Antimicrobial peptides

TECHNICAL FIELD
The field of this invention is the area of antimicrobial peptides with activity against a broad range of Gram-negative and Gram-positive bacteria and fungi. The antimicrobial peptides of this invention are useful for inhibiting microbial growth and in pharmaceutical compositions for treatment or prevention of infections and for the treatment and/or prevention of gingivitis.
BACKGROUND OF THE INVENTION
Microbes which invade the human body are challenged by several defense mechanisms. The nature of the defense mechanisms which any given microbe faces depends on the genetic makeup and the physiologic state of the host as well as the portal of entry of the invading microorganism.
Host defenses include mechanical factors and chemical factors. Mechanical factors which help protect epithelial surfaces include the washing action of bodily fluids, including tears, saliva and urine, trapping on mucous layers, removal by cilia and elimination by coughing, sneezing or desquamation. A further mechanical defense is offered by the physical integrity of the skin, although mucous membranes can be penetrated by some pathogens.
Chemical defense factors include the acidity of gastric secretions, unsaturated fatty acids on the skin which kill certain bacterial species, lysozyme in tears, saliva and nasal secretions, iron-binding proteins at the mucosal surface, transferrin in serum, and spermine in semen. Secretions of the mucous membranes also contain antibodies, especially those of the IgA class. Microbial antagonism between different potentially pathogenic bacteria, fungi and yeast strains occurs at the level of competition for nutrients and through the production of inhibitory substances; this antagonism affords further protection to the host.
If the barriers of the skin or mucous membranes are crossed, immunological factors (e.g., antibodies) which are specific to the microorganism as well as nonspecific cellular defenses come into play. In addition, there are some chemical factors which also play a role in host defense, especially transferrin, which chelates available iron on which microorganisms are dependent.
Nonspecific cellular defenses in the form of phagocytic white blood cells from local tissues and the bloodstream respond to an invading microbe. Polymorphonuclear leukocytes (PMNs) actively phagocytize particulates such as bacterial or fungal cells. PMNs are the first class of phagocytic cells recruited to the site of infection or inflammation. The PMNs contain azurophilic or primary granules, which contain lysosomal proteases, myeloperoxidase, lysozyme and certain antimicrobial proteins. Secondary granules within these cells contain alkaline phosphatase, lactoferrin and lysozyme. Stores of glycogen within the PMNs provides for energy through glycolysis so that the cell can function in an anaerobic environment.
Adherence of a particle to the surface of a phagocytic cell initiates phagocytosis; the particle enters the cytoplasm in a phagocytic vacuole. This triggers a respiratory burst and the generation of microbicidal metabolites; the primary granule fuses with the phagocytic vacuole to form a digestive vacuole called the phagolysosome. Intracellular killing of the ingested microorganism occurs as a result of oxygen-dependent and oxygen-independent mechanisms. The oxygen-dependent bactericidal halogenating system uses granule myeloperoxidase, hydrogen peroxide and chloride ion to kill bacteria and viruses via either halogenation of cellular or viral constituents or via reactive oxygen intermediates. Oxygen-dependent killing can also proceed by direct reduction of molecular oxygen via the cytochrome b-oxidase system (reviewed by Orkin (1989) Ann. Rev. Immunol. 7:277-308). Oxygen-dependent killing mechanisms are reviewed by Beaman and Beaman (1984) Ann. Rev. Microbiol. 38:27-48.
The primary granules contain three major groups of antibacterial proteins. The first group includes catalytically active proteins which are only weakly antibacterial when tested individually in purified form. Examples from this group include lysozyme, elastase and collagenase. These enzymes probably participate in the digestion of microorganisms killed by other mechanisms, but elastase, for example, is believed to potentiate killing by the halogenating system. The second category of granule proteins includes those with catalytic activity and bactericidal activity which is independent of the catalytic activity. An example is the chymotrypsin-like neutral protease of human neutrophils. A third group contains bactericidal members which lack known catalytic activity; such a protein class has been purified from rabbit neutrophils. Included in this class are defensins and cationic antibacterial proteins.
Some cationic antibacterial proteins are of relatively high molecular weight (greater than about 25 kDa) and kill certain Gram negative bacteria such as Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa by damaging the cytoplasmic membrane, leading to increased membrane permeability. Human bactericidal/permeability increasing protein (BPI) is a strongly basic protein with molecular weight of about 59 kDa. It is believed that when bound to the outer membrane of susceptible bacterial cells, hydrophobic channels through the outer envelope are exposed, and as a secondary effect, there is a selective activation of autolytic enzymes including phospholipase and peptidoglycan hydrolases. Gram positive bacteria, certain Gram negative bacteria and fungi are not affected by BPI in vitro.
Low molecular weight cationic proteins (10 kDa to 25 kDa) have been reported which inhibit the multiplication of such Gram positive bacteria as Staphylococcus aureus (Root and Cohen (1981) Rev. Infect. Dis. 3:565-598). In addition, cationic proteins with fungicidal activity have been identified in alveolar macrophages. It is believed that cationic proteins are most efficient in killing phagocytized microorganisms in combination with other microbicidal defense mechanisms (Elsbach and Weiss (1983) supra).
Generally defensins are relatively small polypeptides of about 3-4 kDa, rich in cysteine and arginine. Gabay et al. (1989) Proc. Natl. Acad. Sci. USA 86:5610-5614, used reverse phase HPLC to purify 12 major polypeptides from the azurophil granules of human PMNs; purified proteins were analyzed individually for antimicrobial activity and for N-terminal amino acid sequence. A 4 kDa defensin (HNP-4) and a 29 kDa polypeptide named azurocidin were purified and shown to possess broad spectrum antimicrobial activity. Defensins as a class have activity against some bacteria, fungi and viruses. They are also reported to have cytotoxic activity against transformed cells. Selsted et al. (1985) J. Clin. Invest. 76:1436-1439, presents a sequence comparison of human and rabbit defensins. The defensins are believed to have molecular conformations stabilized by cystine infrastructure, which are essential for biological activity.
Granzymes are a family of serine proteases in the granules of cytolytic lymphocytes. Proteolytic enzymes are believed to function in cell-mediated cytoxicity; some of the genes have been cloned, and sequence information is available. Within the granzyme family there is at least 38% amino acid sequence identity. Human lymphocyte protease has 73% amino acid sequence identity to mouse granzyme B (Jenne and Tschopp (1988) Immunol. Reviews 103:53-71).
Cathepsin G (Cat G) is a granule protein with chymotrypsin-like activity; it is also known as chymotrypsin-like cationic protein. Cat G (Odeberg and Olsson (1975) J. Clin. Invest. 56:1118-1124) and three other mutually homologous polypeptides called defensins are active against a broad spectrum of gram positive bacteria, gram negative bacteria and fungi (Shafer et al. (1986) Infect. Immun. 54:184-188; Shafer et al. (1988) Infect. Immun. 56:51-53; Drazin and Lehrer (1977) Infect. Immun. 17:382-388; Ganz et al. (1986) Semin. Respir. Infect. 1:107-117). Sensitive bacteria include Capnocytophaga sputigena, Escherichia coli, Listeria monocytogenes, Neisseria gonorrhoeae, Pseudomonas aeruginosa and S. aureus. All of these pathogens, with the notable exceptions of P. aeruginosa and C. sputigena, are only sensitive to both enzymatically-active and -inactive cathepsin G (Miyasaki and Bodeau (1991) J. Clin. Invest 87:1585-1593; Wasiluk et al. (1991) Infect. Immun. 59:4193-4200 and Table 11 herein). P. aeruginosa and C. sputigena are only sensitive to enzymatically-active cathepsin G. It is not clear, however, if cathepsin G-killing of these two pathogens requires degradation of bacterial proteins or whether an intact active site is needed to align antibacterial domains of cathepsin G with the bacterial target.
Gabay et al. (1989) supra, has reported antibacterial activities of a number of proteins isolated from human PMNs, including cathepsin G and elastase, and has given the amino terminal sequence of these and other proteins. The N-terminal five amino acids of elastase and Cat G are identical; further sequences have significant relatedness. Cat G also exhibits significant sequence similarity to chymotrypsin, which is not known to exhibit antimicrobial activity similar to that of Cat G.
The sequence of human Cat G is known, and the gene has been cloned from human leukemic cell line U937 (Salvesen et al. (1987) Biochemistry 26:2289-2293). Sequence analysis of the cDNA revealed significant sequence identity to rat mast cell proteinase (47%) and to an activated mouse cytotoxic lymphocyte product (56%).
Another class of antimicrobial polypeptides are those known as magainins; at least five proteins can be isolated from the skin of the African clawed frog (Xenopus laevis). The natural proteins are active against a broad range of microorganisms including bacteria, fungi and protozoans (Zasloff (1987) Proc. Natl. Acad. Sci. USA 84:5449-5453). The broad spectrum antimicrobial activity is present in synthetic peptides and in certain truncated analogs of the natural proteins. Derivatives of about 19 to about 23 amino acids have antibacterial activity as measured using Escherichia coli. In the protozoan Paramecium caudatum treated with the magainin peptides, there is disruption of membrane functions. The configurations of the bioactive peptides can be modeled as amphiphilic alpha-helices and are sufficiently long to span a lipid bilayer. (Zasloff et al. (1988) Proc. Natl. Acad. Sci. USA 85:910-913). Spanning a lipid bilayer is believed to require at least 20 amino acid residues in an alpha-helical configuration (Kaiser and Kennedy (1987) Ann. Rev. Biophys. Chem. 16:562-581). The sequence of a representative magainin peptide is GIGKFLHSAKKFKAFVGEIMN (SEQ ID NO:48) (Zasloff et al. (1988) supra).
SUMMARY OF THE INVENTION
It is an object of this invention to provide peptides with antimicrobial activity. The antimicrobial peptides of the present invention contain from about five to about twenty-six amino acids joined in a linear array by peptide bonds.
An object of the present invention is a broad spectrum bactericidal peptide, termed CG 117-136 herein, which has the sequence RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22). Further objects are additional bactericidal peptides, particularly effective for P. aeruginosa but not for S. aureus or N. gonorrhoeae, which peptides are termed CG 61-80 and CG 198-223, herein. CG 61-80 has the sequence RRENTQQHITARRAIRHPQY (SEQ ID NO:19) and CG 196-223 has the sequence GKSSGVPPEVFTRVSSFLPWIRTTMR (SEQ ID NO:26).
In other embodiments, the peptides comprise the amino acid sequences IIGGR (SEQ ID NO:1), IVGGR (SEQ ID NO:2), IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16) and HPQYNQR (SEQ ID NO:3). The peptide of the sequence IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16) is particularly preferred. A consensus sequence for peptides related to HPQYNQR (SEQ ID NO:3) has been formulated: HX.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6, where X.sub.1 is proline, histidine or alanine; X.sub.2 is aspartic acid, asparagine, glutamic acid, glutamine, alanine, serine, threonine, isoleucine, valine, histidine, tyrosine, arginine, methionine oxide or methionine sulfone; X.sub.3 is tyrosine, phenylalanine, tryptophan or beta-naphthyl-alanine; X.sub.4 is asparagine or alanine; X.sub.5 is glutamine, proline, N-methyl alanine, or alanine; and X.sub.6 is arginine, lysine, alanine or NH.sub.2 or OH (SEQ ID NO:4).
A peptide fitting the consensus sequence preferably has at position 3 (as X.sub.2 above) a nonbulky, hydrophilic amino acid capable of hydrogen bonding, glutamine or proline at position 6 (as X.sub.5 above) and lysine or arginine at position 7 (as X.sub.6 above). Preferably amino acid 3 is glutamine, alanine, glutamate, asparagine, or aspartate. Antimicrobial peptide sequences whose sequences fall within the consensus sequence include, but are not limited to, HPQYNQR (SEQ ID NO:3), HPAYNPK (SEQ ID NO:5), HPAYNPR (SEQ ID NO:6) and HPAYNQR (SEQ ID NO:7). Additional antimicrobial peptide sequences related to HPQYNQR include, but are not limited to, HPQYAQR (SEQ ID NO:8), HPQYNQA (SEQ ID NO:9), HPQYNAR (SEQ ID NO:10), HPAYNPR (SEQ ID NO:6), HAQYNQR (SEQ ID NO:11), and HPQYNQ (SEQ ID NO:12) and RHPQYNQR (SEQ ID NO:13). These peptides possess microbicidal activity for Gram positive and Gram negative bacteria. The oligopeptide corresponding in sequence to amino acid 77-96 of mature cathepsin G, HPQYNQRTIQNDIMLLQLSR (SEQ ID NO:14), is not significantly bactericidal, however, for P. aeruginosa, N. gonorrhoeae or S. aureus.
An object of the present invention is to provide antimicrobial peptides which are useful as bactericides and/or bacteriostats, useful, for example, for inhibiting microbial growth in a variety of solutions and sterile solutions, such as contact lens solutions, herbicidal solutions, hazardous or refuse waste streams, surface disinfectant solutions and oil recovery fluids.
A further object of the invention is to provide therapeutic compositions, suitable for human, veterinary, agricultural or pharmaceutical use, comprising one or more of the antimicrobial peptides of the present invention and a suitable pharmacological carrier. Such therapeutic compositions can be formulated as understood in the art, e.g., for topical or aerosol application, for controlling and/or preventing infection by Gram positive or Gram negative bacteria or fungi. Preferably, the antimicrobial peptides of the present invention are used in the treatment of infections by Gram-negative or Gram-positive bacteria. The antimicrobial peptides of the present invention, when used in therapeutic compositions, will not have significant immunogenic activity. In vitro antimicrobial activity of the oligopeptides of the present invention is an accurate predictor of in vivo antimicrobial activity.
Pharmaceutical compositions contain a therapeutically effective amount of an antimicrobial peptide. A therapeutically effective amount of an antimicrobial peptide can be readily determined according to methods known in the art. Pharmaceutical compositions are formulated to contain the therapeutically effective amount of an antimicrobial peptide and a pharmaceutically acceptable carrier appropriate for the route of administration (topical, gingival, intravenous, aerosol, local injection) as known to the art. For agricultural use, the composition comprises a therapeutically effective amount of an antimicrobial peptide and an agriculturally acceptable carrier suitable for the organism (e.g., plant) to be treated. Preferably for use in a pharmaceutical composition, the antimicrobial peptide will have an ED.sub.50 in vitro less than about 10.sup.-3 M. The skilled artisan can readily determine a therapeutically effective amount against a target bacterial strain, for example, based on the ED.sub.50 using the methods disclosed herein and the teachings of the art.
Therapeutic compositions may be administered by topical, dental rinse, aerosol or intravenous application, or by local injection for the control or prevention of infection or control of tumor cell growth, by any means known to the art.
The IIGGR-related (SEQ ID NO: 1) antimicrobial oligopeptides, including IIGGR (SEQ ID NO:1), IVGGR (SEQ ID NO:2) and IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16), of the present invention may also be used to kill or control the growth of tumor cells or virus-infected cells. In such applications, these peptides will be particularly useful when coupled to antibodies or other molecules which are specific for the target tumor cell or virus-infected cell so that the peptide acts specifically on the tumor or virus-infected cell.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the bactericidal activity of clostripain digests of human neutrophil cathepsin G and elastase. Proteinases were digested as described in the Examples, lyophilized, dissolved in HBSS (pH 7.5), and tested (50 .mu.g/ml) for antibacterial activity against S. aureus strain 8325-4. Each data point represents the mean of three samples with <5% variance between each.
FIG. 2 shows Reverse Phase-HPLC fractionation of cathepsin G peptides obtained from clostripain fragmentation. Low e peptides obtained by gel filtration chromatography were lyophilized, dissolved in 250 microliters of 0.1% (v/v) TFA and subjected to RP-HPLC as described in the Examples. Peptides were eluted in several peaks (1-10). These were further purified, separately, by RP-HPLC as described herein. Only peptides in peaks 6 and 7 were found to exert antibacterial action in vitro against S. aureus and N. gonorrhoeae. FIG. 2(A), cathepsin G digest fractionation; FIG. 2(B), rechromatography of peak 6; FIG. 2(C), rechromatography of peak 7.
FIG. 3 illustrates the bactericidal activity of synthetic peptides derived from cathepsin G. Synthetic peptides (0-100 .mu.g) were tested against N. gonorrhoeae and S. aureus as described in the Examples. Each data point represents the mean of three determinations from three separate experiments using the same lot of synthetic peptide. FIG. 3(A), peptides IIGGR (SEQ ID NO:1) vs. IVGGR (SEQ ID NO:2); FIG. 3(B), peptides HPQYNQR (SEQ ID NO:10) vs. APQYNQR (SEQ ID NO:15).
FIG. 4 illustrates the pH dependency for the bactericidal activity of HPQYNQR (SEQ ID NO:3). Aliquots of peptide (100 .mu.g) were assayed for antibacterial activity in incubation with either of the two test bacteria at different pH values, as described herein. Each data point is the average of two determinations from three separate experiments.
FIG. 5 compares the bactericidal capacities of Cat G synthetic peptides and certain antibiotics. N. gonorrhoeae strain FA 102 was exposed to 5.0.times.10.sup.-4 M of IIGGR (SEQ ID NO:1), HPQYNQR (SEQ ID NO:3), penicillin G, chloramphenicol, tetracycline, streptomycin, kanamycin, and a mixture of three partially purified human defensins in HBSS (pH 7.5). After 30 min at 37.degree. C. the samples were plated onto GCB agar, incubated for 48 hr, and colonies counted. There were no gonococcal colonies after 48 hr of incubation.
FIG. 6 illustrates the bactericidal action of Cat G synthetic peptides (HPQYNQR (SEQ ID NO:3), Ac-HPQYNQR) (see Table 2), and human granzyme B-derived peptide (HPAYNPK (SEQ ID NO:5)) under conditions of different pH, FIG. 6(A) and ionic strength, FIG. 6, (B), and at different time points FIG. 6(C). The synthetic peptides (5.times.10.sup.-4 M) and purified, enzymatically inactive Cat G (1.8.times.10.sup.-6 M), were tested against S. aureus in HBSS modified to different pH (A) and ionic strengths (B). Standard ionic strength HBSS was used in FIG. 6(A), while pH 7.5 HBSS was used in FIG. 6(B). The peptides and Cat G preparations are identified in the inserts. The results are mean values from three separate experiments. FIG. 6(C) Time course of killing S. aureus by Cat G and granzyme B-derived synthetic peptides. Synthetic peptides (5.times.10.sup.-4 M) were tested for bactericidal action over a 120 min period in optimal HBSS. The results are from three separate experiments and mean values.
FIG. 7 graphically illustrates the in vitro bactericidal activity of various Cat G-related oligopeptides against C. sputigena ATCC 33123. 10.sup.8 cells/ml were incubated 2 h in 1/100 strength HBSS at a concentration of 100 .mu.g/ml for cathepsin G, or at 500 .mu.g/ml for peptides derived from cathepsin G. The y-axis gives bactericidal activity, as measured by logs (base 10) of lost viability.
FIG. 8 illustrates the complete amino acid sequence (SEQ ID NO:41) for the mature human cathepsin G, as deduced from analysis of its cDNA. In the chymotrypsin nomenclature, it displays the charge relay profile of His57 Asp102 Ser195 that is typical of serine proteases; the charge relay system amino acids are identified with an asterisk (*). The Ser195 residue (residue 181 in the mature cathepsin G protein) is the target of phosphorylation by DFP, resulting in irreversible inhibition of chymotryptic activity. The residues lining the primary specificity pocket of cathepsin G are marked with #.
FIG. 9 presents hydrophobicity analysis for peptide CG 117-136 (SEQ ID NO:22) and the corresponding hydrophobicity-hydrophilicity plot for the amino acid sequence.
FIG. 10 presents a comparison of the amino acid sequence of the broad spectrum antibacterial peptide CG 117-136 (SEQ ID NO:22) with related sequences in other antibacterial proteins and serine proteases. The dots represent amino acid residue identity with the CG 117-136 sequence. The amino acid sequences of the referenced portions of human granzume B, mouse granzyme B, CAP 37, human elastase, proteinase 3 and bovine chymotrypsin are given in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, respectively.

DETAILED DESCRIPTION OF THE INVENTION
As used herein, an oligopeptide is composed of from about five to about twenty-seven amino acids linked together by peptide bonds in a linear array. The peptide may be in a linear conformation or it may assume secondary structure. A cyclic peptide derivative can also have antimicrobial activity, and thus is a functional equivalent of the antimicrobial peptides of the present invention. Sequences are conventionally given from the amino terminus to the carboxyl terminus. The peptides of the present invention have antimicrobial activity by themselves or when coupled to another molecule, e.g., polyethylene glycol or a carrier protein such as bovine serum albumin, so long as the peptides are positioned such that they can come into effective contact with the target cell.
Table 1 presents most abbreviations used in this application. Other abbreviations are as commonly used in the art.
TABLE 1______________________________________Abbreviations______________________________________A = Ala = Alanine M = Met = MethionineC = Cys = Cysteine N = Asn = AsparagineD = Asp = Aspartic Acid P = Pro = ProlineE = Glu = Glutamic Acid Q = Gln = GlutamineF = Phe = Phenylalanine R = Arg = ArginineG = Gly = Glycine S = Ser = SerineH = His = Histidine T = Thr = ThreonineI = Ile = Isoleucine V = Val = ValineK = Lys = Lysine W = Try = TryptophanL = Leu = Leucine Y = Tyr = TyrosineBoc = tert-butyloxycarbonylCFU = colony forming unitDFP = diisopropylfluorophosphateHLE = human leukocyte elastasePam = (phenylacetamido) methyl______________________________________
ED.sub.50 is the concentration of an antimicrobial agent which kills (or otherwise inhibits growth) 50% of the input indicator microorganism or cell under particular test conditions.
For convenience, the peptides newly disclosed herein are named according to the amino acid positions in mature Cat G (FIG. 8) CG 1-20 represents amino acid residues 1-20 of the mature Cat G sequence and has the sequence IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16).
CG 21-40 corresponds in sequence to amino acids 21-40 of Cat G, QSPAGQSRCGGFLVREDFVL (SEQ ID NO:17).
CG 41-60, corresponding to amino acids 41-60 of Cat G, has the sequence TAAHCWGSNINVTLGAHNIQ (SEQ ID NO:18).
CG 61-80, corresponding to amino acids 61-80 of Cat G, has the sequence RRENTQQHITARRAIRHPQY (SEQ ID NO:19).
CG 77-96, corresponding to amino acids 77-96 of Cat G, has the amino acid sequence HPQYNQRTIQNDIMLLQLSR (SEQ ID NO:20).
CG 97-116, corresponding to amino acids 97-116 of Cat G, has the sequence RVRRNRNVNPVALPRAQEGL (SEQ ID NO:21).
CG 117-136, corresponding to amino acids 117-136 of Cat G, has the sequence RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22).
CG 137-156, corresponding to amino acids 137-156 of Cat G, has the sequence DTLREVQLRVQRDRQCLRIF (SEQ ID NO:23).
CG 157-176, corresponding to amino acids 157-176 of Cat G, has the sequence GSYDPRRQICVGDRRERKAA (SEQ ID NO:24).
CG 177-197, corresponding to amino acids 177-197 of Cat G, has the sequence FKGDSGGPLLCNNVAHGIVSY (SEQ ID NO:25).
CT 198-223, corresponding to amino acids 198-223 of Cat G, has the sequence GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:26).
Antimicrobial activity, as used herein, refers to the ability of a peptide of the present invention to kill at least one species selected from the group consisting of Gram positive bacteria, Gram negative bacteria, fungi, and protozoans. It is increasingly preferred that the peptide kill at least 50%, 60%, 70%, 80%, 90% or all cells of at lest one species of gram positive or gram negative bacteria, fungi, or protozoans. Sensitive Gram positive bacteria can include, but are not limited to, Staphylococcus aureus. Sensitive Gram negative bacteria include, but are not limited to, Escherichia coli, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. Periodontal disease-associated bacteria include Capnocytophaga sputigena, Actinobacillus actinomycetemcomitans and Eikenella corrodens. Capnocytophaga sputigena ATCC 33123 is sensitive to IIGGR (SEQ ID NO:1), IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16) and HPQYNQR (SEQ ID NO:3). A. acetinomycetemcomitans is sensitive to IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3). E. corrodens is more sensitive to IIGGR than to HPQYNQR. Sensitive fungi can include, but are not limited to, Candida albicans. Antimicrobial activity can also refer to the ability to kill or inhibit the growth of other cells, in particular, those which are tumor cells or virus-infected cells.
The antimicrobial peptides of the present invention are oligopeptides which possess antimicrobial activity, as defined herein. These antimicrobial peptides may contain modifications such as acetylation, provided that the antimicrobial activity is not destroyed. Chemical modifications which do not destroy antimicrobial activity are those which do not substantially decrease the hydrophilicity of the antimicrobial peptide and those which are not bulky hydrophobic chemical groups, particularly for antimicrobial peptides related in sequence to HPQYNQR. Modified peptides with antimicrobial activity are functionally equivalent to the antimicrobial peptides of the present invention. Such modified peptides with antimicrobial activity include, but are not limited to, (1-methyl-H)QYNQR, (3-methyl-H)PQYNQR, (Ac-H)PQYNQR and HPAYNA.sup.M K.
Antibacterial pharmaceutical compositions, as defined herein, comprise a pharmaceutically acceptable carrier and one or more antibacterial peptides of the present invention. Such antimicrobial pharmaceutical compositions may be formulated in ways, as understood in the art, for use for topical application, for gingival application (for gingivitis or periodontal disease) or for local or systemic injection. For use in the treatment or prevention of gingivitis, the peptides of the present invention can be incorporated in effective amounts in a dental rinse for application to the buccal area, or they may be incorporated in other suitable compositions for topical application. The antibacterial peptides of the present invention may also be incorporated in effective amounts in chewing gum, lozenges for sucking, toothpowder or toothpaste. The antibacterial peptides of the present invention can comprise from 0.001% to 50% by weight of such compositions. It will be understood that a composition for systemic injection will contain an antimicrobial peptide, e.g., an antibacterial peptide such as HPQYNQR, in a therapeutically effective amount or a therapeutically effective amount of an antimicrobial peptide can be conjugated to an antibody, or any other compound as understood in the art, with specificity for the target cell type. The choice of the peptide will be made with consideration of immunogenicity and toxicity to the infected host, effective dose of the peptide, and the sensitivity of the target microbe to the peptide, as well-understood in the art.
The Cat G protein was analyzed to determine whether the same portions of the protein were responsible for the enzymatic and antibacterial activity. Human Cat G was purified and digested with the proteolytic enzyme clostripain. Peptides resulting from that digestion were purified and individually tested for antibacterial and enzymatic activity. None of the peptides tested exhibited the chymotrypsin-like activity of the intact molecule. However, two Cat G-derived peptides exhibited antibacterial activity using Staphylococcus aureus or Neisseria gonorrhoeae as the indicator organism. Those peptides were IIGGR (SEQ ID NO:1) (peptide 1; amino acids 1-5) and HPQYNQR (peptide 2; amino acids 77-83) (SEQ ID NO:3). Similar antibacterial activities were observed for synthetic peptides identical in sequence to the above-noted peptides. Similarly, the oligopeptide corresponding in amino acid sequence to amino acids 1-20 of Cat G (i.e., SEQ ID NO:16) exhibited strong bactericidal activity against Pseudomonas aeruginosa. Even more effective as an antimicrobial agent is the oligopeptide corresponding in sequence to amino acids 1-20 of Cat G, but which retains some blocking groups from the component derivatized amino acids in chemical peptide synthesis or an artifactual reaction product. The chemical identity of the substituents on that oligopeptide or reaction product have not yet been identified. FIG. 4(A) illustrates killing of S. aureus and N. gonorrhoeae by these two peptides.
A peptide with a one amino acid substitution (V for I at position 2 of IIGGR (SEQ ID NO:1)) was synthesized and tested for antibacterial activity. This sequence is identical with the first five amino acids of human leukocyte elastase, CAP37 and azurocidin. Killing of both the Gram positive and the Gram negative test organism was less efficient with the IVGGR sequence (SEQ ID NO:2) (ED.sub.50 >8.75.times.10.sup.-4 M) than with the IIGGR sequence (ED.sub.50 =4.3.times.10.sup.-4 M), although the IVGGR sequence was somewhat more effective against gonococci than staphylococci (see FIG. 3A).
Killing of staphylococci and gonococci by HPQYNQR (SEQ ID NO:3) and APQYNQR (SEQ ID NO:27) was also tested. The results are shown in FIG. 3(B); for HPQYNQR (SEQ ID NO:3 the ED.sub.50 =1.19.times.10.sup.-4 M for S. aureus, and the ED.sub.50 for N. gonorrhoeae was 5.0.times.10.sup.-5 M. The replacement of histidine with alanine (APQYNQR) abolished its bactericidal activity for both microorganisms.
Antimicrobial activity of the Cat G-derived peptides was found to be dependent on assay temperature, pH and ionic strength. As for full-length Cat G, effective killing of S. aureus by the synthetic peptides was optimal at 37.degree. C. with activity from about 30.degree. to about 39.degree. C., and was optimal at pH values of 7.0-7.5 for the range of pH values tested (Results for HPQYNQR (SEQ ID NO:3) are shown in FIGS. 4 and 6B). The antimicrobial action of these peptides was optimal in standard HBSS (0.12M NaCl); conditions of increased ionic strength (0.17-0.21M NaCl) completely inhibited the antibacterial action (FIG. 6B) except for peptides HPQYNPK (SEQ ID NO:49) or Ac-HPQYNQR.
Because the peptides tested individually showed significantly less activity than the natural Cat G protein or the clostripain digest, IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3) were tested for synergistic activity. Under optimal conditions of temperature, pH and ionic strength, IIGGR and HPQYNQR together were about twice as active against S. aureus than when tested individually, but still less active than native Cat G or a clostripain digest of Cat G. A synthetic peptide consisting of the peptides 1 and 2 joined by a bridge (AIR) had activity similar to that of HPQYNQR alone (ED.sub.50 =5.0.times.10.sup.-5 M). Thus, these two peptides together were less effective against S. aureus than natural Cat G (ED.sub.50 =4.0.times.10.sup.-6 M), suggesting that other antimicrobial domains or sequences flanking peptides 1 and 2 in the full-length molecule contribute to the total antimicrobial activity of full-length Cat G. Alternatively, other peptides in the clostripain-Cat G digestion mixture may have potentiated the activity of IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3).
The antibacterial activities of IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3) were compared with other known antimicrobial agents (FIG. 5). These peptides exhibited activities roughly comparable to those of a pool of defensins and to the clinically useful antibiotics chloramphenicol and tetracycline (test concentration of each compound 5.0.times.10.sup.-4 M). For the heptapeptides, this corresponds to about 100 micrograms per ml. Chloramphenicol was more effective at this concentration than the defensin pool or the test peptides, and these peptides from Cat G were more active than tetracycline, streptomycin, kanamycin or the defensin pool.
IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3) were used to search known protein sequences of other cytotoxic serine proteases present in the cytolytic lymphocytes of mice and humans for partial amino acid sequence identities. Proteins containing the IIGGR sequence at the N-termini include Cat G and eosinophil cationic protein (Gleich et al. (1986) Proc. Natl. Acad. Sci. USA 83:3146-3150). Human leukocyte elastase (Gabay et al. (1989) supra; azurocidin (Sinha et al. (1987) Proc. Natl. Acad. Sci. USA 84:2228-2232); and CAP 37 (Pereira et al. (1990) J. Clin. Invest. 85:1468-1476) begin with the sequence IVGGR (SEQ ID NO:2). Human leukocyte elastase does not have antimicrobial activity in vitro. CCCP I and human lymphocyte granzymes B, D, E and G begin with IIGGH (SEQ ID NO:28) while RMCP II begins with the sequence IIGGV (SEQ ID NO:29) (Salvesen et al. (1987) supra). It may be assumed that the IIGGR (SEQ ID NO:1) of Cat G is located on the interior of the protein because it forms a salt bridge with Asp-170, a residue which has been invariably identified to be a part of the binding site of other chymotrypsin-like serine proteases. This Cat G residue corresponds to Asp-102 in the catalytic triad of chymotrypsin. This hypothesis is consistent with the X-ray crystallographic structure of chymase (Remington et al. (1988) Biochemistry 27:8097-8115), which has significant sequence similarity with Cat G; a similar domain is buried in the interior of the molecule. It is probable that HPQYNQR is oriented toward the surface of Cat G because of the positioning of an analogous sequence in chymase, the structure of which has been predicted.
Human granzymes A and B (granzyme B is also known as human lymphocyte protease) both contain internal sequences related to HPQYNQR (SEQ ID NO:3). HPAYNPK (SEQ ID NO:5), corresponding to an internal region of human leukocyte protease, possesses broad spectrum antimicrobial activity in vitro. The related sequences of mouse granzymes A, C, E, F and G do not possess such activity (see Table 5).
Variant peptides related to the sequences IIGGR and HPQYNQR were tested for antimicrobial activity. The results of testing variant sequences of the HPQYNQR peptide sequence for antimicrobial activity has led to the formulation of a consensus sequence for an antimicrobial peptide: HX.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 (SEQ ID NO:4).
Preferably X.sub.1 is proline, alanine or histidine; X.sub.2 is one of asparagine, aspartic acid, glutamine, glutamic acid or alanine; X.sub.3 is tyrosine or phenylalanine; X.sub.4 is asparagine or alanine; X.sub.5 is proline, glutamine, alanine or N-methyl alanine; and X.sub.6 is lysine, arginine, alanine, --OH or --NH.sub.2.
Antimicrobial hexa- and heptapeptide sequences falling within the consensus sequence include, but are not limited to, HPQYNQR (SEQ ID NO:3), HPAYNPK (SEQ ID NO:5), HPAYNPR (SEQ ID NO:6) and HPQYNQR (SEQ ID NO:3). Additional antimicrobial peptide sequences related to HPQYNQR include, but are not limited to, HPQYAQR (SEQ ID NO:8), HPQYNQA (SEQ ID NO:9), HPQYNAR (SEQ ID NO:10), HPQYNPR (SEQ ID NO:30), HAQYNQR (SEQ ID NO:11), HPQYNQ (SEQ ID NO:12), HHQYNQR (SEQ ID NO:31) and HPQYNQ.sup.M K. Preferably, the oligopeptides consist essentially of the foregoing sequences. CG 77-96 (HPQYNQRTIQNDIMLLQLSR (SEQ ID NO:14)) does not exhibit antimicrobial activity.
To determine the amino acids essential for the broad spectrum activity of HPQYNQR (SEQ ID NO:3), synthetic peptides with single alanine substitutions were prepared and tested for their antibacterial activity in vitro. The parental peptide sequence HPQYNQR and the following alanine-substituted derivatives (HPAYNQR (SEQ ID NO:7), HPQYAQR (SEQ ID NO:8) and HPQYNQA (SEQ ID NO:9) exerted antibacterial action in vitro against S. aureus strain 8325-4 at a concentration of about 5.times.10.sup.-4 M, while certain other alanine-substituted derivatives (HPQYNAR (SEQ ID NO:10) and HPAYNPR (SEQ ID NO:6)) had reduced antibacterial activity, and still others (APQYNQR (SEQ ID NO:27) and HPQANQR (SEQ ID NO:32)) had no antibacterial activity in this assay. Similar activities or lack of activity was also observed with E. coli and with N. gonorrhoeae. These results are given in Table 2.
TABLE 2______________________________________Bactericidal Action of Synthetic PeptideVariants of Antimicrobial HPQYNQRPeptide Sequence.sup.1 % Survival of S. aureus N______________________________________HPQYNQR (SEQ ID NO: 3) 28.6 .+-.1.57 15APQYNQR (SEQ ID NO: 27) 98.5 .+-.2.1 12HAQYNQR (SEQ ID NO: 11) 31.2 .+-.2.4 9HPAYNQR (SEQ ID NO: 7) 43.6 .+-.2.54 6HPQANQR (SEQ ID NO: 32) 96.7 .+-.4.34 6HPQYAQR (SEQ ID NO: 8) 30.6 .+-.0.85 6HPQYNAR (SEQ ID NO: 10) 51.2 .+-.3.6 6HPQYNQA (SEQ ID NO: 9) 39.8 .+-.1.96 6HPQKNTY (SEQ ID NO: 33) 98.6 .+-.2.13 3______________________________________ .sup.1 Antimicrobial synthetic peptide HPQYNOR, its derivatives with single alanine substitutions at each position, and a control 403A peptide (HPQKNTY) were synthesized on an Applied Biosystem Model 403A peptide synthesizer (0.1 mmol scale) using phenylacetamidomethyl or pmethylbenzyhydrylamine copoly(styrene/divinyl benzene) resins (Applied Biosystems, Inc.) and tertbutyloxycarbonyl (Boc)protected amino acids. Th data are presented as % survival .+-. SEM and represent results from at least 3 separate experiments for each peptide with S. aureus as the indicator organism.
These results suggest that the N-terminal histidine and the tyrosine residue at position 4 may be important determinants for the antibacterial activity of HPQYNQR (SEQ ID NO:3). To more closely examine the structural requirements of this peptide sequence needed for microbicidal activity, several additional peptides (see Table 4) containing alterations in either His-1 or Tyr-4 and a truncated peptide lacking Arg-7 were prepared and tested for killing of the S. aureus indicator organism. Methylation of nitrogen at position 1 or 3 of the imidazole ring of His-1 had little effect on the antibacterial activity, but derivatives with the imidazole modified at nitrogen-3 with more bulky benzyl or dinitrophenyl groups were inactive. Moreover, a synthetic peptide containing the D-stereoisomer of His was also inactive. Thus it appears that major modifications of the imidazole ring of His-1 abolish antibacterial activity, perhaps due to impaired hydrogen bonding with other amino acid side chains. While replacement of Tyr-4 by alanine inhibited antibacterial activity (Table 2), partial activity was observed where phenylalanine was present as the fourth residue, suggesting that an aromatic amino acid at position 4 is critical to antibacterial activity. The truncated pentapeptide HPQYN-amide was inactive, while the hexapeptide HPQYNQ-amide had antibacterial activity, suggesting that at least six amino acid residues are necessary to form a structure effective for antimicrobial activity. (See Table 3.) Surprisingly, an oligopeptide of the sequence HPQYNQRTIQNDIMLLQLSR (SEQ ID NO: 14) did not exhibit significant antimicrobial activity against either S. aureus or P. aeruginosa.
TABLE 3______________________________________Bactericidal Action of Synthetic Derivativesof Antimicrobial Peptide HPQYNQRPeptide Sequence.sup.1 % Survival of S. aureus N______________________________________HPQYNQR (SEQ ID NO: 3) 28.6 .+-.1.57 15APQYNQR (SEQ ID NO: 27) 98.5 .+-.2.1 12H.sup.1m QYNQR 37.3 .+-.2.25 3H.sup.3m PQYNQR.sup.2 18.3 .+-.3.82 3H.sup.Dnp PQYNQR.sup.2 102.2 .+-.2.92 8H.sup.Benzyl PQYNQR.sup.2 99.0 .+-.2.6 6.sup.Ac HPQYNQR 31.0 .+-.1.6 5.sup.Hep HPQYNQR 97.0 .+-.2.8 3D-HPQYNQR 98.6 .+-.1.6 5HPAYNQR (SEQ ID NO: 7) 43.6 .+-.2.54 6HPQANQR (SEQ ID NO: 32) 96.7 .+-.4.34 6HPQFNQR (SEQ ID NO: 34) 45.0 .+-.4.91 6HPQYNQA (SEQ ID NO: 9) 39.8 .+-.1.96 6HPQYNQ-amide 32.8 .+-.1.83 3HPQYN-amide 98.5 .+-.2.85 5______________________________________ .sup.1 All peptides were tested at 5 .times. 10.sup.-4 M as described in Table 2. .sup.2 Modified histidines are 1methyl (1 m), 3methyl (3 m), 2,4dinitrophenyl (Dnp), benzyl, acetyl (Ac), and heptanoyl (Hep), and D stereoisomer (DHPQYNQR) as described in the text.
To learn whether the hydrophilic nature of HPQYNQR is an important feature for antibacterial action, derivatives containing an acetyl or an heptanoyl moiety attached to the N-terminal amino group were synthesized and tested. The small increase in hydrophobicity imparted by the acetyl group has no significant effect, while the more bulky and hydrophobic heptanoyl group rendered the HPQYNQR (SEQ ID NO:3) derivative inactive.
Peptides corresponding or related to sequences within mouse granzymes A, B, C, D, E, F and G and human granzymes A and B were synthesized. As shown in Table 4, only the synthetic peptides corresponding to mouse granzyme B and human granzyme B had some antibacterial activity.
The human granzyme B-derived peptide differs from the Cat G peptide at positions 3, 6 and 7. To test how these differences affected antibacterial activity, variants of the Cat G and granzyme B peptides containing some of these heterologous amino acids or with alanine substitution at position 3 or 6 were synthesized and tested (Table 5). Placement of the Ala-3 residue in the Cat G only had a slight effect on antibacterial action (Tables 2 and 4). Conversely, placement of the Gln-3 residue in the Granzyme B peptide severely inhibited antibacterial activity. The Pro-6 residue of the granzyme B peptide also appeared to be crucial, because the replacement of Pro-6 with alanine abolished microbicidal activity. To further test the importance of Pro-6, a derivative containing N-methylalanine, which lacks the ring structure of proline but might mimic its hydrogen bonding potential, was synthesized and found to have antibacterial activity. The contribution of the Pro-6 residue to antibacterial activity in the granzyme B-derived peptide appeared to be unique for this peptide, because substitution at position 6 in the Cat G-derived peptide suppressed antibacterial activity. Further derivatives of HPQYNQR (SEQ ID NO:3) with amino acid replacements at the third position were synthesized and tested for antimicrobial activity. HPNYNQR (SEQ ID NO:35) and HPEYNQR (SEQ ID NO:36) had antimicrobial activity comparable to that of HPAYNQR (SEQ ID NO:7) while HPLYNQR (SEQ ID NO:37) lacked activity in the assay. (Table 4) HPDYNQR (SEQ ID NO:53) will have activity comparable to that of HPAYNQR.
TABLE 4______________________________________Activity of Cat G Peptides with Third Position ReplacementsPeptide Percent Survival______________________________________HPQYNQR (SEQ ID NO: 3) 21.2%HPAYNQR (SEQ ID NO: 7) 40.3%HPEYNQR (SEQ ID NO: 36) 45.3%HPNYNQR (SEQ ID NO: 35) 42.9%HPLYNQR (SEQ ID NO: 37) 100.8%______________________________________
TABLE 5______________________________________Bactericidal Action of Synthetic Human and Mouse Granzyme Peptides.sup.1 % SurvivalPeptide Sequence of S. Aureus N______________________________________Cat G HPQYNQR 28.6 .+-.1.57 15 (SEQ ID NO: 3)Cat G (Pro-6) HPQYNPR 78.7 .+-.2.8 4 (SEQ ID NO: 50)Human YPCYDPA 102 .+-.2.6 3 (SEQ ID NO: 51)granzyme AHuman HPAYNPK 36 .+-.2.5 9 (SEQ ID NO: 5)granzyme BHuman HPAYNAK 97 .+-.1.8 3 (SEQ ID NO: 52)granzyme B'.sup.2Human HPQYNPK 95.1 .+-.3.4 5 (SEQ ID NO: 49)granzyme B''Human HPAYNPR 87.5 .+-.4.3 5 (SEQ ID NO: 6)granzyme B'''Human HPAYNA.sup.m K.sup.3 40.5 .+-.1.6 3granzyme B''''Human HPAYNP-amide 42.5 .+-.3.6 5granzyme B'''''Mouse YPCYDEY 99.5 .+-.1.7 3 (SEQ ID NO: 54)granzyme AMouse HPDYNPK 62 .+-.3.4 3 (SEQ ID NO: 55)granzyme BMouse HPDYNPD 104 .+-.3.8 3 (SEQ ID NO: 56)granzyme CMouse HPDYNAT 98 .+-.2.7 3 (SEQ ID NO: 57)granzyme D,E.sup.2Mouse HPAYDDK 98 .+-.1.9 3 (SEQ ID NO: 58)granzyme FMouse HPAFDRK 102 .+-.2.8 3 (SEQ ID NO: 59)granzyme G______________________________________ .sup.1 All peptides were tested at 5 .times. 10.sup.-4 M as described in Table 2. .sup.2 Peptide sequences for mouse granzymes D and E are identical. Human granzyme B variants are designated B'-B'''''. .sup.3 A.sup.M, Nmethyl alanine.
While the deletion of the C-terminal lysine-7 residue from HPAYNPK (SEQ ID NO:5) had no effect on the bactericidal action of the granzyme B-related peptide, its replacement with arginine as in the Cat G peptide severely inhibited antibacterial activity.
As for the full-length Cat G, the antibacterial action of the granzyme B-derived peptide was found to be optimal at slightly basic pH (FIG. 6A) although, like the acetylated derivative of HPQYNQR, significant antibacterial activity was observed at a pH of 6.0. As for full-length Cat G, the antibacterial action of human granzyme B was also sensitive to increasing concentrations of NaCl in the incubation mixture. Surprisingly, the Cat G-derived peptide HPQYNQR (SEQ ID NO:3) and its acetylated derivative retained antibacterial activity even at the highest NaCl concentration (0.21M) tested (FIG. 6B).
As seen with the Cat G-derived peptide, the antibacterial action of human granzyme B extended to E. coli and N. gonorrhoeae as well as S. aureus. Killing of the S. aureus indicator was more rapid with the Cat G-derived peptide than with the human granzyme B-derived peptide (FIG. 6C).
Results with variant peptides related to HPQYNQR (SEQ ID NO:3) suggest that the N-terminal histidine and the internal tyrosine residues were important in generating antimicrobial activity. A substitution of alanine for histidine, D-histidine for the L-isomer or the attachment of a 2, 4-dinitrophenyl group to the imidazole group of L-histidine resulted in a loss of activity. Similarly, a substitution of alanine for tyrosine at position 4 led to a loss of microbicidal activity (see Table 4).
To determine whether hydrophilicity of the antimicrobial peptide was determinant of activity, HPQYNQR (SEQ ID NO:3) was modified by the addition of an acetyl or a heptanoyl group to the N-terminal amino group. Acetylation did not affect activity, while the heptanoyl derivative lacked antimicrobial activity.
The antibacterial activity of the HLP-derived peptide (HPAYNPK) (SEQ ID NO:5) cannot be readily explained using the Cat G sequence (HPQYNQR) (SEQ ID NO:3) as a canonical model. The alanine residue at position 3 and the proline at position 6 of the HLP-derived peptide cannot result from conservative amino acid changes in HPQYNQR, although the lysine for arginine at position 7 is a conservative change. The ability of a position 7 substitution variant (alanine for arginine) of the Cat G sequence to retain significant killing activity might suggest that this is not a key position in determining activity. An alanine substitution at position 3 (for glutamine) decreased activity of the Cat G-related peptide.
The result of testing variant sequences of the HPQYNQR peptide sequence for antimicrobial activity has led to the formulation of a consensus sequence for an antimicrobial peptide. HX.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6, where X.sub.1 is proline, histidine or alanine; X.sub.2 is aspartic acid, asparagine, glutamic acid, glutamine, alanine, serine, threonine, isoleucine, valine, histidine, tyrosine, arginine, methionine oxide or methionine sulfone; X.sub.3 is tyrosine, phenylalanine, tryptophan or naphthyl-alanine; X.sub.4 is asparagine or alanine; X.sub.5 is glutamine, proline, alanine, or N-methyl alanine; and X.sub.6 is arginine, lysine, alanine, --OH or --NH.sub.2 (SEQ ID NO:4).
Cat G-derived peptides were tested for antimicrobial activity against Capnocytophaga sputigena ATCC 33123, which is the same as that now available as C. sputigena ATCC 33612 (American Type Culture Collection, Rockville, Md.). C. sputigena is representative of oral pathogens associated with periodontal disease and/or gingivitis. As shown in FIG. 7, IIGGR (SEQ ID NO:1) and CG 1-20 (SEQ ID NO:16) are effective against C. sputigena ATCC 33123 in vitro. When incorporated in pharmaceutical compositions, one or more of the peptides related in sequence to Cat G can be used to ameliorate gingivitis and/or treat periodontal disease. Compositions for oral use include oral rinses, lozenges and formulations for topical application to the gums. The skilled artisan can use the teachings of the present specification and knowledge readily accessible to the art to prepare pharmaceutically useful formulations for oral application, topical or other applications, particularly after animal studies to confirm that these peptides are not toxic to the human or animal host and are effective in vivo.
Because the antimicrobial activity resulting from the IIGGR and HPQYNQR peptides recovered after clostripain digestion was less than 1% of the activity of intact Cat G, an alternate approach was pursued. Eleven peptides that span the entire 223 amino acid cathepsin G protein were synthesized. These eleven peptides were tested for antibacterial action against N. gonorrhoeae, P. aeruginosa and S. aureus. Of the eleven peptides, only the peptide corresponding to residues 117-136 in the full-length cathepsin G (CG 117-136) (SEQ ID NO:22) displayed antibacterial action against all three pathogens; 500 .mu.g of peptide per ml killed 5-6 logs of P. aeruginosa and S. aureus. See Table 6 for the peptide sequences and activities, as measured using a crude peptide concentration of 500 .mu.g/ml with an input viable cell concentration of about 10.sup.7 colony forming units/ml (CFU/ml) in 1/100 strength HBSS. CB117-136 will be useful as an antibacterial agent against a wide range of bacteria, including pathogens.
TABLE 6__________________________________________________________________________Antibactericidal Activity of Crude Peptides.sup.1 Log Kill (500 .mu.g/ml) P. aeruginosa N. gonorrhoeaePeptide Sequence ATCC 27853 strain WS1__________________________________________________________________________1 20 5.507 2.57 (SEQ ID NO: 16)IIGGRESRPHSRPYMAYLQI21 40 -0.034 0.05 (SEQ ID NO: 17)QSPAGQSRCGGFLVREDFVL41 60 0.5 -0.09 (SEQ ID NO: 18)TAAHCWGSNINVTLGAHNIQ61 80 5.15 0 (SEQ ID NO: 19)RRENTQQHITARRAIRHPQY77 96 0.06 0.24 (SEQ ID NO: 20)HPQYNQRTIQNDIMLLQLSR97 116 5.93 -0.21 (SEQ ID NO: 21)RVRRNRNVNPVALPRAQEGL117 136 5.88 2.38 (SEQ ID NO: 22)RPGTLCTVAGWGRVSMRRGT137 156 0.01 0.43 (SEQ ID NO: 23)DTLREVQLRVQRDRQCLRIF157 176 1.02 -0.18 (SEQ ID NO: 24)GSYDPRRQICVGDRRERKAA177 197 -0.79 0.68 (SEQ ID NO: 25)FKGDSGGPLLCNNVAHGIVSY198 223 5.73 0.78 (SEQ ID NO: 26)GKSSGVPPEVFTRFVSSFLPWIRTTMRBuffer -0.139 -0.07__________________________________________________________________________ .sup.1 Peptides shown in Bold were purified by RPHPLC and tested vs. P. aeruginosa, N. gonorrhoeae and S. aureus (see Table 7A).
Table 7A provides antimicrobial activities of peptides purified by RP-HPLC, determined as above. Only CG 117-136 (SEQ ID NO:22) exhibits high activity against P. aeruginosa, S. aureus and N. gonorrhoeae. Table 7B discloses the ED.sub.90 in .mu.g/ml for the antibacterial peptides, as measured with P. aeruginosa ATCC 27853 as above. ED90 is the dose required to kill 90% of the input cells in 2 h at 37.degree. C. in 1/100 strength HBSS, where the input viable cell concentration is about 10.sup.7 CFU/ml.
TABLE 7A______________________________________Antibacterial Action of RP-HPLC-Purified Peptides Log Kill (500 .mu.g/ml S. aureus P. aeruginosa N. gonorrhoeae strain SEQPeptide ATCC 278533 strain WSI 8325-4 ID NO.______________________________________CG 1-20 1.0 0.43 0.3 16CG 61-80 5.15.sup.1 .sup. ND.sup.2 0.58 19CG 97-116 0.22 0.2 -0.1 21CG 117-136 5.88.sup.1 4.20 5.34 22CG 198-223 5.73.sup.1 ND 0.26 26Buffer Control -0.139 -0.07 -0.25______________________________________ .sup.1 Also active against four clinical isolates of P. aeruginosa .sup.2 Not determined
TABLE 7B______________________________________Potency of Synthetic Cathepsin G Peptides Against P. aeruginosa ED.sub.90Synthetic Peptides (.mu.g/ml) SEQ ID NO:______________________________________CG 1-20 500 16CG 61-80 75 19CG 97-116 >500 21CG 117-136 15 22CG 198-223 115 26______________________________________
It was noted that CG 1-20 (SEQ ID NO:16) and CG 97-116 (SEQ ID NO:21) were highly active as crude peptides, but exhibited much lower activity when purified by RP-HPLC. Without wishing to be bound by any particular hypothesis, the inventors suggest that the high activity in the crude peptide preparation is due to one or more residual blocking or substituent groups or some unidentified side reaction product generated during the hydrogen fluoride cleavage and/or post-synthetic work-up.
Those peptides which exhibited significant activity against P. aeruginosa ATCC 27853, were also tested against four independent clinical isolates. P. aeruginosa ATCC 27853 (American Type Culture Collection, Rockville, Md.) is the strain used for testing antimicrobial activity against P. aeruginosa unless otherwise noted. It is a standardized strain for antibiotic-susceptibility testing of pseudomonads (see, e.g., Code of Federal Regulations, Title 21, Part 460, 1987). CG 61-80 (SEQ ID NO:19) and CG 198-223 (SEQ ID NO:26) exhibited some variability in effectiveness for killing of clinical strains, but so far as tested, CG 117-136 (SEQ ID NO:22) appeared to be a highly effective bactericidal peptide for P. aeruginosa as well as N. gonorrhoeae and S. aureus (see Tables 7A, 8). Agents effective against P. aeruginosa are needed in the art because multiple antibiotic resistance is quite common among clinical strains, and therefore resultant infections are often difficult to treat.
TABLE 8______________________________________Bactericidal Activity of HPLC-Purified PeptidesAgainst P. aeruginosa Clinical Isolates Log Reduction in Viability.sup.1P. aeruginosa Control CG 61-80 CG 117-136 CG 198-223______________________________________ATCC 27853 -0.14 5.49 5.95 5.35#385128 -1.1 1.91 6.17 4.71#36152 -0.89 4.86 5.38 4.28#27853 -0.58 5.80 5.79 4.80#A-91-330-0347 -0.41 4.11 6.40 1.17______________________________________ .sup.1 3-5 .times. 10.sup.7 CFU/ml of the designated strains were exposed to 500 .mu.g/ml of HPLCpurified peptides in 1/100 strength BHSS (10 mm sodium phosphate, pH 7.0, 10 mM NaCl.
To further characterize the activity of CG 117-136, a "D- enantiomer," composed only of D-amino acids but in the same sequence, was synthesized and tested for antimicrobial activity. The L- and D-forms of this same amino acid sequence had equivalent bactericidal activity against both P. aeruginosa and N. gonorrhoeae (Table 9). This result suggests that killing does not require the recognition of a microbial target with a chiral center.
TABLE 9______________________________________The Antibacterial Capacity of CG 117-136 (SEQ ID NO: 22)is Independent of Stereochemistry Log Kill.sup.1 P. aeruginosa N. gonorrhoeaePeptide ATCC 27853 strain WS1______________________________________L-enantiomer 5.62 4.20D-enantiomer 5.92 4.20______________________________________ .sup.1 P. aeruginosa (3 .times. 10.sup.7 CFU/ml) was exposed to 125 .mu.g/ml of each peptide, while N. gonorrhoeae (5 .times. 10.sup.6 CFU/ml was exposed to 500 .mu.g/ml of each peptide.
As shown in FIG. 2B, CG 117-136 (SEQ ID NO:22) is predicted to have a hydrophobic domain in the N-terminal portion of the peptide and a cationic, hydrophilic domain in the C-terminal portion. The contribution(s) of these domains to antimicrobial activity was assessed by synthesizing truncated versions of CG 117-136. Both domains were required for full activity. Omission of either C- or N-terminal residues destroyed activity against S. aureus, while omission of the five N-terminal residues resulted in only about a 10-fold drop in activity for both P. aeruginosa and N. gonorrhoeae. Omission of the ten N-terminal residues caused nearly total loss of activity against P. aeruginosa and a lesser reduction in activity as measured against N. gonorrhoeae (see Table 10A, 10B).
TABLE 10A______________________________________Summary of Domains in CG 117-136 and inTruncated CG 117-136 Variants Hydrophobic HydrophilicPeptide Domain Domain______________________________________CG 117-136 (SEQ ID NO: 22) + +CG 117-129 (SEQ ID NO: 38) + -CG 122-136 (SEQ ID NO: 39) .+-. +CG 127-136 (SEQ ID NO: 40) - +______________________________________
TABLE 10B______________________________________Antibacterial Action of CG 117-136 andTruncated Variants of CG 117-136 Log Kill (500 .mu.g/ml SEQ P. aeruginosa N. gonorrhoeae S. aureusPeptide ID NO: ATCC 27853 strain WS1 strain 8325-4______________________________________CG 117-136 22 5.8 4.20 5.34CG 117-129 38 0.86 0.05 0CG 122-136 39 4.75 4.18 0CG 127-136 40 0.27 3.20 0______________________________________
The secondary structure of CG 117-136 (SEQ ID NO:22) has been predicted by computer analysis to exhibit .beta.-Sheet structure. By contrast, several peptides known to interrupt the gram-negative envelope are .alpha.-helical peptides (See, e.g., Vaara, M. (1992) Microbiological Rev. 56:395-411).
The potent antibacterial peptide CG 117-136 (SEQ ID NO:22) displays partial significant sequence identities with other serine proteases or serine protease-like proteins (FIG. 10), termed serpocidins, based on their toxic action against bacteria and eucaryotic cells. The mechanism of cytotoxicity has not been defined, but it is likely that the serpocidins behave as membrane-disorganizing agents. Electron microscopic analysis of CG 117-136-treated P. aeruginosa cells revealed similar effects on cell morphology to those seen after treatment with polycationic agents. Lysis did not result from CG 117-136 treatment.
A comparison of the amino acid sequence of CG 117-136 (SEQ ID NO:22) to partial sequences of other antimicrobial proteins and serine proteases is given in FIG. 10. The dots represent amino acid identity. The skilled artisan can readily identify variants of the CG 117-136 sequence (or of other antibacterial peptides disclosed herein) by synthesizing defined variants and testing as taught herein.
The sensitivities of the P. aeruginosa and S. aureus indicator cells to enzymatically-active and inactive Cat G were determined (see Table 11). Surprisingly, peptides (CG 61-80, CG 117-136, CG 198-223 (SEQ ID NOS:19, 21, 26, respectively)) from cathepsin G display potent bactericidal action in vitro even against a pathogen (P. aeruginosa) that is killed only by enzymatically active Cat G.
TABLE 11______________________________________Bactericidal Capacities of Enzymatically-Active and -Inactive Cathepsin Log Reduction in CFU/ml.sup.1 S. aureus P. aeruginosa______________________________________Control -0.18 -0.7650 .mu.g/ml enzymatically active Cat G 2.82 2.4150 .mu.g/ml DFP-treated Cat G 2.93 0.125______________________________________ .sup.1 Approximately 5 .times. 106 CFU/ml of the test bacteria were incubated with the cathepsin G samples in 1% (w/v) TSB (tryticase soy broth) for 2 h at 37.degree. C. The results are average values from 2 determinations for each strain and preparation of cathepsin G.
Only enzymatically-active cathepsin G kills P. aeruginosa, while other pathogens are readily killed by both active and inactive cathepsin G. It is theorized that in order for the antibacterial peptides to properly interact with the pseudomonad cell envelope, the structure of the active site must be in its native state in order to allow accessibility of bactericidal domains in the full-length molecule or promote liberation of bactericidal fragments the full-length molecule by a mechanism such as bacterial protease action or by autoproteolysis, but the inventors do not wish to be bound by this theory. The results disclosed herein support the notion that bactericidal serine esterases possess broad spectrum antibacterial action due to the presence of internal antibacterial domains and that multiple, distinct domains exist within cathepsin G for the purpose of killing different pathogens.
The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention. Because modification of the examples below will be apparent to those of skill in the art, it is intended that this invention be limited only by the scope of the appended claims. All references cited in this application are hereby incorporated by reference herein.
EXAMPLES
Example 1.1
Purification of Cat G and Human Neutrophil Elastase
Cat G and human neutrophil elastase (HLE) were purified from extracts of human PMN granules as described previously (Baugh and Travis (1976) Biochemistry 15:836-841). After purification, each enzyme was stored at -20.degree. C. in 50 mM sodium acetate (pH 5.5) 0.5M NaCl prior to use.
Example 1.2
Clostripain Digestion of Cathepsin G
Cat G (600 micrograms) was incubated with purified clostripain (Sigma Chemical Company, St. Louis, Mo.) at a molar ratio of 50:1 in 50 mM Tris-HCl (pH 7.5) 10 mM CaCl.sub.2 0.16M NaCl 2.5 mM Dithiothreitol at 37.degree. C. for 24 hours. During the incubation period samples were removed at various times and assayed for enzymatic and/or bacterial activity.
The enzymatic digestion of Cat G was monitored by loss of esterase activity using the synthetic substrate (Suc-L-Ala-L-Ala-Pro-Phe-pNA (Nakajima et al. (1979) J. Biol. Chem. 254:4027-4032). In some instances digestion was monitored by SDS polyacrylamide gel electrophoresis (Laemmli (1970) Nature (London) 227:680-685). Cat G was tested for antimicrobial activity as described in Shafer et al. (1986) Infect. Immun. 54:184-188.
Example 1.3
Isolation of Cat G Peptides from Clostripain Digest
Clostripain (Mr 30 kDa) and undigested Cat G were separated from the degradation peptides by loading the 24 h digestion mixture (1.0 ml) on a Sephadex G-50.TM. (Pharmacia Fine Chemicals, Piscataway, N.J.) column which was equilibrated with 1.0M NH.sub.4 OH. Absorbances at 220 nm and 280 nm were monitored and those fractions containing peptides were pooled, lyophilized and dissolved in 0.1% trifluoroacetic acid (TFA).
The peptide mixture was then applied to a reverse phase HPLC (RP-HPLC) C-18-10 column which had been previously equilibrated with 0.1% TFA. Bound peptides were then eluted using a linear gradient of acetonitrile (0 to 70% (v/v)) in 0.1% TFA at a flow rate of 0.5 ml/min. Fractions were lyophilized for subsequent testing in antimicrobial assays. Fractions with bactericidal activity were reapplied to the RP-HPLC C-18-10 column. The bound peptides were eluted with non-linear gradient of acetonitrile (0 to 50% v/v) in 0.1% TFA with a flow rate of 0.5 ml/h. The elution of peptides was monitored by following A.sub.220. Peptide concentrations were measured using ninhydrin as described (Rosen et al. (1962) Anal. Biochem. 4:213-221).
Example 1.4
Antimicrobial Activity Testing
Neisseria gonorrhoeae strain FA 102 and Staphylococcus aureus strain 83251-4 were the test bacteria used in many experiments; these strains have been described previously (Shafer et al. (1986) supra; Shafer and Onunka (1989) J. Gen. Microbiol. 135:825-830). N. gonorrhoeae were passaged on clear typing agar as nonpiliated, transparent variants. For testing, cultures were grown with shaking at 37.degree. C. in GC broth containing glucose, iron and sodium bicarbonate supplements. S. aureus was grown at 37.degree. C. with shaking in LB broth. At midlogarithmic phase (OD.sub.550 of 0.35) the cultures were diluted in Hanks Balanced Salt Solution (HBSS) (Gibco Laboratories, Grand Island, N.Y.) (pH 7.5) to give approximately 10.sup.5 CFU/ml. In other experiments, P. aeruginosa ATCC 27853, a standard antibiotic tester strain, was used.
Peptides were dissolved in HBSS (pH 7.5) and added in various amounts (0 to 100 micrograms) to sterile microtiter wells. After UV sterilization of the wells, 0.1 ml samples of the bacterial were added and the volumes in each well were adjusted with HBSS to 0.2 ml. The bacteria-peptide mixtures were incubated at 37.degree. C. for 45-60 min unless otherwise noted. For N. gonorrhoeae, incubation was carried out under an atmosphere of 5% CO.sub.2. In other experiments, as noted, 1/100 strength HBSS was used. For at least some strains, the use of 1/100 HBSS resulted in greater sensitivity to the bactericidal activity of the peptides disclosed herein.
Viability was determined after incubation by plating 10 and 100 microliter samples on LB agar (S. aureus) or GCB agar (N. gonorrhoeae). All assays were done in duplicate or triplicate, and the results given are the means of three independent experiments. The % survival of the test bacteria was calculated as 100.times.(# CFU in the presence of peptide)/(# CFU in the absence of peptide); standard error of the mean for each data point was never greater than 5%.
Certain antibiotics were tested for antibacterial activity using the procedure described above. Penicillin G, tetracycline, chloraphenicol, streptomycin and kanamycin (Sigma Chemical Company, St. Louis, Mo.) were each dissolved in distilled water or 50% (v/v) methanol to give a 1 mg/ml solution. All antibiotic solutions were stored at -20.degree. C.
A partially purified preparation containing the three human defensins described by Selsted et al. (1985) J. Clin. Invest. 76:1436-1439, was also used in the antibacterial activity testing protocol described above. This preparation was provided by Dr. John Spitznagel, Emory University School of Medicine, Atlanta, Ga.). The preparation was obtained by Sephadex G-75.TM. (Pharmacia, Piscataway, N.J.) chromatography of a crude acid extract of human PMN granules (Shafer et al. (1988) Infect. Immun. 56:51-53). The mixture of the three defensins eluted from the column after lysozyme (Mr 14.4 kDa), as is consistent with their molecular weights of approximately 5 kDa. Before use in the antibacterial activity test, the pooled defensins were dialyzed against 4 liters of distilled water at 4.degree. C. using dialysis tubing with an exclusion limit of 3 kDa.
Example 1.5
Sequence analysis of Antibacterial Peptides
The sequences of the antibacterial peptides from clostripain digestion and HPLC resolution were determined by automated Edman degradation in a Model 477A Pulse Liquid Sequencer (Applied Biosystems, Inc., Foster City, Calif.), and the PTH-amino acids were identified on-line in a 120A PTH Analyzer (Applied Biosystems, Inc.).
Example 2
Preparation of Synthetic Peptides
Oligopeptides were synthesized using an Applied Biosystems Model 430A peptide synthesizer (0.1-0.5 mmol scale) using phenylacetamidomethyl (Pam) or p-methylbenzyhydrylamine copoly(styrene/divinylbenzene) resins (Applied Biosystems, Inc., Foster City, Calif.) and tert-butyloxycarbonyl (Boc)-protected amino acids (Applied Biosystems, Inc. or Bachem, Inc., Torrance, Calif.). Boc-N-methyl-Ala, Boc-Arg (tosyl) or Boc-Arg (mesitylenesulfonyl), Boc-Asp (benzyl), Boc-Cys(4-methoxybenzyl), Boc-Glu(benzyl), Boc-His(benzyloxycarbonyl) or Boc-His(2,4-dinitrophenyl), Boc-D-His(4-toluenesulfonyl), Boc-Lys(chlorobenzyloxycarbonyl), Boc-Met, Boc-Ser(benzyl), Boc-Thr(benzyl), Boc-Trp or Boc-Trp(formyl), and Boc-Tyr(2-bromobenzyloxycarbonyl) were used for the incorporation of the respective amino acid residues. Boc-His(methyl) was incorporated in a manual mode on a 0.02 mmol scale using the N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole coupling protocol. All amino acids (except glycine) used herein have the L configuration unless otherwise noted.
Peptides were cleaved from the resin and deprotected in liquid HF/R-cresol/dimethyl sulfide (10:1:0.5) at -5.degree. C. for 90 min, or in liquid HF/anisole (9:1, v/v) at 0.degree. C. for 90 min. The resins were washed with cold diethyl ether, and the peptides were extracted into 1.0H acetic acid and lyophilized. Peptides were then purified by RP-HPLC on an Aquapore.TM. RP-300 C18 silica column (1.times.10 cm, Applied Biosystems, Inc.), or on an MRPH-Gel.TM. polystyrene column (1.times.10 cm, The Nest Group, Scarborough, Mass.) using a 0-60% linear gradient of acetonitrile in 0.1% TFA. The purity of each synthetic peptide preparation was confirmed by microbore HPLC on Aquapore.TM. OD-300 columns of C18 silica (1.times.250 mm, Applied Biosystems, Inc.), quantitative amino acid analysis and sequencing, as described above. Peptides were generally stored in the lyophilized form at 4.degree. C. prior to use in the antibacterial assays.
It is understood in the art that there are other suitable peptide synthetic devices or that manual peptide synthesis could be carried out to produce the peptides of the present invention. Automated solid phase peptide synthesis is described, e.g., in Stewart et al. (1984) Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill.).
Example 3
Search for Related Peptide Sequences
A search was done on protein computer databases using the HPQYNQR sequence for similar sequences in other proteins. Sequences with some similarity to this Cat G sequence were recognized in certain other cytotoxic proteases. Searches were also done using the IIGGR sequence (SEQ ID NO:1) and the CG 117-136 sequence (SEQ ID NO:22).
The antimicrobial peptides of the present invention will be useful for inhibiting bacterial growth for research, including in vitro culture applications, and, when formulated into therapeutic compositions, will be useful in the treatment of infections, especially bacterial infections. The antimicrobial peptides can be administered by any mechanism known to the art, as appropriate for a particular type of infection.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 59(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:IleIleGlyGlyArg15(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:IleValGlyGlyArg15(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:HisProGlnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Region(B) LOCATION: 2..3(D) OTHER INFORMATION: /label=Xaa/note= "X at position 2 is Pro, His or Ala."(ix) FEATURE:(A) NAME/KEY: Peptide(B) LOCATION: 1..3(D) OTHER INFORMATION: /product="OTHER"/label= Xaa/note= "X at position 3 is Asp, Asn, Glu, Gln, Ala, Ser,Thr, Ile, Val, Tyr, Arg, Methionin Oxide or MethionineSulfone."(ix) FEATURE:(A) NAME/KEY: Region(B) LOCATION: 3..4(D) OTHER INFORMATION: /product="OTHER"/label= Xaa/note= "X at position 4is Tyr, Phe, Trp orBeta- naphthyl-alanine."(ix) FEATURE:(A) NAME/KEY: Region(B) LOCATION: 4..5(D) OTHER INFORMATION: /product="OTHER"/label= Xaa/note= "X at position 5 is Ala or Asn."(ix) FEATURE:(A) NAME/KEY: Region(B) LOCATION: 5..6(D) OTHER INFORMATION: /product="OTHER"/label= Xaa/note= "X at position 6 is Gln, Pro, Ala orN-methyl- alanine."(ix) FEATURE:(A) NAME/KEY: Region(B) LOCATION: 6..7(D) OTHER INFORMATION: /product="OTHER"/label= Xaa/note= "X at position 7 is Arg, Lys, Ala, NH2 or OH."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:HisXaaXaaXaaXaaXaaXaa15(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:HisProAlaTyrAsnProLys15(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:HisProAlaTyrAsnProArg15(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:HisProAlaTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:HisProGlnTyrAlaGlnArg15(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:HisProGlnTyrAsnGlnAla15(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:HisProGlnTyrAsnAlaArg15(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:HisAlaGlnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:HisProGlnTyrAsnGln15(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:ArgHisProGlnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:HisProGlnTyrAsnGlnArgThrIleGlnAsnAspIleMetLeuLeu151015GlnLeuSerArg20(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:AlaProGlnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:IleIleGlyGlyArgGluSerArgProHisSerArgProTyrMetAla151015TyrLeuGlnIle20(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GlnSerProAlaGlyGlnSerArgCysGlyGlyPheLeuValArgGlu151015AspPheValLeu20(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:ThrAlaAlaHisCysTrpGlySerAsnIleAsnValThrLeuGlyAla151015HisAsnIleGln20(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:ArgArgGluAsnThrGlnGlnHisIleThrAlaArgArgAlaIleArg151015HisProGlnTyr20(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:HisProGlnTyrAsnGlnArgThrIleGlnAsnAspIleMetLeuLeu151015GlnLeuSerArg20(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:ArgValArgArgAsnArgAsnValAsnProValAlaLeuProArgAla151015GlnGluGlyLeu20(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:ArgProGlyThrLeuCysThrValAlaGlyTrpGlyArgValSerMet151015ArgArgGlyThr20(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:AspThrLeuArgGluValGlnLeuArgValGlnArgAspArgGlnCys151015LeuArgIlePhe20(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GlySerTyrAspProArgArgGlnIleCysValGlyAspArgArgGlu151015ArgLysAlaAla20(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:PheLysGlyAspSerGlyGlyProLeuLeuCysAsnAsnValAlaHis151015GlyIleValSerTyr20(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:GlyLysSerSerGlyValProProGluValPheThrArgPheValSer151015SerPheLeuProTrpIleArgThrThrMetArg2025(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:AlaProGlnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:IleIleGlyGlyHis15(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:IleIleGlyGlyVal15(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:HisProGlnTyrAsnProGln15(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:HisHisGlnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:HisProGlnAlaAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:HisProGlnLysAsnThrTyr15(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:HisProGlnPheAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:HisProAsnTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:HisProGluTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:HisProLeuTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 13 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:ArgProGlyLeuThrLeuCysThrValAlaGlyTrpGly1510(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:CysThrValAlaGlyTrpGlyArgValSerMetArgArgGlyThr151015(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:TrpGlyArgValSerMetArgArgGlyThr1510(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 223 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:IleIleGlyGlyArgGluSerArgProHisSerArgProTyrMetAla151015TyrLeuGlnIleGlnSerProAlaGlyGlnSerArgCysGlyGlyPhe202530LeuValArgGluAspPheValLeuThrAlaAlaHisCysTrpGlySer354045AsnIleAsnValThrLeuGlyAlaHisAsnIleAspArgArgGluAsn505560ThrGlnGlnHisIleThrAlaArgArgAlaIleArgHisProGlnTyr65707580AsnGlnArgThrIleGlnAsnAspIleMetLeuLeuGlnLeuSerArg859095ArgValArgArgAsnArgAsnValAsnProValAlaLeuProArgAla100105110GlnGluGlyLeuArgProGlyThrLeuCysThrValAlaGlyTrpGly115120125ArgValSerMetArgArgGlyThrAspThrLeuArgGluValGlnLeu130135140ArgValGlnArgAspArgGlnCysLeuArgIlePheGlySerTyrAsp145150155160ProArgArgGlnIleCysValGlyAspArgArgGluArgLysAlaAla165170175PheLysGlyAspSerGlyGlyProLeuLeuCysAsnAsnValAlaHis180185190GlyIleValSerTyrGlyLysSerSerGlyValProProGluValPhe195200205ThrArgValSerSerPheLeuProTrpIleArgThrThrMetArg210215220(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:LysProGlyGlnThrCysSerValAlaGlyTrpGlyGlnThrAlaPro151015LeuGlyLysSer20(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:LysProGlnAspValCysTyrValAlaGlyTrpGlyArgMetAlaPro151015MetGlyLysTyr20(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:GluAlaGlnThrArgCysGlnValAlaGlyTrpGlySerGlnSerArg151015SerGlyGlyArg20(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:GlyAsnGlyValGlnCysLeuAlaMetGlyTrpGlyLeuLeuGlyArg151015AsnArgGlyIle20(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:ProHisGlyThrGlnCysLeuAlaMetGlyTrpGlyArgValGlyAla151015HisProProPro20(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:AlaAlaGlyThrThrCysValThrThrGlyTrpGlyLeuThrArgTyr151015ThrAsnAlaAsn20(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:GlyIleGlyLysPheLeuHisSerAlaLysLysPheLysAlaPheVal151015GlyGluIleMetAsn20(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:HisProGlnTyrAsnProLys15(2) INFORMATION FOR SEQ ID NO:50:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:HisProGlnTyrAsnProArg15(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:TyrProCysTyrAspProAla15(2) INFORMATION FOR SEQ ID NO:52:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:HisProAlaTyrAsnAlaLys15(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:HisProAspTyrAsnGlnArg15(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:TyrProCysTyrAspGluTyr15(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:HisProAspTyrAsnProLys15(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:HisProAspTyrAsnProAsp15(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:HisProAspTyrAsnAlaThr15(2) INFORMATION FOR SEQ ID NO:58:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:HisProAlaTyrAspAspLys15(2) INFORMATION FOR SEQ ID NO:59:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:HisProAlaPheAspArgLys15__________________________________________________________________________

Antimicrobial peptides

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