Inducible defensin peptide from mammalian epithelia

The present invention relates to an inducible antimicrobial peptide designated lingual antimicrobial peptide (LAP) which has antibacterial and antifungal activity and which can be obtained from mammalian epithelium. The prepro- and the pro- precursors of LAP are also provided. The present invention also relates to cDNA encoding LAP, the prepro- precursor or the pro-lingual precursor. In addition, methods of treating microbial infection of the epithelia are provided. Such infections can be treated by contacting the epithelia with an antimicrobially effective amount of a purified mammalian epithelial LAP or by administering a component which cause endogenous production or up-regulation of LAP.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to inducible antimicrobial and antifungal 
peptides of the mammalian epithelial tissue. In particular, the present 
invention relates to a mammalian epithelial peptide designated lingual 
antimicrobial peptide (LAP) and to its precursor peptides. The invention 
present invention also relates to cDNA segments encoding LAP and its 
precursor peptides, and to methods of treating microbial infection of the 
epithelium. 
2. Description of the Prior Art 
Epithelium is a complex tissue responsible for forming an initial, physical 
barrier protecting the body against potentially harmful environments. 
Epithelial tissue covers the outer body surfaces and lines the luminal 
surface of the respiratory tract, the gastrointestinal tract, and the 
genitourinary system to protect these surfaces from exposure to the 
outside environment. Epithelial surfaces, therefore, serve a "defensive" 
function, protecting the host from the environment (Jacob and Zasloff, 
Ciba Foundation Symposium 186, 1994). 
Antimicrobial peptides provide a second, chemical line of defense 
supplementing the physical barrier of the epithelial tissue surfaces. 
Antimicrobial peptides, produced by various tissues in the body, have 
antibacterial, antifungal, and antiviral activity. These peptides, which 
can be classified into several families, have been found in a variety of 
tissues from diverse species. For example, magainins have been isolated 
from frogs (Zasloff, M., Proc. Natl. Acad. Sci. USA 84: 5449-5453, 1987) 
and cecropins have been found in insects (Boman, H. G., Cell 65: 205-207, 
1991). In addition, two groups of peptides within the defensin family have 
been identified. .beta.-defensins have been isolated from neutrophils of 
cows (Selsted et al., J. of Biol. Chem. 268: 6641-6648, 1993) and from 
tracheal mucosa of cows (Diamond et al., Proc. Natl. Acad. Sci. USA 88: 
3952-3956, 1991; and Diamond et al., Proc. Natl. Acad. Sci. USA 90: 
4596-4600, 1993), while .alpha.-defensins have been isolated from 
neutrophils of humans (Lehrer et al., Annual Rev. Immunol. 11: 105-128, 
1993) and from the epithelial-derived Paneth cells at the base of the 
crypts of the small intestine in murine and human GT tracts (Ouellette et 
al., J. Cell Biol. 108: 1687-1695, 1989; and Jones and Bevins, J. Biol. 
Chem. 267: 23215-23225, 1992). The antimicrobial peptides provide a second 
line of defense, killing bacteria and fungus pathogens which penetrate the 
physical barrier. 
One example of epithelial tissue is the mammalian tongue which is covered 
by a dense stratified epithelium. The tongue is in an environment 
constantly exposed to various microorganisms that are part of the 
microbial flora of the mouth. Despite is constant exposure to microbials, 
invasive infections of the tongue rarely ensue even when abrasions occur 
on the tongue's surface. In investigating the infection resistance 
property of the mammalian tongue, a novel antibacterial and antifungal 
peptide was isolated from the extracts of bovine tongue epithelial tissue. 
SUMMARY OF THE INVENTION 
Accordingly, it is one object of the present invention to provide an 
inducible antimicrobial peptide having antibacterial and antifungal 
activity which can be obtained from mammalian epithelium, such as bovine 
tongue epithelium. 
It is a further object of the present invention to provide the 
prepro-peptide and the pro-peptide precursors of the antimicrobial 
peptide. 
It is another object of the present invention to provide cDNA that encodes 
the inducible mammalian epithelium antimicrobial peptide, the 
prepro-peptide and the pro-peptide. 
It is yet a further object of the present invention to provide a method of 
treating microbial infections of the epithelium and microbial infections 
that extend through, beyond, or deeper in the epitheli, such as into 
connective tissue or the subdermal region. 
Various other objects and advantages of the present invention will be 
apparent from the drawings and the following description of the invention. 
In one embodiment, the present invention relates to a purified inducible 
mammalian epithelial lingual antimicrobial peptide (LAP) having an ion 
mass of about 4627.5 daltons, and having antimicrobial and antifungal 
activity. 
In another embodiment, the present invention relates to a purified 
prepro-lingual antimicrobial peptide (prepro-LAP) or a purified 
pro-lingual antimicrobial peptide (pro-LAP). 
In a further embodiment, the present invention relates to a cDNA encoding a 
lingual antimicrobial peptide, a prepro-lingual antimicrobial peptide, or 
a pro-lingual antimicrobial peptide. 
In yet another embodiment, the present invention relates to a method of 
treating microbial infection of the epithelia. The method comprises 
contacting the epithelia with an antimicrobially effective amount of a 
purified mammalian epithelial lingual antimicrobial peptide (LAP) having 
an ion mass of about 4627.5 daltons, and having antimicrobial and 
antifungal activity so that the microbial infection is inhibited. 
In yet a further embodiment, the present invention relates to a method of 
inducing endogenous expression of lingual antimicrobial peptide (LAP) to 
treat microbial infections. Endogenous expression is induced by 
administering to a patient in need thereof, an effective amount of a 
component which induces the production of LAP by epithelial tissue. 
In another embodiment, the present invention relates to a method of 
identifying endogenous up-regulators of lingual antimicrobial peptide 
(LAP). The method comprises contacting an epithelial cell culture with a 
test substance and measuring the level of mRNA to determine whether the 
test substance is an up-regulator. 
In a further embodiment, the present invention relates to another method of 
identifying endogenous up-regulators of lingual antimicrobial peptide 
(LAP). Up-regulators of LAP can be identified by constructing an 
expression vector containing a .beta.-defensin gene promoter operably 
linked to a reporter gene, infecting a host cell with the expression 
vector, and culturing the host cell in the presence of test substances. 
Whether the test substance is an up-regulator is then determined by 
measuring the level of mRNA or reporter gene expression.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to an inducible antimicrobial peptide 
designated lingual antimicrobial peptide (LAP). LAP is a mammalian 
antimicrobial peptide which has an ion mass of about 4627.5 daltons and 
possesses both antimicrobial and antifungal activity. In one embodiment of 
the present invention, LAP has amino acid sequence (SEQ ID NO:1): 
QGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK. This peptide, obtainable from 
bovine epithelial tissue, is a member of the defensin family of 
antimicrobial peptides. LAP belongs to the .beta.-defensin group of 
peptides as LAP contains the 11 conserved amino acid residues shared by 
all .beta.-defensins. In addition, the signal sequence of LAP is similar 
to the signal sequence of the tracheal mucosa antimicrobial peptide (TAP), 
a .beta.-defensin described by Diamond et al. (Diamond et al., Proc. Natl. 
Acad. Sci. USA 88: 3952-3956, 1991). 
Antimicrobial peptides of the defensin family have been found in several 
species including humans, rabbits, rats, mice, and guinea pigs (Ganz et 
al., Med. Microbiol. Immunol. 181: 99-105, 1992; and Lehrer et al., Annual 
Rev. Immunol. 11: 105-128, 1993). Defensins of bovine origin have been 
placed in the .beta.-defensin group while homologous defensins of human 
origin are designated .alpha.-defensins. As defensin peptides exist in 
many mammalian species, the present invention relates to all mammalian LAP 
including, but not limited to, LAP of bovine origin and LAP of human 
origin. Homologous LAPs from species other than cows can be obtained, for 
example, using the isolation strategy employed with the bovine tongue 
extracts or using cDNA probes. For example, epithelial tissue from humans 
could be probed using either the LAP 48-mer probe (SEQ ID NO:1): 
5'-CCT-CCT-GCA-GCA-TTT-TAC-TTG-GGC-TCC-GAG-ACA-GGT-GCC-AAT-CTG-TCT-3', or 
the signal sequence 51-mer probe (SEQ ID NO:2): 
5'-AGC-AGA-CAG-GAC-CAG-GAA-GAG-GAG-CGC-(AG)AG-GAG-CAG-GTG-ATG-GAG-CCT-CAT- 
3', or the human .alpha.-defensin signal sequence which is highly conserved 
(Jones and Bevins, J. Biol. Chem. 267: 23215-23225, 1992). This would 
identify tissue that would contain either .alpha. or .beta. defensin. One 
could purify the defensin peptide from this tissue or clone the 
corresponding cDNA by reverse transcribing the poly-A RNA message obtained 
from these tissues. Alternatively, one could make a cDNA library from 
these tissues and then clone the corresponding cDNA from the library using 
the probes described above and standard molecular biology techniques 
(Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor 
Laboratory (New York, 1989)). 
LAP has broad spectrum antimicrobial activity against Gram-negative 
bacteria, Gram-positive bacterial and fungal pathogens. The peptide may 
also have antiviral activity. For example, LAP has a specific activity 
against Escherichia coli of 16-32 .mu.g/ml, Pseudomonas aeruginosa of 
63-125 .mu.g/ml, Staphylococcus aureus of 63-125 .mu.g/ml, Candida 
albicans of 32-63 .mu.g/ml, and Candida tropicalis of 16-32 .mu.g/ml. 
When translated from mRNA the peptide of the present invention, LAP, begins 
as a prepro-precusor peptide, designated prepro-LAP. This precursor 
peptide contains a signal sequence consisting of about 20 amino acids 
followed by a short putative pro sequence consisting of about 2 amino 
acids. Thus, the present invention relates to the prepro-LAP and the 
pro-LAP precursor peptides as well as to LAP. Indeed, in one embodiment of 
the present invention, prepro-LAP has amino acid sequence (SEQ ID NO:3): 
MRLHHLLLALLFLVLSAGSGFTQGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK, and in a 
further embodiment, pro-LAP has amino acid sequence (SEQ ID NO:4): 
FTQGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK. 
While the present invention is exemplified with the purification of LAP 
from bovine tongue epithelia, the skilled artisan will understand that the 
peptides of the present invention can be purified, that is isolated from 
proteins with which they are normally associated, from other epithelial 
tissues. Suitable epithelial tissues include, but are not limited to, 
epithelia from the respiratory tract, such as trachea, bronchi, and lung 
tissue, the gastrointestinal tract, such as cecum, colon, and rectum 
tissue, the genitourinary tract, such as bladder tissue, the reproductive 
tract including testes, and facial epithelia, such as conjunctiva. 
Further, in addition to using peptide purification methods, peptides of 
the present invention can be chemically synthesized or recombinantly 
produced using standard techniques in the art. 
The present invention also relates to cDNA which encode the prepro-LAP, 
pro-LAP and/or LAP peptides. In particular, cDNA of the present invention 
include nucleotide sequences which code for an amino acid sequence 
selected from the group consisting of: 
QGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK, 
MRLHHLLLALLFLVLSAGSGFTQGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK, and 
FTQGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK (SEQ ID NOs:1, 4, and 5, 
respectively). Examples of cDNA of the present invention include, but are 
not limited, the nucleotide sequences of FIG. 2B. 
The present invention further relates to recombinant DNA molecules 
comprising a vector and a cDNA encoding prepro-LAP, pro-LAP or LAP. 
Possible vectors include, but are not limited to, Bluescript, Bluescript 
II, pGEM, pRIT, and PET vectors. Host cells transformed with these 
recombinant DNA molecules using standard techniques can be cultured to 
provide a source of LAP or its precursor peptides. Suitable host cells 
include eukaryotic and prokaryotic cells such as yeast, E. coli, 
DH5.alpha., and HB101. 
While LAP is constitutively expressed at low levels in mammalian epithelia, 
high levels of mRNA expression are induced in response to epithelia injury 
and/or infection. For example, increased concentrations of LAP mRNA are 
found in epithelia surrounding acute and chronic areas of infection or 
inflammation. This suggests that LAP plays a role in innate immunity. 
According to Janeway (Janeway, C. A. J. Jr., Immunology Today 13: 11-16, 
1992), innate immunity is characterized by three properties: 
polyspecificity, ability to discern self from nonself, and rapid response 
kinetics. LAP of the present invention is a broad spectrum antibiotic 
which is polyspecific and inducible upon infection, with induction 
occurring rapidly enough to be present in areas of acute inflammation. 
These findings are consistent with each of Janeway's hypotheses and 
suggests that LAP plays a role in innate immunity protecting epithelia 
from injury and infection. 
Accordingly, peptides of the present invention can be used to treat 
epithelial diseases and microbial infections. LAPs can be used to treat 
epithelial diseases such as, diseases occurring in any immunodeficiency 
state, cystic fibrosis, and gum diseases and wounds, as well as microbial 
infections of the epithelia such as, bacterial and viral infection, and 
infections that extend through, beyond, or deeper in the epithelial such 
as into the connective tissue or subdermal regions. To treat such 
conditions, the diseased or infected epithelial tissue is contacted an 
antimicrobially effective amount of LAP or a precursor of LAP, either 
alone or in a pharmaceutically acceptable carrier. Suitable carriers 
include cremes, gels, saline, water, paste, and liposomes made of 
phospholipids. The LAP administered in this manner can be purified from 
epithelial tissue, recombinantly produced using the recombinant vector of 
the present invention or chemically synthesized. 
The effective amount of LAP will vary depending on several factors such as, 
for example, the severity of the disease or infection, the causative 
organism and the type of epithelial tissue being treated, but the amount 
required for a particular patient given the patient's history and symptoms 
is easily determinable by one skilled in the art. For example, LAP could 
be applied to gums with gingivitis in micromolar amounts greater than the 
minimum inhibitory concentration (MIC) of LAP for Staphylococcus. LAP 
could be used as an antifungal in the mouth or GI tract since it has 
activity against Candida albicans and Candida tropicalis, in vitro and can 
be administered in a dose that provides a local tissue level greater than 
the MIC for that organism or in several smaller doses that can be 
repeated. 
In addition, in the respiratory tract one could use LAP to treat pneumonia, 
bronchitis, or cystic fibrosis. For example, LAP could be inhaled, 
aerosolized, placed in a liposome and inhaled, or lavaged into the 
respiratory tract. These formulations could also be used to place the LAP 
in contact with the genitourinary or reproductive tracts. LAP could also 
be applied directly to a skin wound, burn or infection. 
Lingual antimicrobial peptides or other .alpha. and .beta. defensins can 
also play a role in preventing or treating diseases by inducing endogenous 
defenses. Components of infection, such as bacterial cell wall 
lipopolysaccharides, inactivated microbes, glycolipids, glycoproteins, 
sugars, or viral components, can be identified which induce the expression 
of LAP mRNA in epithelial tissues. Such components can be identified using 
standard techniques such as those employed by Brey et al. (Proc. Natl. 
Acad. Sci. USA 90: 6275-6279, 1993), and Diamond and Bevins (Chest. 1994 
March 105(3 Suppl) 51s-52s, 1994). Accordingly, the present invention also 
provides methods of screening test substances to determine whether they 
are up-regulators of LAP. For example, cultures of epithelial cells 
capable of expressing LAP can be exposed to various components and the 
amount of mRNA produced by the cells measured to determine whether 
exposure to a given component increased the mRNA expression. 
Alternatively, an expression vector system could be designed with a 
.beta.-defensin promoter operably linked to a reporter gene. Suitable 
reporter genes included chlorampherical acetyl transferase or 
.beta.-galactosidase. Host cells infected with such an expression vector 
could be cultured in the presence of test substances and the ability of 
these substances to up-regulate LAP or other .alpha. and .beta. defensins 
determined by measuring the level of mRNA produced by the host cell or by 
measuring the increase in message as a function of reporter gene 
expression. 
Components which are shown to induce the expression of LAP mRNA can then be 
administered to a patient to induce therapeutic endogenous expression of 
LAP. The induction of endogenous LAP production can be used to treat, for 
example, patients with AIDS, severe microbial infections, inflammatory 
skin or gum lesions, or infections of any epithelial surface or infections 
that extend through, beyond, or deeper in the epithelial, such as into the 
connective tissue or subdermal regions. 
For the purposes of illustrating a preferred embodiment of the present 
invention, in the following non-limiting examples, the lingual 
antimicrobial peptide (LAP) was isolated from bovine tongue epithelial 
tissue, the cDNA encoding LAP was isolated and sequence, and mRNA 
expression and tissue distribution analyzed. It is, however, to be 
understood that the discussion generally applies to the isolation of LAP 
or other defensins from any mammalian epithelium. 
EXAMPLES 
Purification of LAP Peptide 
Using a purification scheme that involved organic extraction, gel 
filtration, reverse phase HPLC, and strong cation exchange HPLC, the 
lingual antimicrobial peptide (LAP) was purified from bovine tongue 
epithelial tissue. 
Approximately 500 g of anterior tongue epithelial tissue was dissected from 
5 freshly killed cows and frozen in liquid nitrogen. The tissue was 
pulverized in a blender using liquid nitrogen and extracted for 3 days at 
4.degree. C. with 5 volumes of 60% acetonitrile, 1% Trifluoroacetic Acid 
(TFA). The sample was then centrifuged at 4.degree. C. for 15 minutes and 
the supernatant was extracted using 15 volumes chloroform:methanol (2:1). 
The upper aqueous phase was pooled, lyophilized, and resuspended in 15 ml 
of 25% acetonitrile, 1% TFA. The sample was then centrifuged at 4000 RPM 
for 15 minutes and the remaining supernatant was loaded on a 120 ml P-30 
gel filtration column (Biorad, Richmond, Calif.). 
The active antimicrobial fractions were pooled and loaded onto a reverse 
phase HPLC C-18 column (Poly LC, Columbia, Md.). The active fractions were 
the loaded onto a strong cation exchange HPLC column-PSEA (Poly LC, 
Columbia, Md.) (FIG. 1A) and each fraction was desalted using a C-18 
Sep-pak cartridge (Waters, Milford, Mass.), dried overnight and assayed 
for activity against E. coli D31 and C. tropicalis as described below. 
This peptide was the most abundant of several antimicrobial activities 
isolated from the bovine tongue epithelium. The minimal inhibitory 
concentrations (MIC's) demonstrated broad spectrum antimicrobial activity 
against gram negative and gram positive bacteria, and fungal pathogens 
with a potency similar to magainin-II amide (FIG. 1C) and comparable to 
other defensins previously isolated. (Diamond et al., Proc. Natl. Acad. 
Sci. USA 88: 3952-3956, 1991.) 
Antimicrobial Assaying of LAP Peptide 
Antimicrobial activity was determined during the purification process. 
Approximately 2.5 ml fractions from the P-30 gel filtration column were 
assessed after drawing fractions and taking an aliquot of the fraction and 
spotting that fraction on a radial diffusion plate as described by 
Zasloff, M. (Proc. Natl. Acad. Sci USA 84: 5449-5453, 1987) against E. 
coli D31 or fungal pathogens such as Candida albicans, Candida tropicalis, 
or Staphylococcus aureus. (See also, Zasloff et al., Proc. Natl. Acad. 
Sci. USA 85: 910-913, 1988.) (FIGS. 1B and 1C.) Briefly, the minimal 
inhibitory concentrations (MIC's) were assessed using a 96 well microtitre 
plate (Corning Glass Works, Corning, N.Y.). Microorganisms were grown in 
log phase at 1/4 strength tryptics soy broth (TSB) at a density of 
1.times.10.sup.5 /ml. The assays used 1/4 strength TSB. For each organism, 
dilutions of peptide were made ranging from &gt;500 .mu.g/ml to 1 .mu.g/ml 
using 1/4 strength TSB as a dilution buffer. Zones of bacterial growth or 
lack of growth were assessed under the microscope. MIC's were calculated 
based the lowest concentration of peptide that inhibited growth. 
The results set forth below in Table 1 demonstrate that LAP has broad 
spectrum antimicrobial activity against Gram-negative bacteria, 
Gram-positive bacteria, and fungal pathogens. Indeed, the MIC's 
demonstrated broad spectrum antimicrobial activity against gram negative 
and gram positive bacteria, and fungal pathogens with a potency similar to 
magainin-II amide and comparable to other defensins previously isolated 
(Diamond et al., Proc. Natl. Acad. Sci. USA 88: 3952-3956, 1991). 
TABLE 1 
______________________________________ 
Antimicrobial Activity of LAP and Magainin II-amide 
Minimum Inhibitory 
Concentration 
(.mu.g/ml) 
Microorganism (ATCC) 
LAP Magainin II 
______________________________________ 
Escherichia coli (D31) 
16-32 13-25 
Pseudomonas aeruginosa (27853) 
63-125 13-25 
Staphylococcus aureus (29213) 
63-125 50-100 
Candida albicans (14053) 
32-63 50-100 
Candida tropicalis (13803) 
16-32 13-25 
______________________________________ 
Sequencing LAP Peptide 
The mass ion of LAP is 4627.5, consistent with the size and amino acid 
composition of a .beta.-defensin (FIG. 2A) (Selsted et al., J. of Biol. 
Chem. 268: 6641-6648, 1993.) The carboxyl (C) terminal sequence of 
approximately 20 amino acids of LAP were determined using microsequencing 
after digestion of the purified peptide with trypsin, followed by 
reduction and alkylation of cysteine residues (FIG. 2). Briefly, the 
peptide fragments were sequenced using Edman degradation, a standard 
sequencing technique. The order of the sequenced fragments was determined 
with overlapping fragments or identifying homologous regions to TAP. 
A polymerase chain reaction (PCR) based strategy was designed to complete 
the N-terminal sequence. After microsequencing, degenerate PCR primers 
were designed from the carboxyterminal region of the LAP amino acid 
sequence where there was no sequence homology to TAP and codon assignment 
of TAP was used for homologous amino acids. A non-degenerate primer was 
designed from the first six amino acids of the signal sequence derived 
from the cloning of the cDNA of TAP. 
The primers were sense strand (SEQ ID NO:6) 5'-ATGAGGCTCCATCACCTG 
(non-degenerate) and 5'-(AG)CA(AG)CA(TC)TT(ACGT)AC(TC)TG(ACGT)GC-antisense 
strand (SEQ ID NO:7) (1:256 degeneracy). PCR conditions were 95.degree. C. 
for 1 minute, 58.degree. C. for 2 minutes, and 72.degree. C. for 3 
minutes. This was followed by 72.degree. C. for 15 minutes. PCR products 
were run on an 1.2% agarose gel, purified with Geneclean II, and subcloned 
into Bluescript vector modified to accept PCR products after linearization 
with EcoRV. The cDNA product was sequenced using dideoxy chain termination 
and was identical with the amino acid sequence of LAP derived from 
microsequencing. 
Cloning and Sequencing of LAP Peptide cDNA 
A cDNA library was generated from bovine tongue epithelial poly A(+) RNA 
(Stratagene Kit for .lambda.ZAP library, La Jolla, Calif.) and the cDNA 
for LAP was cloned and sequenced (FIG. 2B). Briefly, a cDNA lambda 
Zap-cDNA library of tongue epithelial tissue was constructed from (2 
.mu.g) poly A(+) RNA and inserts were size selected from 0.1 kb to 3 kb. 
Approximately 0.5.times.10.sup.6 phage were spread over 10 plates and 
there were approximately 100 positive pfu's per plate. The phage were 
isolated using a LAP cDNA probe derived from PCR containing the signal 
sequence and the peptide coding region (183 bp). Duplicate lifts to detect 
positives were used with Genescreen II nylon membranes (Dupont NEN, 
Boston, Mass.). The phage were plaque purified and approximately 6 
positive phage were isolated, subcloned into Bluescript and sequenced 
using T3 and T7 primers and dideoxy chain termination. The sequence was 
confirmed in triplicate using sequences derived from multiple clones. 
The cloned message encodes a 64 amino acid precursor, structurally similar 
to the prepro .beta.-defensin, TAP (Diamond et al., Proc. Natl. Acad. Sci. 
USA 88: 3952-3956, 1991). The signal sequence consists of 20 amino acids 
followed by a short putative pro sequence consisting of 2 amino acids 
which could be cleaved by a dipeptidase as described for the antimicrobial 
mellitin (Boman et al., J. Biol. Chem. 264: 5852-5860, 1989; and Kreil, 
G., TIBS 15: 23-26, 1990). The mature peptide is at the C terminus of the 
precursor and consists of 42 amino acids followed by an in frame stop 
codon. The polyadenylation signal is 14 nucleotides from the poly A tail. 
Expression and Distribution of LAP mRNA in Epithelia Tongue Tissue 
The bovine tongue is covered by a dense parakeratinized stratified 
epithelium (FIG. 3A). The upper surface of the epithelium is comprised of 
senescent cells while the middle and basal layers represent 
transcriptionally active cells (Fuchs, E., J. Cell Biol. 111: 2807-2814, 
1990). The basal layer of the epithelium is comprised of germinal cells. 
The epithelium is nourished by a connective tissue layer which forms 
papillae within the epithelium, and contains blood vessels and nerves. 
There is a striated muscular layer inferior to the connective tissue. To 
determine the expression and distribution pattern of LAP mRNA, bovine 
tongue tissue was hybridized with the LAP antisense probe. 
Bovine tongue was obtained from Moyer Packing Company (MO) (Souderton, 
Pa.) using freshly slaughtered cows (Jersey Holstein and black angus 
species). The anterior epithelium was dissected from the underlying 
connective and muscle tissue and the epithelial tissue was fixed 
immediately using 4% paraformaldehyde, 1.times.PBS. The tissue was 
embedded in a paraffin block and 6-8 .mu.icron thick sections were cut and 
mounted on sialanated slides. The slides were maintained at -70.degree. C. 
Riboprobes were made with a full length cDNA of LAP subcloned into 
Bluescript, and linearlized with Sma and Kpn I enzymes for sense and 
antisense transcripts, respectively. 
The slides were dried and fixed using standard in-situ conditions (Young et 
al, Neurosci. Lett. 70: 198-203, 1986) and hybridizations were carried out 
at 37.degree. C. with overnight incubations using 2.times.10.sup.6 
cpm/slide. The slides were washed at high stringency of 65.degree. C., 
with .beta.-mercaptoethanol and the slides were exposed to 
autoradiographic film. The slides were dipped in photographic emulsion, 
Kodak NTB-2 prior to exposure of the emulsion to the slides for 41/2 weeks 
at 4.degree. C. The slides were developed under standard conditions, then 
stained with hematoxalin and eosin, and photographed at 20-40.times. 
magnification. 
Intense hybridization to the middle layers of the epithelium was seen in 
FIG. 3B. (FIG. 3A represents the sense-negative control.) The sense probe 
yielded no hybridization signal. In contrast, the mRNA for .beta. tubulin 
appeared to be distributed uniformly throughout the entire tissue section. 
Although tongue epithelium has been identified as the major site of LAP 
tissue expression, the cellular pathways of processing or secretion have 
not yet been determined. Since the LAP precursor contains a signal 
sequence, it should be secreted from individual epithelium cells or into 
intracellular granules. LAP could be secreted in the pro form and 
processed post translationally as suggested for human defensins (Ganz et 
al., Blood 82: 641-650, 1993). 
To discern the role of LAP in innate immunity, three cows with naturally 
occurring tongue lesions were selected. In all three cases, the lesions 
represented areas consisting of both acute and chronic infection and 
inflammation (FIGS. 3C-3F). In each case, destruction of the normal 
epithelium was noted. There were areas of acute inflammation characterized 
by hemorrhage and erythrocyte accumulation, infiltration of 
polymorphonuclear leukocytes, along with areas of more chronic 
inflammation characterized by infiltration of mononuclear cells. The area 
surrounding and including the tongue lesions were excised from the three 
cows and fixed in 4% paraformaldehyde/PBS prior to in-situ hybridization. 
In-situ hybridization was performed as described above. The lesions were 
hybridized with either full length riboprobes for LAP (sense and 
antisense) or .beta.-tubulin (sense and antisense). All slides were 
exposed to emulsion for 41/2 weeks prior to developing. 
An increase in the concentration of LAP mRNA was found in the remaining 
epithelia surrounding both acute and chronic areas of infection. The 
pattern of expression is consistent with induction of LAP mRNA in the 
existing cells of the epithelium surrounding the infection. 
These observations parallel the experimental data of Brey et al. who showed 
induction of cecropin mRNA in the epithelial cell layer of silkworm larvae 
after epicuticular and cuticular wounding (Brey et al., Proc. Natl. Acad. 
Sci. USA 90: 6275-6279, 1993). Induction only occurs when the abraded 
larvae are challenged with live bacteria or bacterial cell wall 
components. Diamond et al. showed in an in vitro system that TAP mRNA from 
primary cultured bovine tracheal epithelial cells was induced 5-fold by 
adding LPS to the culture medium (Diamond and Bevins, Chest 105(3 Suppl) 
51s-52s, 1994). The sequences of the gene from the bovine defensin TAP, 
and both the cecropin and a dipthericin loci from drosophila, contain an 
nF.kappa.B site in the 5' region implicated in the LPS responsiveness of 
these genes (Diamond et al., Proc. Natl. Acad. Sci. USA 90: 4596-4600, 
1993; Kapper et al., EMBO J. 12: 1561-1569, 1993; and Sun and Faye, Europ. 
J. Biochem. 204: 885-892, 1992). 
For tissue distribution studies, epithelia from the gastrointestinal, 
respiratory, genitourinary, male and female reproductive tracts of cows 
and facial cow epithelia was employed. Northern blot were performed on 
bovine tissues. RNA was prepared from bovine epithelial tissue specimens 
taken from freshly killed cows. Tongue RNA was also obtained from mixed 
gestation aged fetal tongue (Moyer Packing Company, Souderton, Pa.) and 
from 4 month old milk fed veal calves (March Farms, Souderton, Pa.). The 
tissue was immediately frozen in liquid nitrogen. RNA was prepared after 
quanidinium isothiocyanate extraction followed by centrifugation of the 
RNA on a cesium chloride cushion. For the poly A(+) blot, RNA was isolated 
from 200 .mu.g of total RNA, followed by isolation of poly (A)+ RNA using 
oligo dT push columns. 4 .mu.g of poly (A)+ RNA from several tissues were 
electrophoresed on a 1.2% formaldehyde gel using 1.times. MOPS as a 
running buffer. Approximately 15 .mu.g of total RNA was used from each 
specimen. The tissues were also run on a 1.2% formaldehyde gel. The gels 
were blotted using Zetabind positively charged nylon membranes, 
transferring the RNA using 10.times. SSC at pH 7.4. Hybridizations were 
carried out at 42.degree. C., using standard hybridization conditions of 
6.times. SSC, 5.times. Denhardt's, 20% formamide, 200 .mu.g/ml of yeast 
RNA, 0.5% SDS. Probes were designed as follows: 
LAP (48 mer) (SEQ ID NO:2): 
5'-CCT-CCT-GCA-GCA-TTT-TAC-TTG-GGC-TCC-GAG-ACA-GGT-GCC-AAT-CTG-TCT-3'. 
Signal sequence (51 mer) (SEQ ID NO:3): 
5'-AGC-AGA-CAG-GAC-CAG-GAA-GAG-GAG-CGC-(AG)AG-GAG-CAG-GTG-ATG-GAG-CCT-CAT- 
3'. 
The probes were each end labelled using .UPSILON..sup.32 ATP to a specific 
activity of 1.times.10.sup.8 CPM/.mu.g DNA. The .beta.-tubulin probe was 
the full length cDNA bovine clone and was labelled with .alpha..sup.32 P 
dCTP using random priming to a specific activity of 1.times.10.sup.9 
CPM/.mu.g DNA. The blots were hybridized overnight and washed at the 
following conditions: 
LAP--65.degree. C., 1.times. SSC, 0.1% SDS; and 
.beta.tubulin--65.degree. C., 0.1.times. SSC, 0.1% SDS. 
LAP mRNA (or closely homologous messages) were widely expressed in the 
numerous epithelial tissues of the bovine respiratory tract including 
trachea, bronchi, and bronchi/lung; lower gastrointestinal tract including 
cecum, colon, and rectum; reproductive system including testes; and facial 
epithelium including conjunctiva (FIG. 4). The finding that LAP or a 
closely related message is expressed in so many epithelial tissues 
suggests that LAP plays a role in epithelial defense in sites in addition 
to the tongue. 
LAP message was not expressed in the fetal tongue but was expressed after 
birth (FIG. 4). This pattern of expression supports induction or 
developmental regulation. Thus, LAP mRNA appears to be expressed at a low 
constitutive level in normal bovine tongue after birth (FIG. 3A), and is 
induced to higher levels of expression in response to injury and 
infection. It is possible that LAP contributes to wound healing and/or 
playing a role in limiting the physical area of the infection and 
sterilize the tissue. Both mechanisms have been suggested previously for 
other defensins (Lehrer et al., Annual Rev. Immunol. 11: 105-128, 1993). 
All publications mentioned hereinabove are hereby incorporated in their 
entirety by reference. 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of this disclosure that various changes 
in form and detail can be made without departing from the true scope of 
the invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 12 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlnGlyValArgAsnSerGlnSerCysArgArgAsnLysGlyIleCys 
151015 
ValProIleArgCysProGlySerMetArgGlnIleGlyThrCysLeu 
202530 
GlyAlaGlnValLysCysCysArgArgLys 
3540 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 48 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CCTCCTGCAGCATTTTACTTGGGCTCCGAGACAGGTGCCAATCTGTCT48 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 28 
(D) OTHER INFORMATION: /note="N is either A or G." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AGCAGACAGGACCAGGAAGAGGAGCGCNAGGAGCAGGTGATGGAGCCTCAT51 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 64 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetArgLeuHisHisLeuLeuLeuAlaLeuLeuPheLeuValLeuSer 
151015 
AlaGlySerGlyPheThrGlnGlyValArgAsnSerGlnSerCysArg 
202530 
ArgAsnLysGlyIleCysValProIleArgCysProGlySerMetArg 
354045 
GlnIleGlyThrCysLeuGlyAlaGlnValLysCysCysArgArgLys 
505560 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
PheThrGlnGlyValArgAsnSerGlnSerCysArgArgAsnLysGly 
151015 
IleCysValProIleArgCysProGlySerMetArgGlnIleGlyThr 
202530 
CysLeuGlyAlaGlnValLysCysCysArgArgLys 
3540 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ATGAGGCTCCATCACCTG18 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: one-of(1, 4) 
(D) OTHER INFORMATION: /note="N is A or G." 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: one-of(7, 13) 
(D) OTHER INFORMATION: /note="N is T or C." 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: one-of(10, 16) 
(D) OTHER INFORMATION: /note="N is A,C,G, or T." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
NCANCANTTNACNTGNGC18 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 127 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CAAGGAGTAAGAAATTCTCAAAGCTGCCGTAGGAATAAAGGCATCTGTGTGCCGATCAGG60 
TGCCCTGGAAGCATGAGACAGATTGGCACCTGTCTCGGAGCCCAAGTAAAATGCTGCAGG120 
AGGAAGT127 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 133 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTTACTCAAGGAGTAAGAAATTCTCAAAGCTGCCGTAGGAATAAAGGCATCTGTGTGCCG60 
ATCAGGTGCCCTGGAAGCATGAGACAGATTGGCACCTGTCTCGGAGCCCAAGTAAAATGC120 
TGCAGGAGGAAGT133 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AsnProValSerCysValArgAsnLysGlyIleCysValProIleArg 
151015 
CysProGlySerMetLysGlnIleGlyThrCysValGlyArgAlaVal 
202530 
LysCysCysArgLysLys 
35 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 350 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CTCGTGCATTCGGCACCGACAGCATGAGGCTCCATCACCTGCTCCTTGCGCTCCTCTTCC60 
TGGTCCTGTCTGCTGGGTCAGGATTTACTCAAGGAGTAAGAAATTCTCAAAGCTGCCGTA120 
GGAATAAAGGCATCTGTGTGCCGATCAGGTGCCCTGGAAGCATGAGACAGATTGGCACCT180 
GTCTCGGAGCCCAAGTAAAATGCTGCAGGAGGAAGTAAAAGAAGGCGAAGACGTGGCCAG240 
ACTGGATGCGGAGTCAGAAACTGTGCCCTTGGACAGAGAGTTTAAAATTTAAACCAGAAT300 
AAATTTTGTTCAAAGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA350 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 65 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
MetArgLeuHisHisLeuLeuLeuAlaLeuLeuPheLeuValLeuSer 
151015 
AlaGlySerGlyPheThrGlnGlyValArgAsnSerGlnSerCysArg 
202530 
ArgAsnLysGlyIleCysValProIleArgCysProGlySerMetArg 
354045 
GlnIleGlyThrCysLeuGlyAlaGlnValLysCysCysCysArgArg 
505560 
Lys 
65 
__________________________________________________________________________