, Beauerton, Oregon 97006

The structures and hemolytic and bactericidal activities of three bombinin-like peptides, or BLP-1-3, from the skin of Bombina orientalis are described. The peptides were isolated from the skin of B. orientalis and sequenced by tandem mass spectrometry and are amphipathic, cationic peptides of 25-27 amino acids in length. The sequence of the most abundant member (BLP-1) is: Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly- Lys-Ser-Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala- Glu-His-Phe-Ala-Asn-NHa.

All three peptides were found to share considerable, but not complete, homology with bombinin, an antimicrobial, hemolytic peptide first isolated by Michl and Csordas (Csordas, A., and Michl, A. (1970) Monatsh. Chem. 101, 182-189) from the skin of Bombina uariegata. The BLPs have been assayed for antibiotic and hemolytic activity and found to be more potent than magainin 2 (a related antimicrobial peptide from Xen- opus laeuis) in their ability to kill bacteria. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic over eukaryotic membranes. The molecular basis for antibacterial activity is presumed to be due to their predicted amphipathic a-helical structures which is supported by circular dichroism measurements that found significant helical content (63-69% a-helix) in 40% trifluoroethanol. Last, a cDNA library was con- structed from the skin of B. orientalis and screened with an oligonucleotide probe complementary to the COOH terminus of BLP- 1. Several clones were isolated and sequenced that encode BLP-1 and BLP-3, as well as an additional peptide (BLP-4) that differs by two amino acid substitutions from BLP-3.

In recent years, a number of peptides have been isolated from various biological sources with potent antimicrobial properties. Some of the better studied members include the cecropins from Cecropia moth (l), the defensins from human

of Health (RR01614 and CA39237) and National Science Foundation * This work was supported by grants from the National Institutes

(DIR 8700766). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

M76483 and M76484. to the GenBankTM/EMBL Data Bank with accession number(s)

To whom all correspondence should be addressed.

neutrophils (2), and the magainins (or PGS’ peptides) from the South African clawed toad, Xenopus laevis (3,4). All these peptides are cationic but otherwise differ considerably in such basic features as their size (Mr ~2300-4000), presence of disulfide bonds, and structural motifs. For example, the cecropins appear to have at least three separate domains, an amino-terminal amphipathic and carboxyl-terminal hydrophobic stretch separated by a short flexible (3-turn sequence that have potential for forming a-helices ( 5 ) . The magainins are the smallest members and appear to exist as single amphipathic a-helices. The defensins take on a more complicated structure that is largely determined by six cysteines that form three internal disulfide bonds (6).

The amphipathic nature of these peptides presumably underlies their biological activities which enables them to asso- ciate with lipid membranes and disrupt normal membrane functions (7). For the magainins and cecropins, evidence points to the formation of ion-selective membrane channels or pores (5, 8). For example, magainin 1 has been found to induce leakage of negatively charged lipid vesicles (9), but there is still considerable debate on the precise mechanism. All three of these peptide families have more than one member, and this sequence diversity may provide a basis for a wide range of antimicrobial activities within each family of peptides. This certainly appears to be the case for the defensins where some members have rather broad activities while others are quite narrow and target a particular organism (10). One intriguing biological feature of the cecropins and the magainins is their reported selectivity for prokaryote over eukaryote cell membranes ( 5 ) . The molecular basis for this selectivity is unknown but may be related to the presence of cholesterol in the eukaryotic cell membrane.

To date, the majority of structural and biological studies on the amphibian members of these antimicrobial peptides has been limited to the magainins and various synthetic analogs, some of which have enhanced antimicrobial activities (11- 13). However, the magainins belong to a much larger family of amphibian peptides with lytic and/or antimicrobial properties (14, 15). Indeed, this group consists of at least four distinct classes from X . laeuis alone, whose overall structure and biological properties are very similar. Two of these classes originate from precursor polypeptides that also encode peptide-like hormones, such as the caerulein precursor fragments (CPFs) (16, 17) and the structurally related xenopsin and

The abbreviations used are: PGS, peptide glycine-serine; CPFs, caerulein precursor fragments; PGLa, peptide glycine-leucine-amide; BLP, bombinin-like peptide; CID, collision-induced dissociation; LSIMS, liquid secondary mass ion spectrometry; MH‘, protonated molecular ion; MS/MS, tandem mass spectrometry; XPF, xenopsin precursor fragment; LPS, lipopolysaccharide(s); HPLC, high pressure liquid chromatography.

23103

23104 Antimicrobial Peptides from B. orientalis Sk in P G L a G M A S K A G A I A G K I A K V A L K A L - N H 2

P G S / r n a g a i n i n - 2 G I G K F L H S A K K F G K A F V G E I M N S B o r n b i n i n G I G A L S A K G A L K G L A K G L A Q H F A N - N H 2

X P F G W A S K I G Q T L G K I A K V G L K E L I Q P K

PGK G W A S K I G Q T L G K I A K V G L Q G L M Q P K

C P F ( 1 + 5 1 G F G S F L G K A L K A A L K I G A N A L G G S P Q Q

FIG. 1. Sequence of PGLa, bombinin, PGS (or magainin 2), XPF, and one representative member of the CPFs family.

levitide precursor fragments (XPF and peptide glycine-lysine, respectively) (16, 18). The remaining class of this larger peptide family consists of a single member, peptide glycine- leucine-amide or PGLa (19). PGLa was the first peptide from X . laevis skin to be identified with these general properties, and it is also the smallest member of this group, consisting of only 21 amino acids. The other members contain 23 (magainins or PGS), 25 (XPF and peptide glycine-lysine), or 27 (CPFs) amino acids each. All these amphibian peptides are cationic, containing between 3 and 5 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. Their primary sequences are thought to endow these peptides with the ability to form a-helices, as would be expected in an anisotropic environment such as a membrane interface.

The literature on amphibian peptides with antimicrobial activity, however, goes back over a decade earlier. In the 1960s, Csordas and Michl published a series of papers that culminated with a report in 1970 (20) of a 24-amino acid peptide from the European toad, Bombina variegata, with hemolytic and antibiotic properties. This peptide, called “bombinin,” shares many of the general structural features of the larger group of amphibian peptides from Xenopus skin (see Fig. 1). In this paper, we report the isolation, character- ization, and corresponding cDNA precursor sequences for a series of peptides from the skin of a closely related amphibian, Bombina orientalis (or Asian toad). These peptides share considerable homology with bombinin and are called bombinin-like peptides, or BLPs. Furthermore, these peptides were found to possess potent antibacterial activities but, unlike that reported for bombinin, have no appreciable hemolytic activity.

MATERIALS AND METHODS AND RESULTS’

DISCUSSION

Three peptides have been isolated and characterized from the skin of B. orientalis that closely resemble the sequence of a n antibiotic, hemolytic peptide (bombinin) previously isolated from the skin of B. variegata. The sequences of these three peptides and a fourth peptide predicted from a partial cDNA clone, called bombinin-like peptides (or BLPs), are shown in Fig. 14A, along with that for bombinin. The two larger peptides, BLP-1 and BLP-2, have the same length (27 amino acids), molecular weight (Mr 2580.5),3 and amino acid composition but differ from one another only by the inversion of residues 4-5, i.e. Ala-Ser versus Ser-Ala. The shorter peptides, BLP-3 and -4, are both 25 amino acids long (Mr 2379.4 and 2406.4, respectively) and are also highly homologous to the other two BLPs but differ by possessing an Ala-Ala

* Portions of this paper (including “Materials and Methods,” “Re- sults,” Figs. 2-13, and Tables 1-5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. ’ All masses and molecular weights are reported based on the exact

mass of the mass spectrometrically measured ‘*C-containing component of the isotopic distribution.

sequence at positions 4-5 and a truncated COOH terminus that now ends in Phe-amide rather than Asn-amide. BLP-4 differs further by amino acid substitutions at positions 13 (Ala 3 Ile) and 19 (Lys + Asn). All four BLPs contain 2-3 lysine residues and a single acidic amino acid, glutamic acid, near the COOH terminus. By virtue of their size (>20 amino acids), all four peptides are also capable of spanning a bilayer lipid membrane as are all the members of the larger family of amphibian lytic peptides (see Fig. 1).

The overall homology between the BLPs and bombinin is not quite as high as that found among the BLPs. Several deleted sequences exist in bombinin relative to the BLPs that include positions 5-7 and 10. There is also an additional substitution (Ser12 + Gly’) in bombinin not observed in any of the BLPs. In fact, it is possible that the sequence for bombinin may be incorrect due to the rather primitive tech- niques used in the original sequencing work along with the likelihood of multiple peptide homologs. Unfortunately, we have not been able to confirm this possibility as this once prevalent European toad is now an endangered species. None- theless, it is clear that the bombinin peptides from B. variegata and B. orientalis are highly homologous and have a common origin. There is also clear homology between the BLPs and the magainins, but the homology is even more pronounced between the BLPs and the CPFs, both of which have members that are 27 amino acids in length (see Fig. 14B).

Based on their primary sequences, all three BLPs can be fitted to a well behaved a-helix using an Edmundson wheel projection as shown in Fig. 15. The predicted a-helical structures are strongly supported by the clear delineation of a hydrophobic and hydrophilic face that places all three lysine residues on the same side. While the CD studies supported a mostly random structure in an aqueous solvent, the addition of the helix-inducing solvent, trifluoroethanol, clearly showed all three peptides have a high a-helical content. At the higher peptide concentrations, helix values ranged from 63 to 69%, i.e. 67.2% (BLP-l), 68.7% (BLP-2), and 63% (BLP-3) (see Fig. 12 and Table 5 in Miniprint). The differences between these values are probably only significant for BLP-3 because of its smaller size. To better define these values, we plan to prepare synthetic amounts for further CD studies.

Substantial antibiotic activity against Gram-negative non- enteric bacteria was observed for the three BLPs tested as shown in Table 4 (Miniprint). Interestingly, very little antibiotic activity was found for the BLPs and PGS (or magainin 2) against several enteric strains from blood isolates, including Escherichia, Pseudomonas, and Klebsiella strains. This is not inconsistent with the antimicrobial activity reported by Zas- loff and others (4, 13) for the magainins but instead reflects the more stringent experimental condition we employed in our antibacterial assay, i.e. bacterial killing versus growth suppression and the much shorter incubation time of 60 min versus 5 h.

The preference for the BLPs on killing Gram-negative non- enteric bacteria is substantial and most likely underlies a key feature in the mechanism of action of these peptides. Re- cently, Rana et al. (32) showed magainin-2 amide to have substantial preference for “rough” mutants of Salmonella typhimurium. These rough enteric strains differ from normal “smooth” enteric strains by the presence of much smaller surface glycolipids or lipopolysaccharides (LPS). The marked preference for BLPs on the Gram-negative non-enteric Neis- seria species, which normally have small LPS of similar size to the enteric rough strains (33), also points to the relative size of surface LPS as a potentially important factor in modulating membrane susceptibility to the BLPs bactericidal

Antimicrobial Peptides from B. orientalis S k i n 23105

1 5 10 15 20 25 27

Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NH2

Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NHZ

Gly-Ile-Gly Ser Ala-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NHZ El Gly-Ile-Gly-Ala-Ala-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe~NH2

Gly-Ile-Gly-Ala-Ala-Ile-Leu-Ser-Ala-Gly-Lys-Ser le-Lys-Gly-Leu-Ala Gly-Leu-Ala-Glu-His-Phe..NHZ

A.

Bombinin

BLP-1:

BLP-2 :

BLP-3:

BLP-4:

B.

PGS :

BLP-2 :

CPF* :

1 5 10 15 20 25 27

Gly-Ile-Gly Lys-Phe-Leu-His Ser-Ala --------------------Phe-Gly Ala-Phe-Val-Gly-Glu-Ile-Met

Gly-Ile-Gly-Ses-Ala-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-Hls-Phe-Ala-Asn-NHZ

Gly-Ser Phe-Leu-Gly-Lys Ala-Leu-Lys Ile-Gly Gly-Gly-Ser-Pro-Gln-Gln

FIG. 14. Sequences and homology of bombinin-like peptides (BLPs-1-4) and bombinin (A) and BLP- 2 and PGS (or magainin 2) and BLP-2 and CPF ( B ) . The numbering refers only to the BLPs.

FIG. 15. Edmundson wheel repre- sentation of BLP- 1 and -2 and BLP- 3. BLP-1 and BLP-2 differ only by the inversion of amino acids 4 and 5.

BLP-l(2) BLP-3

Le” 14 - A 7

action. Whether or not LPS acts as a “molecular sponge to protect the plasma membrane” as suggested by Rana and his colleagues in an earlier paper (32) remains to be determined. Nonetheless, the marked preference we observed for Gram- negative bacteria that contains “rough-like” LPS is consistent with this earlier observation and provides a basis for which the antibiotic action of these peptides might be better ex- ploited. Indeed, we are currently investigating the susceptibility of other Gram-negative non-enteric bacteria, such as the pathogenic Haemophilus influenzae and Haemophilus du- creyi, that also possess small or rough-like LPS.

In addition to the three major BLPs, a number of earlier eluting peptides were also found in the skin secretions that appeared to be degraded fragments of the BLPs. For example, the peptides with MH’ peaks at m / z 973 and 957 (see Table 1, Miniprint) match the masses expected for the COOH- terminal fragments of BLP-1 and BLP-2 that would be expected to arise from cleavage of the third internal lysine residue at position 19. The sequences of these two peptides were subsequently confirmed by tandem mass spectrometry (data not shown). It is worth pointing out that bombinin was reported to undergo rapid proteolyic degradation within the first few minutes after secretion (20). We have have also observed this phenomenon in the skin secretions of X . laeuis, which contained proteases that degrade various antimicrobial peptides, e.g. PGS and the CPFs, on the NH2-terminal side of several internal lysine residues (3, 16). The subfragments of the BLPs, however, appear to originate from proteolysis

on the COOH-terminal side of lysine, more reminiscent of a trypsin-like activity. The rate of proteolysis also seems to be significantly slower in the skin secretions of B. orientalis when compared with X . laeuis, as much larger intact BLPs were isolated relative to these fragments than the analogous peptides in Xenopus skin. This difference in the rate of proteolysis is also consistent with the reported lack of antimicrobial activity in the skin secretions of Xenopus in the early studies by Bachmayer et at. (34). These early reports of antibacteriostatic activity in Bombina but not X . laeuis skin have always appeared anomalous in light of the high peptide content in Xenopus skin, many with antimicrobial properties (3, 4, 16). However, this is almost certainly due, at least in part, to the much faster rate of proteolytic degradation in Xenopus secretion which then renders these antimicrobial peptides inactive (3, 16). Alternatively, the reported lack of antibiotic action in X . laeuis skin may also have been due to lower antibactericidal activities of magainin when compared with the BLPs (see Table 4, Miniprint).

No appreciable hemolytic activity was observed for any of the BLPs when tested against human blood cells. This result is consistent with what has been reported previously for XPF, PGLa, and magainin 1 and 2 (4, 13,35), all originally isolated from the skin of Xenopus. The lack of hemolytic activity is in marked contrast to the reported hemolytic activity for bombinin (20). Nonetheless, the lack of hemolytic activity in the BLPs suggests a closer evolutionary relationship to the related peptides from X . laeuis and may serve the amphibian primar-

23106 Antimicrobial Peptides from B. orientalis Skin

ily as antimicrobial agents rather than as toxins. This evolutionary relationship is supported by the amino acid homologies shown in Fig. 14. These properties also make the BLPs much more interesting candidates for the development of therapeutic antimicrobial agents, possibly with a higher level of bactericidal activity than the magainins.

Sequence analysis of cDNA clones encoding BLP revealed that the BLP precursors were approximately 200 amino acids long and each encoded two copies of the BLP plus three acidic peptides, two copies of an octapeptide, and a COOH-terminal hydrophobic peptide. These multiple peptides are flanked by single and/or dibasic amino acids as shown in Fig. 16A. The structures of the precursors for BLPs-1-4 are all highly related as described under "Results" (see Miniprint). The BLP precursor contained a striking tandem repeat of nucleic acids (residues 106-292 were repeated perfectly to form residues 295-481 in the precursor to BLP-1). From this repeat came the two identical copies of the BLP found in each precursor as well as repeats of the octapeptide and strong homologies between acidic peptides 1 and 2 (Fig. 16). The long tandem repeat is evidence of recent gene duplication and conversion. Conversion and duplication is also likely responsible for the homology between the different BLP precursors. The homology between the bombinin precursors themselves is suffi- ciently high that it is not clear if they arise from multiple genes or from alternate splicing of a single gene. By homology with the caerulein precursors, however, origin from multiple genes is most likely. Definitive analysis awaits cloning of the genes encoding the BLPs.

Data base analysis showed strongest homology between the

FIG. 16. A , nucleic and amino acid sequences of cDNAs encoding BLP-1 and BLP-3. The sequence of BLP-3 is shown only where it differs from that of BLP-1. The asterisks indicate gaps of one sequence relative to the other. Ver- tical arrows indicate the two most likely sites of signal peptide cleavage based on consensus cleavage sites (36). A l ine is drawn over the sequence of the encoded BLP, and heavy lines are drawn over the dibasic amino acid cleavage points. A dashed line is drawn over the polyade- nylation signal. The other regions of the prohormone are as designated. Note that nucleic acids 106-292 of BLP-1 are repeated perfectly to form residues 295- 481. B, block diagram of the BLP cDNAs. The acidic peptides contain 25-30% Glu or Asp residues. The region of the nucleic acid repeat is as shown.

A .

6.

BLPl

BLP3

BLPl

BLP3

BLPl

BLP3

BLPl

BLP3

BLPl

BLP3

BLPl

BLP3

BLPl

BLP3

BLPl

BLP precursor and the magainin and caerulein precursors. This homology was not only between the conserved antimicrobial peptides (Fig. 14B) but was also apparent in the polypeptide structure of these precursors (Fig. 16B). The relative spacing of the BLP peptides in the BLP precursors is similar to the spacing seen between the caerulein repeats in the caerulein-4 precursor (17).

The likely processing steps giving rise to the BLPs appear to be similar to the ones seen for the other antimicrobial or lytic peptides from Xenopus. Cleavage at the COOH-terminal side of the Arg would liberate Gly as the NH, terminus. The sequence surrounding these single Arg residues, Arg-Glu-Ile- Arg-Gly, is similar to the consensus sequence Arg-Xaa-Val- Arg-Gly found in the precursors to the lytic peptides in X . luevis, i.e. magainins (or PGS peptides), PGLa, CPFs, etc., that is cleaved by a specific endoprotease recently isolated by Kuks et al. (37). Furthermore, the COOH-terminal Gly moie- ties are all processed to an amide as indicated by the sequences established from the mass spectrometry data. This latter processing event more closely aligns the BLPs with PGLa, which is the only other peptide in this larger amphibian class that ends in a COOH-terminal amide (19). The function of this modification is not clear but would likely stabilize these peptides to carboxypeptidase-type digestion in vivo. As men- tioned previously, the precursors to BLP-1 and BLP-3 both contain two identical copies of their respective BLP sequences separated by an intervening sequence that contains a short octapeptide sequence that is flanked by two dibasic residues, (KR)TAEDHEVM(KR), and another longer sequence with a highly acidic COOH terminus. Terminal to the second copy

TA """""""""

~ ~ l u ~ ~ u I l ~ ~ ~ ~ u V ~ ~ ~ u a l u ~ l u I l ~ v I l ~ v ~ ~ r Acidic Peptide 1

"""""""""""""" - N a

e... t. T " " " " " " " " " _ - * * " " " "

" " " " " _ " _ " " " " " " " "

1 8 87 89 1 8

177 48

179 48

267 78

263 76

108 357 353 106

138 447 437 134

168 537 527 164

198 627 617 194

204 717 707 200

signal acidic 1

tandem repeat tandem repeat

Antimicrobial Peptides from B. orientalis Skin 23107

of BLP-1 and BLP-3 is a second octapeptide sequence that is highly homologous to the first one, (KR)TAEEHEVM(R) or (KR)TAEEHEMM(R), respectively. However, unlike the previous octapeptide, these two sequences are flanked at the COOH terminus by only a single basic residue that continues for another 36-39 amino acids before reaching potential dibasic processing sites (Lys-Lys or Lys-Arg). The masses of these latter octapeptides were not observed in the LSIMS/ HPLC analyses (see Table 1, Miniprint), but it should be pointed out that these peptides are highly hydrophilic and would be predicted to give rather poor molecular ion abundances under LSIMS conditions and/or elute in the void volume of the HPLC column.

In the final COOH-terminal end, both precursors terminate past this Lys-Arg pair in a 19-amino acid hydrophobic seg- ment ending in Gly, i.e. ILGPILGLVSNALGGLLG. If this latter peptide were to be processed to a amide, a MH' ion at m/z 1619.0 would be predicted. Such an ion was in fact observed in the HPLC/LSIMS analyses (MH' 1618.8, peak 45), although at relatively low abundance. Preliminary sequence analysis has indicated that the peptide is similar, if not identical, to the predicted structure (data not shown). The functional significance of these other peptides encoded within the BLP precursor is not known, and no strong homology was found in the data base for either the octapeptide sequences or for the COOH-terminal hydrophobic peptide. Given the hydrophobic nature of the COOH-terminal product, it is possible that these peptides may also act at membrane surfaces, perhaps even in a synergistic or concerted mechanism with the BLPs antimicrobial action.

It has been previously pointed out that these precursors are derived from common ancestral genes and that the preporo- caerulein and preproxenopsin are mosaic genes that have arisen via exon shuffling at their 3'-ends (38). One of us has additionally commented on the homology of these precursors at their 5'-ends (39), which is very high (76-94%) between the various cDNA sequences relative to that for preproxenopsin. Kriel and colleagues (40) have analyzed the genomic DNA that encodes these precursors and have found a homologous export exon in genes encoding PGLa, xenopsin, caerulein, and levitide (40). The significance of this distinction is not known but may underlie some important differences in their evolution or biology.

Why multiple copies are found of the BLPs, as has generally been found for other families of antimicrobial peptides, may lie in providing a broader activity range against infection. Alternatively, the structural diversity of the BLPs may reflect other functions, such as in the gastrointestinal tract or central nervous system, as suggested by the Northern blot analyses that found low but potentially significant binding of BLP RNA in Bornbinu brain and stomach. It is also possible that a synergism may exist between different members of the BLPs or between other peptide members that may be present in the skin of B. orientalis such as the COOH-terminal hydrophobic peptides encoded in the BLP precursors. Indeed, such a synergism has been previously reported, at least in vitro, between magainin 2-amide and PGLa (41). Whether another family of antimicrobial peptides exists in B. orientalis, similar to that found in X . laevis, is not yet known.

Note Added in Proof-After submission of this paper, Kreil et al. (Simmaco, M., Barra, D., Chiarini, F., Noviello, L., Melchiorri, P., Kreil, G., and Richter, K. (1991) Eur. J. Biochem. 199, 217-222) published the sequences of a family of bombinin-related peptides from the skin of Bombina uariegata. These peptides, as predicted from their corresponding cDNA sequences, are all 27 amino acids long. They are most similar to BLP1-2, but none has the same sequence as the original bombinin.

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27. White, L., and Kellogg, D. S. (1965) Appl. Microbiol. 13, 171-174 28. Ausbel, F. M., Brent, R., Kingston, R. E., Moore, D. M., Seidman,

J. G., Smith, J . A., and Struhl, K. (1987) in Current Protocols in Molecular Biology, John Wiley and Sons, New York

29. Chen, E. Y., and Seeburg, P. H. (1985) DNA 4, 165-170 30. Johnson, R. S., Martin, S. A., and Biemann, K. (1988) Int. J.

31. Morrisett, J . D., David, S. K., Pownall, H. J., and Gotto, A. M.,

32. Rana, F. R., Sultany, C. M., and Blazyk, J. (1990) FEBS Lett.

33. Phillips, N. J., John, C. M., Reinders, L. G., Gibson, B. W., Apicella, M. A., and Griffiss, J. M. (1990) Biomed. Enuiron. Mass Spectrom. 19, 731-745

34. Bachmayer, H., Michl, H., and Roos, B. (1967) in Animal Toxins (Russell, F. E., and Saunders, P. R., eds) pp. 395-399, Pergamon Press, Oxford

35. Soravia, E., Martini, G., and Zasloff, M. (1988) FEBS Lett. 228,

36. Perlman, D., and Halvorson, H. (1983) J. Mol. Biol. 167, 391-

Mass Spectrom. Ion Proc. 86, 136-154

Jr. (1973) Biochemistry 12, 1290-1299

26 1,464-467

337-340

409

23 108 Antimicrobial Peptides from B. orientalis Sk in

37. Kuks, P. F. M., Creminon, C., Leseney, A.-M., Bourdais, J., Chem. 263, 5745-5751 Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 14609- 40. Kuchler, K., Kreil, G., and Sures, I. (1989) Eur. J . Biochem. 179, 14612 281-285

38. Hunt, L. T., and Barker, W. C . (1988) FEBS Lett. 233, 282-288 41. Williams, R. W., Starman, R., Taylor, K. M., Gable, K., Beeler, 39. Terry, A. S., Poulter, L., Williams, D. H., Nutkins, J. C., Gio- T., Zasloff, M., and Covell, D. (1990) Biochemistry 29, 4490-

vannini, M. G . , Moore, C. H., and Gibson, B. W. (1988) J . Biol. 4496

Supplcmmtll MatcrlP1 to Bombln(n-Uke Pcptldcs aith Anllmlcroblll AcUvIty 6'om the sum Senrtlons of

BomblM a * n t d S

Bradford W. Clbson. Dah1 Tang. Robert Mandrell, Mlchele Kelly and Ellot R. Splndel

MATERIALS

[Santa Ana. CAI. For chromatography. HPLC grade water and acetonitrile was obtalned from Burdlck and Jackson. For anUmlcrabla1 studles. several bactenal stralns were tested lneludlng Neisseria gonarrhoeae. 12 1,221 Neasseria meningil$dis. 1231 Nelsserla lacrarnrco I231 and Neisseria clnem 1231 and haw been descrlbed previously. Other Cram-ncgauve strams were human blood Isolates.

Adult frogs IBomblna orlenlalis) wcrc obtained from the Callfornia Zoologcal Supply

METHODS

Rcparatlon m d Fwificntlon of Skln Secrctlons. To ~tltnulate the release of peptldes

noreplnephrlne was InJected Into the dorsal Sacs of Bombina orienfallF.124.25] The frogs through adrenerglc medlaled granular gland Secretlon. approximately 0.5 mL of a 1 m~

were then emersed in a 0.9 % NaCl Solullon dunng the 5-10 mln penod of granular secretion. After removlng the frog. the solutions were lmmedlately frozen to mmirnlze prateolysls and IyophllIzd to dryness

desalted over a S e p h a d u G-10 column (1.5 x 25 cml with refraetlve index detection

appearance of the first maJor salt peak. Thls de-salted fractlon was then IyophllIzed and (Knauerl. A l l matedal was pooled startlng with the column void v o l ~ m e and up to the

dlssolved ln 0.1% tnfluoroacetlr a d d for subsequent HPLC separauon.

The lyophlllzed crude skln Secretions were then dissolved In 1% acetle acld and

SeparaUon of the componenfs contalned In the add-soluble fraction was carded out an a M n l n gradlent HPK system uslng a Vydac C-18 analyucal column (25 cm x 4.4 mm.

CH3CN/0.08% TFA in 1 0 0 mln. A flow rate of 1 mLlmin was used and the eluting 1.d.l. Peptlde were eluted with a h e a r gradlent of 0.1% TFAIH20 to 70% TFA/30%

components were momtored at 215 n m with a Kratos 783 vanable UV detector

M O l e C v l u Welght Dctemhtions and Segucnec AUdysls of Bomblnh-Uke Peptldea (BLP.). All peaks In the HPLC chromatogram of the de-salted acld soluble fracuon were analyzed by llquid secondary lo" mass spectromeoy ILSIMS). Molecular wrights of the indlvidual

fracuons were dned down onto a ~talnless steel probe tlp and dlssolved In a mlxture of components were determlned as prevlousiy descnbed 1251. Brlefly. allquots of HPLC

glyceral/thloglyceraI (2/11. The samples were then acldlfled wlth 0.1 pL of I % TFA and Inserted mto the source of a Kralos MS 50s Or VG 70VSE double focusmg mass

Scan were taken at elther 300 sldecade [KratoS MS 50s) or 10 s/decade WG 70VSE) and spectrometer. Samples were lonlzed and sputtered uslng a Cs+ Source at an energv of 8 kv.

calibrated manually with Ullran~ark 1620 or automaucally with a VAX Opus data system to an overall accuracy or t0.2 ~ a .

The three malor late eluong peaks (peaks 40. 41 and 441 were subsequently analyzed by LSlMS and tandem mass spectrometry. both before and after dlgesuon ulth cndoprotemase Lys-C 1Boeringer-Mannheiml. For tandem fMS/MSI analysis. a Kratas Concept I I H H Mass spectrometer was used and has been described I" detall elsewhere 1261 Brlefly. pepUde samplcs were dlssolved In glycerollthloglyceroI 1211) and m n m d

selected in M S ~ I a n d vibronlcally activated an a hellum CDIIISIOII cell adJustcd lo attenuate using a Cs' source as descnbed above. The protonated molecular ions (MH*) specks were

separated In MS-2 uslng a llnked B/E scan. These fragment Ions were mass assgned via a the molecular ion beam by -65%. T h e resulting fragnlent (or daughter] Ions were then

Mach 2 data system aperatlng On a Sun SparkVorks ta tm

Amho Acid Analpla me three maJor late eluting peaks were collected and lyophillzed

converted to their phenylthlocamayl derlvallves. Samples were then analy'ed using a prior to amino acld analysls. Peptldes were hydrolyzed in 6 N HCI for 24 h at 110°C and

n o - T a g analysls systcm on a Beckman 121 MB amino acld analper.

EndOprOteh*e Lys-c Westion ~ I B ~ m b l d n - L i k e Peptldes. To obtain complete sequences a1 the BLP 1-3. each pepude was separately dlgested with endoprotemase Lys-C. Samples were dissolved in 70 mM NH4HC03 buffer (pH 8.31 at a relauve e n w e ' s u b s t r a t e rauo of I f 10 (wtlwt) and Incubated for 4 h at 37°C. The resulung peptlde fragments were then directly separated by reverse phase HPLC under condltlons ldentlcal l o that used for the separatlon of the total skln pcpllde fraction Peptldes were collected In 1.5 mL polprapylene tubes. lyaphlllzed. and analyzed by both LSIMS and tandem mass spectrometry

Hemolyel. OCH-UI Erythrocflcs To assay for hemolyoc actlvity. a lnodincauon of the hemolysls assay described by Zasloff 141 was used. Human erythrocnes from four lndlviduals were collected In heparlnlred contalners. then pooled. The cells were washed 3 times with VBS. pH 7.2. then dduted l o a final concentratlon of 5% In PBS. Peplldes dlluted In VBS ( 1 0 0 mL1 were added to mlcrofuge tubes conlainmg 200 nll Of 5% cells. The mlxiure was incubated 30 nmules at 3 7 'C: the tubes then were mlcroiuged 110.000 RPM) far 2 mm and the OD350 of the supernalen1 was measured. Maxrmum hemolysis was detemlned by addlng 0.2% Triton X100 10 a sample of c~IIE.

BactcrlcLW &.say 4 t h Peptlder. Various Gram-negauve enterlc and mucosal ~omrnen~a l and pathogenlc bacterla were gown overnlgbt On CC agar base IDIfCo Laboratones Inc.. DctmIt. Mlchlgan) containing 1% supplemcnt 1271. Organisms were collected from the plate Wth a stedle platlnum loop attd mixed in stenle veronal buffered s a l m WSI. pH 7.2. to an OD620 ofapproxlmatrly 0.6. The bacterla were thcn dlluted In VBS 10 5 x IO4 organlsms/ml. The assay mwture conslsted of IO ml of sterlle~flllered 10.45 mml peptide ldlluted In YBSI. 20 ml IIquid GC medla. and 20 ml of dduted bacterla I ~ D D T O W I ~ ~ B ~ ~ Y ID1 organlsmsl The assay mtxture was lncubaled 60 mlnutes at 3 7 "C and 15.ml 01 the mwture were plated on CC ag'u. The plates were Incubated overnight at 37 'C in 5% CO2 and the survlving colonies counted. A control was mcluded using PBS ln place of peplade

Co~u~tructlon of cDNA Ub"ry. Scrccnhg M d Sequence Analysis. Total RNA and polyL4l RNA were prepared from dorsal sklns 01 12 Bombina orientalis after naradrenerglc lnductlon of pepude symthcsls 1241 uslng guanldlnlum thlocyanate and OllgolUTI as prevlously desedbed [25.281. A =DNA llbrary from dorsal S k I n RNA was prepared In the vector UAPII (Stratagene Clonlng Systems. La Jolla. CAI as previously descrlbed 1251. The library was mpllned once and screened with mlxed ollgonucleoude probes complementary to the sequence ofamphiblan bomblnln as descdbed In Results. Hyhridlrlng phage were plaque punned. then exclsed In vim Into the plasmld Bluescrlpt following Stratagene's protocol DNA sequence was obtalned from double stranded template (291 lnlually uslng flanklng vector sequence as pnmcrs and then uslng Internal sequences as prlmers. All sequence was obtalned from at least hvo plasmlds or on both strands. Prlmers for llbrary screenlng and sequenclng were syntheslzed with an Applled BIosystemS 391 DNA synthesler.

For Northern blot analysis. total RNA was prepared from tlssue extracts as descrlbed above. RNA was electrophoresed on formaldehyde-agarose gels 11.75%1. transferred IO a nylon membrane and W crossllnked 1281. 3*P-labeled antisense cRNA was transcrlbed from the cDNA encodlng BLPl usmg TI polymerase as descnbed prevlausly 1251. Hybddlzatlon condluons were 50% fomamide: 5X SSC: 5X Denhardt's soIu11on: 50 mM NaP04. pH 7 . 0 2 5% SDS. sonlcated. denatured salmon sperm I200 pglml). and phenol- extracted yeast RNA (200 pglml) at 65 'C for I 8 hrs. Blots were washed 2 x 15 mln In 2 X ssc lo .2% SDS at 25 "C followed by 2 x 30 mln washes in 0.1x SSC10.596 SDS at 65 'C and then exposed 10 nlm at -70 'C with an lntcnslfylng screen.

RESULTS

Scp-tion of Skin Component.. On Sephadex C- 10. a broad talllng peak was observed thal began with the column "old volume and emended to the eluuon of the. two malor salt peak. When thls peak was furiher separated by reversed-phase HPX. over 40 well denned peaks were observed as shown In Flg. 2 below. The pattern of peaks were reproduclble. although some dlfferences were seen between dlfferent frog preparations. These dlfferences were prlmanly a function of the relatlve abundances of late clutlng components b 4 8 h CHJCN. peaks 40 and larger) to early ones Ic40?+ CHJCN. peaks 39 and smaller).

Lsms ~ n . l y a b of S k h PRctlons. The LSlMS spectra abtalned from HPLC fracuon 1-47 mdlcated a molecular welght dismbuuon of 242 to 2580. In Table 1 below. the mol~cular Ions correspondlng to each peak In the HPLC chromatogram (Fig. 21 are b l e d . Figure 3 shows a representauve LSlMS spectrum of one of the late elut~ng peaks (peak 401 that was subsequently ldenufied as BLP-1

Clrculpr Mcbrolsm Studles. Pcptldes wert Isolated from several repeal analyses by H P E and quantltaled by ammo acld analysls as descnbed above. Samples were dlssolved In a 50 mM sodlum phosphate buffer IpH 7.0) with and without the addition of 40% trlfluoroethanol at three dllutions: 72. 36. and 7.2 nmollml. Peptide SOIU~IO~S were then placed In a 1.0 mL cell and spectra taken on a Jasco 300 CD spectrophotometer. For each peptlde SoIUtlon. 3 scans were taken from 190-360 nm at 20 nmlmln and averaged.

Antimicrobial Peptides from B. orientalis Skin. 23109

1 18 973.6. 1105.5 2 242.3 19 373.6. 1044.6. 1131.7. 36 1322.9

l R R i R

35 1065.5. 1783.3

3 4

5

6 7 8 9

10 1 1

13 12

14

16 15

17

284.3. 363.2

475.3

488.3. 510.3

242.3. 687.4 500.3. 502.3. 596.4 486.4. 506.3 501.3. 613.5

501.4. 687.5 573.3. 595.3. 673.5 659.4. 771.5. 1473.9 1735. I 1169.7. 1301.5. 1850.3. 1980.8 739.4. 849.3

20 21

22

23 24 25 26

27 939.6. 1665.4. 1912.2 28 668.4. 939.6. 1230.8

29 30

31

32 33

34

13x7 x 1736 I

9;;,,-1089.5

903 5. 973.5. 1521.7 630.3. 682.4. 772.5.

973 5. 1521.7. 1641.6 679.3. 826.5. 904.4.

656.4. 973.5. 2016 6 656.4, 1978.9 842.5. 1976.3 1619.8 (Bombcsln)

990.7. 1504.8. 1922.3 611 4. 1614.8. 1771 I . 1909.4. 2080.6 1909.4

~. ~

625.4. 1267.9 682.5. 720.4. 705.4

2080.5

37 38

39

40

42 41

43

4 4 45

46 47

2284.8 1337 3. 1716.1

1979.9

2580.5 IBLP-1) 2580.9 (ELF-2)

2564.4 1804.9. 2409.3

2319.4 (BLP~3) 1618.8

1917.1 1931.0

Figure 3 LSIMS s p e c h m of BL%l (MH* E 2980.51. Masses for the N-temmai an, b, and

Johnson et ai. 1301. The sequence shown 1s that derived for BLP~l from the c, ions and C-leminal yn ions are labelled accordlng to the nomenclature of

MS/MS spectra Of the cndoprotcmase Lys-C fragmenls as discussed later.

Table 2. Amino Arid Analysis of Bomblnin~l.lke Peptldes IBLPsl I . 2 and 3

Sample &x Glx. Ser Gly 13)s N a Ile Leu Phe Lys

BLP-1 1.00 1.02 2.96 5.39 1.07 6 11 1.83 4.20 1.05 3.12 IMr=2580.51 I 1 1 Ill I31 15) I l l 16) 12) 14) I!) 131

BLP-2 1.00 1.06 2.95 5.29 1.06 599 1.82 4.45 1.06 2.98 (Mr=2580.51 I l l Ill 131 15) ( 1 1 ( 6 ) I21 141 I11 (31

BLP-3 0.05 L O O 1.92 507 1 0 1 584 1.90 4.12 1.01 2.97 lMr=2378.91 - - I l l 121 151 I11 I61 121 141 ( i l ( S I

* Only amlno aclds with relative values >O.OX commred to G h lor mxl arc listed

To properly order lhese p e p d e s . the LSlMS and/or MSIMS spectra of the intact BLPs were used. For cxample. Fig. 3 shows the LSIMS spectrum af BLP-1. and several ion tvpes (indudmg the C~terminal fragments yn and q, and N-terminal fragments an. bo. and fn) were Identrhed. Thhe assrgnments made in the LSIMS spectrum of BLP-1. along With those for B1.P-l and ~2 [data no1 shown) allowrd fat the unamblguous ardellng of Lys.4. Lpl . Lys-2 and Lys-3. from N~ to C~termmus. lo be made lor BLP-1. -2 and ~ 3 .

'I '" 1 1

MH"44183 Ssr - Ala - Leu . LYS

Mast

Figure 5. Tandem CID m a s spectrum of Lys-1 fragment IMH' 418.31 from BLI-1

1 372 315 702 131 Y 388 331 218 147 r 357 244 173

a 143 214 b 171 242 e 7S 188 ZS9 d 101

1 0 0 150 301 h

M

Fqum 6 Tanden? CID mass spertrum of Lvs-2 fragment IMH* 388.31 from BLP~I

23110 Antimicrobial Peptides from B. orientalis Sk in w 741 670 541 186 v 742 542 D 8 4 1 784 611 600 471 334 Y 857 8W 687 616 487 350 203 132 x 826 713 642 513

MH+.857.4 GIY . ku . Ala . Clu . HI8 . Pho . AIS . A ~ . N H ~

II 143 b 171 242 371 508 655 726 840

343 480 627 698 812

525 672 143 d 285 414 551 169 m+

I

Flgure 7 . Tandem C1D mass spectrum of Lys-3 fragment IMH* 857.4) from BLP-I

w 556 485 356

2 656 599 486 415 286 149 y 612 615 502 431 302 165 I 641 528 457 328 191

357 73

MH+d72,) GI7 . Leu . AIS . Glu I His . Phe-Nx,

a 143 343 480 627 b 171 242 371 508 655

259 388 525 d 101 285 414 551 m+

-ra-r* 111-

Flgure 8. Tandem CID mass spectrum of Lys-3 fragment IMH' 672.3) from BLP-3

650

w 871 " 858 2 900 781 730 659 572 459 346 259 188 131 y 973 926 803 746 675 588 475 362 275 204 I47 x 942 829 172 101 614 5001 301 173

729 612 543 416 329 258 643 530 330

143 271 358 411 584 611 142 b 171 228 299 386 499 612 699 770 827 c I88 403 629 716 787 844 d 453

7 0

6 0

S O

40

3 0

10

I O

0

I 871 713 642 543 416 329 258 V 858 114 1 957 9w 187 730 643 512 459 346 259 I88 131

173

530 411 330

Y 973 916 803 146 659 588 475 362 115 2M 147 942 829 772 685 614 SO1 388 301

a 143 287 358 471 584 611 742 b 171 228 351 386 499 612 699 770 821 956 c I 8 8 403 516 629 716 181 844 d 27 I 542

"X1 x79 Xil. m*

I ::/ 70

I D O 2nn 300 400 boo 600 100 800 snn M.*r

Figure 10. Tandem CID mas spcctntm of Lys-4 fragment lMH* 973.6) from BLP-2

w 855 v 842

713 642 543 416 329 258

2 884 111 714 643 512 346 259 188 131 Y 957 903 787 730 659 588 475 362 275 2M 147 I 921 813 756 685 614 301 I13

MH+=951.6 Gly . I Ie . GIy . A h . Ala . Ile . Leu . Ser . Ala . GIy . Lyg

530 330

a I43 100 211 462 455 568 655 126 b 171 228 299 370 483 596 683 754 811 c I 8 8 613 1W 111 828 d 115 342 854 a+

70

60

s o

40

3 0

10

I O

0

Flgure 11. Tandem CID mass spectrum of Lys-4 fragment IMH' 957.61 from BLP-3

Hcmolgtlc A s s a p . In Table 3 below. t h e results of the hemolysis aeuvlty are 11sled for I h c BLPs and PGS Imagalnln 2). Each pepude was run at three dlfferent concentmtlons. In a l l cases. no appreclable hemolyUc acuvlly was measured above background. or as compared IO the endpolnt detemlnauon uslng a 0 2% TTlton X- 100 soiuuon.

Table 3. Hemolysis Assay wlth Peptldes

Pepllde concentratian

fpg/mll OD350

BLP 1 BLP I 100 0017

BLP I 25 0.019

R1.P 2 0.023

100 0.030 6

-

BLP 2 BLP 2

BLP 3 BLP 3 BLP 3 PGS PGS PGS

TrltOn x100

a 5 6

100 2 5

6 100 25

6 1 0 . z ~

F l g ~ r ~ 9. Tandem CID mass spectrum of Lys-4 fragment IMH+ 973.61 from BLP-1.

Antimicrobial Peptides from B. orientalis Sk in 23111

Antlbactedal A n m y . . As s h o w below. The BLPs land PCSl had lltllc c&ct on a sulle of Cram-ncgauve cntcnc baclena. but had a subslantla1 bactcncldal acuwy whcn dlrcctcd against a sencs of non.cnlcnc Gram-ncgauvc baelerla of the Neisseria lamlly.

Table 4. Baclerlcldal Assay wllh Pcplldcs

% KIII’

Bacteria Straln BLP 1 BLP 2 BLP3 PGS

Cltrobacter Klebslella OnJtOca

18172 NK’ NK NK NK

Eseherlehla coli 18173 NK NK NK NK

Escherichia coI1 I8174 NK NK NK NK

Escherichia coli 18175 NK NK NK NK

Pseudomonas aerugbosa 18177 NK NK NK NK 18176 NK NK NK NK

Klebswlla pnetrmoniae 18178 NK NK NK NK Eschcrichlo coll Pseudomonas (Icruglnosa 181 80 NK NK NK NK

I8179 NK NK NK NK

Enlcrobacter C I ~ ~ C C I E 181 R I NK NK NK NK

Nelsseria menlngltldts 118V IC1 82 58 59 0 Nerssena meninglltdb 15240 IAI 98 98 99 0 Ncisscna menlngittdis 126E IC1 85 98 99 0 Nclssena gonorrhoeoe F62 98 97 99 0 Nelsseria Iocfamm 1 5 3 2 3 96 99 97 0 Ncmerla lactomica 15215A 100 100 100 0 Nelsseria clnerea 15461 98 95 97 10

Each peptide was lcslcd at a 20 pg/ml nnal conccnlrauon In the assay mlxturc lscc Methods for dctallsl. ’ NK = No KIII.

Ch* Mehrolsm Studh of BLP 1-3. Tnc CD spectra taken of the BLPs In 10096 aqueous condluons (data no1 shown) showed pnmnnly a random stmclum wl lh Ilttlc cvldcncf of an m-hcllcal Slruclurc. However. In 4% tnfluorocthanol all three pcprldcs lsce Flc. 121 showcd a slpnlfleanl a-helleal component. Using lhc method of Mornset et aI. 1311. the c,. hcllcal conlcnl was calculated at the two hlgher eonecntrallons and are llsted In Table 5.

BLP. 2 72

7.2 36

-41.5 68.7 .22.5 75.2 -5.9 ...

1 2

! - 28s

bombinin-like peptides with antimicrobial activity from ... · three peptides have been isolated...

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