Antimicrobial peptides

Peptides which exhibit antimicrobial activity comparable to certain known antibiotics are provided. These peptides are related in sequence to amino acid sequences within Cathepsin G. A broad spectrum bactericidal peptide disclosed herein is RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22). It is active against Pseudomonas aeruginosa, Neisseria gonorrhoeae and Staphylococcus aureus. RRENTQQHITARRAIRHPQY (SEQ ID NO:19) and GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:26) also exhibited potent activity against P. aeruginosa strains, including clinical isolates. IIGGR (SEQ ID NO:1) and IVGGR (SEQ ID NO:2) act against both gram-negative and gram-positive bacterial strains. HPQYNQR (SEQ ID NO:3) and certain related peptides are also active against both gram-negative and gram-positive bacteria, including, but not limited to, strains of Escherichia coli, Neisseria gonorrhoeae, Staphylococcus aureus, Capnocytophage sputigena and Pseudomonas aeruginosa. The peptides of the present invention will be useful in pharmaceutical compositions useful in the treatment of prophylaxis of infections.

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.

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 = Methionine 
C = Cys = Cysteine N = Asn = Asparagine 
D = Asp = Aspartic Acid 
P = Pro = Proline 
E = Glu = Glutamic Acid 
Q = Gln = Glutamine 
F = Phe = Phenylalanine 
R = Arg = Arginine 
G = Gly = Glycine S = Ser = Serine 
H = His = Histidine T = Thr = Threonine 
I = Ile = Isoleucine 
V = Val = Valine 
K = Lys = Lysine W = Try = Tryptophan 
L = Leu = Leucine Y = Tyr = Tyrosine 
Boc = tert-butyloxycarbonyl 
CFU = colony forming unit 
DFP = diisopropylfluorophosphate 
HLE = human leukocyte elastase 
Pam = (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 &gt;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 Peptide 
Variants of Antimicrobial HPQYNQR 
Peptide Sequence.sup.1 
% Survival of S. aureus 
N 
______________________________________ 
HPQYNQR (SEQ ID NO: 3) 
28.6 .+-.1.57 15 
APQYNQR (SEQ ID NO: 27) 
98.5 .+-.2.1 12 
HAQYNQR (SEQ ID NO: 11) 
31.2 .+-.2.4 9 
HPAYNQR (SEQ ID NO: 7) 
43.6 .+-.2.54 6 
HPQANQR (SEQ ID NO: 32) 
96.7 .+-.4.34 6 
HPQYAQR (SEQ ID NO: 8) 
30.6 .+-.0.85 6 
HPQYNAR (SEQ ID NO: 10) 
51.2 .+-.3.6 6 
HPQYNQA (SEQ ID NO: 9) 
39.8 .+-.1.96 6 
HPQKNTY (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 Derivatives 
of Antimicrobial Peptide HPQYNQR 
Peptide Sequence.sup.1 
% Survival of S. aureus 
N 
______________________________________ 
HPQYNQR (SEQ ID NO: 3) 
28.6 .+-.1.57 15 
APQYNQR (SEQ ID NO: 27) 
98.5 .+-.2.1 12 
H.sup.1m QYNQR 37.3 .+-.2.25 3 
H.sup.3m PQYNQR.sup.2 
18.3 .+-.3.82 3 
H.sup.Dnp PQYNQR.sup.2 
102.2 .+-.2.92 8 
H.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 3 
D-HPQYNQR 98.6 .+-.1.6 5 
HPAYNQR (SEQ ID NO: 7) 
43.6 .+-.2.54 6 
HPQANQR (SEQ ID NO: 32) 
96.7 .+-.4.34 6 
HPQFNQR (SEQ ID NO: 34) 
45.0 .+-.4.91 6 
HPQYNQA (SEQ ID NO: 9) 
39.8 .+-.1.96 6 
HPQYNQ-amide 32.8 .+-.1.83 3 
HPQYN-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 Replacements 
Peptide 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 
% Survival 
Peptide 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 A 
Human HPAYNPK 36 .+-.2.5 
9 (SEQ ID NO: 5) 
granzyme B 
Human HPAYNAK 97 .+-.1.8 
3 (SEQ ID NO: 52) 
granzyme B'.sup.2 
Human 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 
3 
granzyme B'''' 
Human HPAYNP-amide 
42.5 .+-.3.6 
5 
granzyme B''''' 
Mouse YPCYDEY 99.5 .+-.1.7 
3 (SEQ ID NO: 54) 
granzyme A 
Mouse HPDYNPK 62 .+-.3.4 
3 (SEQ ID NO: 55) 
granzyme B 
Mouse HPDYNPD 104 .+-.3.8 
3 (SEQ ID NO: 56) 
granzyme C 
Mouse HPDYNAT 98 .+-.2.7 
3 (SEQ ID NO: 57) 
granzyme D,E.sup.2 
Mouse HPAYDDK 98 .+-.1.9 
3 (SEQ ID NO: 58) 
granzyme F 
Mouse 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. gonorrhoeae 
Peptide Sequence ATCC 27853 
strain WS1 
__________________________________________________________________________ 
1 20 5.507 2.57 
(SEQ ID NO: 16) 
IIGGRESRPHSRPYMAYLQI 
21 40 -0.034 0.05 
(SEQ ID NO: 17) 
QSPAGQSRCGGFLVREDFVL 
41 60 0.5 -0.09 
(SEQ ID NO: 18) 
TAAHCWGSNINVTLGAHNIQ 
61 80 5.15 0 (SEQ ID NO: 19) 
RRENTQQHITARRAIRHPQY 
77 96 0.06 0.24 
(SEQ ID NO: 20) 
HPQYNQRTIQNDIMLLQLSR 
97 116 
5.93 -0.21 
(SEQ ID NO: 21) 
RVRRNRNVNPVALPRAQEGL 
117 136 
5.88 2.38 
(SEQ ID NO: 22) 
RPGTLCTVAGWGRVSMRRGT 
137 156 
0.01 0.43 
(SEQ ID NO: 23) 
DTLREVQLRVQRDRQCLRIF 
157 176 
1.02 -0.18 
(SEQ ID NO: 24) 
GSYDPRRQICVGDRRERKAA 
177 197 
-0.79 0.68 
(SEQ ID NO: 25) 
FKGDSGGPLLCNNVAHGIVSY 
198 223 
5.73 0.78 
(SEQ ID NO: 26) 
GKSSGVPPEVFTRFVSSFLPWIRTTMR 
Buffer -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 SEQ 
Peptide ATCC 278533 
strain WSI 8325-4 ID NO. 
______________________________________ 
CG 1-20 1.0 0.43 0.3 16 
CG 61-80 5.15.sup.1 .sup. ND.sup.2 
0.58 19 
CG 97-116 
0.22 0.2 -0.1 21 
CG 117-136 
5.88.sup.1 4.20 5.34 22 
CG 198-223 
5.73.sup.1 ND 0.26 26 
Buffer 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.90 
Synthetic Peptides 
(.mu.g/ml) 
SEQ ID NO: 
______________________________________ 
CG 1-20 500 16 
CG 61-80 75 19 
CG 97-116 &gt;500 21 
CG 117-136 15 22 
CG 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 Peptides 
Against P. aeruginosa Clinical Isolates 
Log Reduction in Viability.sup.1 
P. 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. gonorrhoeae 
Peptide ATCC 27853 strain WS1 
______________________________________ 
L-enantiomer 5.62 4.20 
D-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 in 
Truncated CG 117-136 Variants 
Hydrophobic 
Hydrophilic 
Peptide 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 and 
Truncated Variants of CG 117-136 
Log Kill 
(500 .mu.g/ml 
SEQ P. aeruginosa 
N. gonorrhoeae 
S. aureus 
Peptide ID NO: ATCC 27853 strain WS1 
strain 8325-4 
______________________________________ 
CG 117-136 
22 5.8 4.20 5.34 
CG 117-129 
38 0.86 0.05 0 
CG 122-136 
39 4.75 4.18 0 
CG 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 -0.76 
50 .mu.g/ml enzymatically active Cat G 
2.82 2.41 
50 .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: 
IleIleGlyGlyArg 
15 
(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: 
IleValGlyGlyArg 
15 
(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: 
HisProGlnTyrAsnGlnArg 
15 
(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 Methionine 
Sulfone." 
(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 or 
Beta- 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 or 
N-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: 
HisXaaXaaXaaXaaXaaXaa 
15 
(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: 
HisProAlaTyrAsnProLys 
15 
(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: 
HisProAlaTyrAsnProArg 
15 
(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: 
HisProAlaTyrAsnGlnArg 
15 
(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: 
HisProGlnTyrAlaGlnArg 
15 
(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: 
HisProGlnTyrAsnGlnAla 
15 
(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: 
HisProGlnTyrAsnAlaArg 
15 
(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: 
HisAlaGlnTyrAsnGlnArg 
15 
(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: 
HisProGlnTyrAsnGln 
15 
(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: 
ArgHisProGlnTyrAsnGlnArg 
15 
(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: 
HisProGlnTyrAsnGlnArgThrIleGlnAsnAspIleMetLeuLeu 
151015 
GlnLeuSerArg 
20 
(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: 
AlaProGlnTyrAsnGlnArg 
15 
(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: 
IleIleGlyGlyArgGluSerArgProHisSerArgProTyrMetAla 
151015 
TyrLeuGlnIle 
20 
(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: 
GlnSerProAlaGlyGlnSerArgCysGlyGlyPheLeuValArgGlu 
151015 
AspPheValLeu 
20 
(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: 
ThrAlaAlaHisCysTrpGlySerAsnIleAsnValThrLeuGlyAla 
151015 
HisAsnIleGln 
20 
(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: 
ArgArgGluAsnThrGlnGlnHisIleThrAlaArgArgAlaIleArg 
151015 
HisProGlnTyr 
20 
(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: 
HisProGlnTyrAsnGlnArgThrIleGlnAsnAspIleMetLeuLeu 
151015 
GlnLeuSerArg 
20 
(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: 
ArgValArgArgAsnArgAsnValAsnProValAlaLeuProArgAla 
151015 
GlnGluGlyLeu 
20 
(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: 
ArgProGlyThrLeuCysThrValAlaGlyTrpGlyArgValSerMet 
151015 
ArgArgGlyThr 
20 
(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: 
AspThrLeuArgGluValGlnLeuArgValGlnArgAspArgGlnCys 
151015 
LeuArgIlePhe 
20 
(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: 
GlySerTyrAspProArgArgGlnIleCysValGlyAspArgArgGlu 
151015 
ArgLysAlaAla 
20 
(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: 
PheLysGlyAspSerGlyGlyProLeuLeuCysAsnAsnValAlaHis 
151015 
GlyIleValSerTyr 
20 
(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: 
GlyLysSerSerGlyValProProGluValPheThrArgPheValSer 
151015 
SerPheLeuProTrpIleArgThrThrMetArg 
2025 
(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: 
AlaProGlnTyrAsnGlnArg 
15 
(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: 
IleIleGlyGlyHis 
15 
(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: 
IleIleGlyGlyVal 
15 
(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: 
HisProGlnTyrAsnProGln 
15 
(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: 
HisHisGlnTyrAsnGlnArg 
15 
(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: 
HisProGlnAlaAsnGlnArg 
15 
(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: 
HisProGlnLysAsnThrTyr 
15 
(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: 
HisProGlnPheAsnGlnArg 
15 
(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: 
HisProAsnTyrAsnGlnArg 
15 
(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: 
HisProGluTyrAsnGlnArg 
15 
(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: 
HisProLeuTyrAsnGlnArg 
15 
(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: 
ArgProGlyLeuThrLeuCysThrValAlaGlyTrpGly 
1510 
(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: 
CysThrValAlaGlyTrpGlyArgValSerMetArgArgGlyThr 
151015 
(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: 
TrpGlyArgValSerMetArgArgGlyThr 
1510 
(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: 
IleIleGlyGlyArgGluSerArgProHisSerArgProTyrMetAla 
151015 
TyrLeuGlnIleGlnSerProAlaGlyGlnSerArgCysGlyGlyPhe 
202530 
LeuValArgGluAspPheValLeuThrAlaAlaHisCysTrpGlySer 
354045 
AsnIleAsnValThrLeuGlyAlaHisAsnIleAspArgArgGluAsn 
505560 
ThrGlnGlnHisIleThrAlaArgArgAlaIleArgHisProGlnTyr 
65707580 
AsnGlnArgThrIleGlnAsnAspIleMetLeuLeuGlnLeuSerArg 
859095 
ArgValArgArgAsnArgAsnValAsnProValAlaLeuProArgAla 
100105110 
GlnGluGlyLeuArgProGlyThrLeuCysThrValAlaGlyTrpGly 
115120125 
ArgValSerMetArgArgGlyThrAspThrLeuArgGluValGlnLeu 
130135140 
ArgValGlnArgAspArgGlnCysLeuArgIlePheGlySerTyrAsp 
145150155160 
ProArgArgGlnIleCysValGlyAspArgArgGluArgLysAlaAla 
165170175 
PheLysGlyAspSerGlyGlyProLeuLeuCysAsnAsnValAlaHis 
180185190 
GlyIleValSerTyrGlyLysSerSerGlyValProProGluValPhe 
195200205 
ThrArgValSerSerPheLeuProTrpIleArgThrThrMetArg 
210215220 
(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: 
LysProGlyGlnThrCysSerValAlaGlyTrpGlyGlnThrAlaPro 
151015 
LeuGlyLysSer 
20 
(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: 
LysProGlnAspValCysTyrValAlaGlyTrpGlyArgMetAlaPro 
151015 
MetGlyLysTyr 
20 
(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: 
GluAlaGlnThrArgCysGlnValAlaGlyTrpGlySerGlnSerArg 
151015 
SerGlyGlyArg 
20 
(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: 
GlyAsnGlyValGlnCysLeuAlaMetGlyTrpGlyLeuLeuGlyArg 
151015 
AsnArgGlyIle 
20 
(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: 
ProHisGlyThrGlnCysLeuAlaMetGlyTrpGlyArgValGlyAla 
151015 
HisProProPro 
20 
(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: 
AlaAlaGlyThrThrCysValThrThrGlyTrpGlyLeuThrArgTyr 
151015 
ThrAsnAlaAsn 
20 
(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: 
GlyIleGlyLysPheLeuHisSerAlaLysLysPheLysAlaPheVal 
151015 
GlyGluIleMetAsn 
20 
(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: 
HisProGlnTyrAsnProLys 
15 
(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: 
HisProGlnTyrAsnProArg 
15 
(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: 
TyrProCysTyrAspProAla 
15 
(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: 
HisProAlaTyrAsnAlaLys 
15 
(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: 
HisProAspTyrAsnGlnArg 
15 
(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: 
TyrProCysTyrAspGluTyr 
15 
(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: 
HisProAspTyrAsnProLys 
15 
(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: 
HisProAspTyrAsnProAsp 
15 
(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: 
HisProAspTyrAsnAlaThr 
15 
(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: 
HisProAlaTyrAspAspLys 
15 
(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: 
HisProAlaPheAspArgLys 
15 
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