(Received for publication, January 22, 1997, and in revised form, March 13, 1997)
From the Cathelicidins are the precursors of potent
antimicrobial peptides that have been identified in several mammalian
species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and
activate mesenchymal cells during wound repair. To permit further study
of these proteins a reverse transcriptase-polymerase chain reaction
approach was used to identify potential mouse homologs. A full-length
562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other
cathelicidins and a unique COOH-terminal peptide. This gene, named
Cramp for cathelin-related antimicrobial peptide, was mapped
to chromosome 9 at a region of conserved synteny to which genes for
cathelicidins have been mapped in pig and man. Northern blot analysis
detected a 1-kilobase transcript that was expressed in adult bone
marrow and during embryogenesis as early as E12, the earliest stage of
blood development. Reverse transcriptase-polymerase chain reaction also
detected CRAMP expression in adult testis, spleen, stomach, and
intestine but not in brain, liver, heart, or skeletal muscle. To
evaluate further the expression and function of CRAMP, a peptide
corresponding to the predicted COOH-terminal region was synthesized. CD
spectral analysis showed that CRAMP will form an amphipathic Endogenous antimicrobial peptides play an important role in innate
immunity (1-3). More than 100 microbicidal peptides have been isolated
from plants and animals (4). The role of these defense peptides in
mammals has been inferred from their expression in neutrophil granules
and at sites exposed to multiple microbes such as the skin and
gastrointestinal tract. To exert their antimicrobial effect these
peptides adhere to and permeabilize the surface membranes of potential
pathogens. This activity is a consequence of several common features
such as a high content of basic residues and the tendency of some to
adopt an amphipathic conformation. However, antimicrobial peptides show
marked diversity in structure and antimicrobial spectrum. One class of
antimicrobial peptides, the cathelicidin-derived peptides, contains a
highly conserved prepro region that is homologous to cathelin, a
putative cysteine-proteinase inhibitor originally isolated from pig
leukocytes (5). Cathelicidins have been identified in several species
including pig, cow, sheep, rabbit, and man (6-13). The high degree of
conservation of their cathelin domain suggests that the members of this
family evolved from a common ancestor gene through duplication and
modification (7). In general, the precursors of these peptides are
stored in neutrophil granules. Upon stimulation, the cathelin domain is
cleaved proteolytically to allow the mature COOH-terminal antimicrobial peptide to be released.
The antimicrobial portion of the cathelicidin-derived gene family is
highly diverse in terms of structure and function. In the pig, the
cathelicidin PR-39 has been found to have potent activity against
Gram-negative bacteria and also to function as a stimulator of
syndecan-1 and -4 expression on fibroblasts and endothelia (14).
Observations of functions beyond antimicrobial activity have also been
made for other host defense peptides (15) and suggest the need for
further study of the role these peptides play in vivo.
The mouse is a highly useful animal model to study the function of the
immune system in events such as wound repair and cutaneous inflammation. Surprisingly, despite the well characterized immune system in the mouse, antimicrobial peptides have only been identified in the mouse intestine (16, 17) and appear to be absent from neutrophils (18). Thus, in this investigation we sought to identify a
mouse member of the cathelicidin gene family and describe its expression and antimicrobial function. We report a full-length cDNA
sequence derived from mouse marrow which is a member of the cathelicidin gene family. This gene, named Cramp, for
cathelin-related antimicrobial peptide, mapped to a
single region on murine chromosome 9, homologous to the map locations
of cathelicidins in man and pig. Transcripts for Cramp were
expressed in multiple mature tissues and during embryogenesis. Finally,
CRAMP protein was identified by immunostaining in murine bone marrow
cells and neutrophils and behaved structurally and functionally as a
potent antimicrobial agent.
Amino acids and coupling reagents for peptide
synthesis were from PerSeptive Biosystems (Framingham, MA) and
Novabiochem (Laufelfingen, Switzerland). HPLC-grade
acetonitrile,1
N-methyl-2-pyrrolidone, dichloromethane, and
N,N-dimethylformamide were from Lab-Scan (Dublin,
Ireland). Trifluoroacetic acid, N-methylmorpholine, and
trifluoroethanol were from Janssen Chimica (Beerse, Belgium). o-Nitrophenyl- Total RNA was extracted from C57BL/6 mouse
femoral marrow cells with guanidinium thiocyanate (19). To isolate
potential murine cDNA homologs to the cathelin-related
antimicrobial peptides, a 5 Northern blot analysis of total RNA
was performed as described previously (14). Approximately 10 µg of
total RNA was extracted from whole C57BL/6 mouse embryos at different
gestational ages and separated by electrophoresis through a 1%
agarose/formaldehyde gel. RNA was transferred to a GeneScreen Plus
membrane (DuPont NEN). Hybridization was carried out at 65 °C in
QuikHyb Solution (Stratagene) and probed using [32P]dCTP
random primer-labeled cDNA, corresponding to base pairs 79-249 of
murine CRAMP cDNA. Filters were washed twice for 15 min in 2 × SSPE (0.18 M NaCl, 0.01 M
Na2H2PO4, 1 mM EDTA, pH 7.7), 0.1%
sodium dodecyl sulfate at room temperature and then twice in 0.2 × SSPE, 0.1% sodium dodecyl sulfate at 55 °C.
Total RNA from marrow, testis,
stomach, small intestine, liver, lung, skeletal muscle, brain, heart,
spleen, kidney, and large intestine of 12-week-old C57BL/6 mice was
prepared with guanidinium thiocyanate (19). Approximately 5 µg of
DNase-treated total RNA from each tissue was annealed at 42 °C with
random hexamers in a solution containing 10 mM Tris-HCl, pH
8.3, 50 mM KCl, and 5 mM MgCl2. The
annealed hexamers were then extended by adding dNTP at a final
concentration of 0.1 mM, 1.5 units of RNasin (Promega, Madison), and 1 unit of Superscript reverse transcriptase (Life Technologies, Inc.) at 42 °C for 90 min. The resulting cDNA was then amplified with the specific primers 5 Solid phase peptide
synthesis of CRAMP-1 was done on a Milligen 9050 synthesizer (Milligen,
Bedford, MA). The synthesis was performed using
Fmoc-L-Glu(OtBu)-PEG-PS resin (0.2mmol/g) and N,N-dimethylformamide as solvent. Couplings were
carried out with a 6-fold excess of an equimolar mixture of Fmoc-amino
acid, N-hydroxybenzotriazole, and
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in the presence of N- methylmorpholine. Deprotection
from the Fmoc group was performed with piperidine,
N-methyl-2-pyrrolidone, and
N,N-dimethylformamide (1:2:2, v/v) in the
presence of 0.7% (v/v) 1,8-diazabicyclo (5,4,0) undec-7-ene. To
improve yield, the column temperature was increased to 45 °C, the
resin was washed with
N-methyl-2-pyrrolidone/N,N-dimethylformamide/dichloromethane (1:1:1, v/v) containing 1% Triton X-100 and 2-methylencarbonate immediately before each coupling step, and 1-hydroxy-7-azabenzotriazole and O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate replaced N-hydroxybenzotriazole and TBTU
for the coupling of residues 7-11, 15-18, and 24. CRAMP-2 was
obtained by elongation of CRAMP-1. Amino acid side chains were
protected with trityl (Gln, Asn), t-butyloxycarbonyl (Lys),
t-butyl (Glu, Ser), and
2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg). Peptide deprotection and
cleavage from the resin were carried out using a mixture of
trifluoroacetic acid/ethandithiol/phenol/water/triisopropylsilane (90:4:2:2:2, v/v) for 2 h at room temperature. After cleavage, both peptides were extracted repeatedly with ethyl ether and purified by reverse phase HPLC on a C18 Delta-Pak column (Waters, Bedford, MA),
using a 0-60% water/acetonitrile gradient in the presence of 0.1%
trifluoroacetic acid.
The peptide concentration was measured by
the absorbance of Phe at 257 nm and using a molar extinction
coefficient of 195 (20). Mass determinations were performed with an API
I ionspray mass spectrometer (PE SCIEX, Toronto, Canada). Circular
dichroism spectra were recorded at room temperature on a Jasco J-600
spectropolarimeter (Jasco Corp., Tokyo, Japan). Peptide samples (10-25
µM) were dissolved in 5 mM sodium phosphate
buffer, pH 7.0, in the absence or presence of 15, 30, and 45% (v/v)
trifluoroethanol. The Antibacterial and antifungal minimum inhibitory
concentrations (MIC) were determined by a microdilution susceptibility
test described previously (9). The following bacterial strains were tested: Escherichia coli ATCC 25922, ML-35 and D21,
Salmonella typhimurium ATCC 14028, Pseuodomonas
aeruginosa ATCC 27853, Serratia marcescens ATCC 1800, Proteus vulgaris ATCC 13315, Staphylococcus aureus ATCC 25923, Cowan 1, and two methicillin-resistant clinical isolates carrying the mecA gene (provided by L. Dolzani,
Department of Biomedical Sciences, University of Trieste, Italy),
Staphylococcus epidermidis ATCC 12228, and Bacillus
megaterium BM 11. The MIC values were determined after incubation
at 37 °C for 18 h. The antifungal activity was determined using
clinical isolates of Candida albicans and Cryptococcus
neoformans. The assay conditions were similar to those used for
bacteria, except that the fungal species were grown and tested in
Sabouraud liquid medium, and the MIC was determined after incubation at
30 °C for 36-48 h.
The bactericidal activity of CRAMP-1 and CRAMP-2 against midlog phase
cultures of E. coli ATCC 25922, P. aeruginosa
ATCC 27853, and S. aureus ATCC 25923 was tested in low ionic
strength buffer (10 mM sodium phosphate buffer, pH 7.4).
Bacteria (0.4-0.6 × 106 colony-forming units/ml)
were incubated in the absence (controls) or in the presence of
different amounts of peptides in a 96-well microtiter plate (final
volume of 150 µl). After a 1-h incubation at 37 °C, samples were
serially diluted with sterile PBS, plated in duplicate on
Mueller-Hinton agar, and incubated for 16-18 h to allow colony counts
(9). Bacterial inner membrane permeabilization was evaluated with the
E. coli ML-35 strain as described previously (22).
Antibodies to synthetic
CRAMP-1 were raised in New Zealand White rabbits by an initial
intramuscular injection of 150 µg of CRAMP-1 in complete Freund's
adjuvant followed by five booster injections of 150 µg of CRAMP in
incomplete Freund's at 3-week intervals. Serum was collected 10 days
after boosting.
4 × 105
Swiss-Webster mouse bone marrow cells in 100 µl of PBS, 1% bovine
serum albumin, or 5 × 106 C57BL/six peripheral blood
cells in Tris-buffered saline, 5 mM EDTA, were attached to
glass slides by cytospin at 400 × g for 5 min in a
Shandon Cytospin 3 cytocentrifuge, fixed in methanol, then blocked for
1 h with a solution of 5% goat serum in PBS, 1% bovine serum
albumin. Slides were then incubated for 1 h with a 1:200 dilution
of rabbit anti-CRAMP antiserum or nonimmune rabbit serum. To confirm
specificity of antibody binding, parallel slides were treated
identically with rabbit-anti CRAMP serum that had been preincubated for
1 h at room temperature with 20 µg/ml synthetic CRAMP-1.
Following primary antibody incubation slides were washed three times
for 5 min in PBS and then incubated for 1 h at room temperature
with a 1:250 dilution of fluorescein-conjugated goat anti-rabbit IgG
(Cappel Research Products, Durham, NC). Each slide was washed three
times for 5 min with Tris-buffered saline. Coverslips were mounted with
Prolong 228 Antifade mounting medium (Molecular Probes, Eugene, OR) and
cells photographed with Kodak Elite II Ektachrome ASA400 film on a
Zeiss Axiophot microscope.
Cramp was mapped by analysis
of the progeny of two multilocus crosses: (NFS/N or C58/J × Mus
m. musculus) × M. m. musculus (23) and (NFS/N × M. spretus) × M. spretus or C58/J (24). Progeny
of these crosses have been typed for over 1000 markers including the
Chr 9 markers TRF (transferrin), Gnatl (guanine nucleotide-binding protein, To isolate potential murine homologs to the family of
antimicrobial peptides related by a cathelin-like domain, primers were designed using sequence information from the conserved cathelin-like domain of the porcine gene PR-39. A nested RACE PCR strategy
was used, capitalizing on the highly invariant cathelin-like prepro domain from all species studied to date. Three independently derived clones were sequenced in both directions to give the cDNA sequence shown in Fig. 1. No other related cDNAs were
identified by this approach despite screening more than 500 clones for
isolates related by the 5
The murine cathelin-related cDNA contains an open reading frame of
516 base pairs and encodes a predicted translation product of 172 amino
acids. Sequence comparisons between the prepro regions of the predicted
murine (CRAMP) pig (PR-39) and human (FALL-39) cathelicidins identify
significant primary sequence similarities; the murine protein maintains
52% identity with PR-39 and FALL-39 in the cathelin-like domain and
80% identity with either individually (Fig. 2). This
sequence similarity includes the conservation of cysteines at positions
83, 94, 105, 122 and potential processing sites at the carboxyl
terminus of the cathelin portion of the peptide. The previously
reported cDNA sequences of human FALL-39 and porcine PR-39 encode
different peptides in the 3
To evaluate the expression of Cramp mRNA
in the mouse, RNA was extracted from whole murine embryos isolated at
various gestational ages and from whole adult mouse tissues. Northern
blot of total RNA from whole embryos demonstrated abundant 1-kilobase
message (Fig. 3, A and B) mRNA
was detectable as early as gestational days 12 and 13 and increased
relative to ribosomal RNA during development. In adult tissues, this
transcript was detectable by Northern blot analysis only in marrow
total RNA (data not shown). To increase the sensitivity for detection
in adult tissues, a reverse transcriptase-PCR strategy was employed.
Total RNA from brain, heart, spleen, kidney, testis, colon, liver,
marrow, stomach, small intestine, lung, and skeletal muscle was
prepared, and reverse transcriptase-PCR was performed using nested
primers to specifically amplify cDNA. As a control for all reverse
transcriptase-PCR experiments, primers selected for
Using the probe
spanning base pairs 79-249 of Cramp, we identified
ScaI fragments of 10.2 kilobases in parental M. musculus DNA, 11.7 in NFS/N and C58/J, and 14.2 in M. spretus. Inheritance of the variant fragments was typed in the
progeny of two sets of genetic crosses and compared with inheritance of
more than 1000 genetic markers previously typed and mapped in these
mice. CRAMP showed linkage to markers on distal Chr 9 (Fig.
4) and was positioned just distal to Gnat1.
This region of mouse Chr 9 is homologous to human 3p23-21, consistent
with the location of the human homolog of this gene,
Fall-39.
The translation
products of the cathelicidins are processed to release
carboxyl-terminal peptides with antimicrobial activity. To investigate
the properties of the carboxyl-terminal peptide predicted by the CRAMP
cDNA, two peptides were synthesized. The first peptide, CRAMP-1,
was prepared based on the predicted elastase cleavage site at position
138-139 and is a 33-amino acid peptide. The second, CRAMP-2, was
prepared based on potential processing at the dibasic site at position
133-134 and is a 38-amino acid peptide. Helical wheel projections of
both CRAMP-1 and CRAMP-2 predict that these peptides will form an
amphipathic helix similar to that for FALL-39 (13) and
non-cathelin-related antimicrobial peptides such as the maganins and
cecropins. To evaluate this directly, circular dichroism (CD) spectral
analysis of CRAMP-1 and CRAMP-2 was done (Fig. 5). The
spectra of 10-25 µM synthetic CRAMP-1 in buffer suggest
that the peptide is in a random coil configuration and that it will
assume a helical structure in the presence of 15-45% (v/v)
trifluoroethanol. The CD spectra of CRAMP-2 (not shown) were similar to
CRAMP-1.
The capacity of
CRAMP-1 and CRAMP-2 to function as antimicrobial agents was evaluated
directly on a variety of microbes (Table I). Both
peptides demonstrated potent antimicrobial activity against
Gram-negative bacteria with MIC values in the range of 0.5-8.0
µM. CRAMP peptides were less active against Gram-positive strains (MIC 32-64 µM) and not active at the
concentrations tested against P. vulgaris, C. albicans, and selected strains of S. aureus. The CRAMP
peptides were also found to have potent direct bactericidal activity
when assayed in low ionic strength buffer (Table II). Under these conditions even the Gram-positive bacterium (S. aureus) was killed at concentrations as low as 1 µM.
Table I.
Antimicrobial activity of CRAMP-1 and CRAMP-2
Table II.
Bactericidal activity of CRAMP-1 and CRAMP-2
Joint Program in Neonatology,
National Institutes of Health, Bethesda, Maryland 20892, the
** Departimento di Scienze e Tecnologie Biomediche,
National
Laboratory Consorzio Interuniversitario Biotechnologie,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helix
similar to other antimicrobial peptides. Functional studies
showed CRAMP to be a potent antibiotic against Gram-negative bacteria
by inhibiting growth of a variety of bacterial strains (minimum
inhibitory concentrations 0.5-8.0 µM) and by
permeabilizing the inner membrane of Escherichia coli
directly at 1 µM. Antiserum against CRAMP revealed
abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the
mouse can use a nonoxidative mechanism for microbial killing and
permit use of the mouse to study the role such peptides play in host
defense and wound repair.
Materials
-D-galactopyranoside was from
Sigma. Mueller-Hinton broth, Bacto-agar, dextrose, mycological peptone,
and yeast extract powder were from Unipath Ltd (Basingstoke, U. K.).
All other chemicals were of analytical grade.
and 3
RACE strategy was applied (Life
Technologies, Inc.; 3
and 5
RACE systems, Gaithersburg, MD). For 3
RACE, cDNA synthesis was carried out using adapter primer
5
-GGCCACGCGTCGACTAGTAC(T)17-3
. Amplification
toward the 3
end was done first with the universal amplification primer 5
-(CUA)4GGCCACGCGTCGACTAGTAC-3
and
with a cathelin-specific primer-1, 5
-TCGGAAGCTAATCTCTAC-3-3
, which was designed based on a base pairs 165-182 sequence of porcine prepro-PR-39. A second nested amplification was then done with a
cathelin-specific primer-2, 5
-(CAU)4CTGGACCAGCCGCCCAAG-3
designed based on base pairs 195-212 of prepro-PR-39 and the universal amplification primer. For amplification toward the 5
end, the gene-specific primer-1, 5
-TTTGCGGAGAAGTCCAGC-3
, based on a sequence derived from 3
RACE, was used for cDNA synthesis. Amplification was done with an anchor primer, provided by Life Technologies, Inc.,
and with a gene-specific primer-2,
5
-(CAU)4GAAATTTTCTTGAACCG-3
. Products of 3
and 5
RACE were cloned into pAMP1 for sequencing by automated sequencer
(model 373A, Perkin-Elmer) in both directions using primers against M13
and T7. Three independent clones were sequenced in both directions.
-GCTGATGTCAAAAGAATCAGCG-3
and 5
-TCCCTCTGGAACTGCATGGTTCC-3
, based on base pairs 10-32 and 357-378, respectively, of the CRAMP cDNA sequence. These primers were used with the following thermal cycle profile: 95 °C for 5 min;
20 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for
1.5 min; and a final extension step of 72 °C for 7 min. A second
round of PCR was performed with nested primers
5
-CTGTGGCGGTCACTATCACT-3
and 5
-GTTCCTTGAAGGCACATTGC-3
based on base
pairs 49-68 and 291-310, respectively, of the CRAMP cDNA
sequence. Amplification conditions were as above, but a higher
annealing temperature of 58 °C was used. Products were separated on
3% agarose gel and photographed using Eagle Eye apparatus
(Stratagene).
-helical content was estimated by the method
of Chen et al. (21) from the [
]222 measurements, using
1,500 and
39,500 (1-2.5/n)
deg-cm2 dmol
1, as the values for 0% and
100% of helix, respectively, with n indicating the number
of residues.
transducing subunit), Scn10a
(peripheral sodium channel 3, subunit 10), and Cck
(cholecystokinin) as described previously (25, 26). Data were stored an
analyzed using the program LOCUS developed by C. E. Buckler (NIAID,
Bethesda, MD). Recombinational distances and S.E. were calculated
according to Ref. 27. Genes were ordered by minimizing the number of
recombinants.
Molecular Cloning of a Cathelin-related cDNA in the
Mouse
cathelin domain but distinct in the 3
region.
Fig. 1.
Nucleotide and predicted amino acid sequence
of CRAMP. The nucleotide sequence is numbered on the
left, and the amino acid sequence is numbered from the first
methionine on the right. The sequence of the predicted
mature protein is underlined. The stop codon is marked with
an asterisk, and the cysteine residues are in
boldface type.
[View Larger Version of this Image (51K GIF file)]
region. Similarly, the predicted
translation product of CRAMP is distinct in this region.
Fig. 2.
Sequence similarities of CRAMP, porcine
PR-39, and human FALL-39. The amino acid sequences of CRAMP,
PR-39, and FALL-39 are deduced from the cDNA. Residues identical
between CRAMP and at least one other protein are boxed.
Small letters denote the sequences corresponding to the
mature antimicrobial peptides.
[View Larger Version of this Image (34K GIF file)]
-actin were
chosen, and reactions were included lacking either RNA or reverse
transcriptase. Cramp transcripts were detectable in spleen,
testis, colon, marrow, stomach, and small intestine (Fig.
3C). Faint bands corresponding to Cramp were also
seen in heart, lung, and skeletal muscle but were not detectable in
brain, kidney, and liver. All samples were positive with actin-specific
primers and negative when performed in the absence of RNA or reverse
transcriptase.
Fig. 3.
CRAMP is expressed during embryogenesis and
in adult mouse tissues. Panel A, Northern analysis of total
RNA from mouse embryos at indicated gestational age. kb,
kilobases. Panel B, graph quanitifying the relative
abundance of CRAMP mRNA/28 S RNA. Panel C, photograph
of reverse transcriptase-PCR products from mRNA isolated from adult
mouse tissues. Lanes: 1, molecular weight standards;
2, brain; 3, heart; 4, spleen;
5, kidney; 6, testis; 7, colon;
8, liver; 9, bone marrow; 10, stomach;
11, small intestine; 12, lung; 13,
skeletal muscle.
[View Larger Version of this Image (48K GIF file)]
Fig. 4.
Map location of Cramp gene on
mouse chromosome 9. To the right of the map are
recombination fractions for adjacent loci: the first fraction
represents data from the M. musculus crosses, and the second
fraction is from the M. spretus crosses. In
parentheses are calculated recombinational distances and
S.E. Map locations of the human homologs are indicated to the
left.
[View Larger Version of this Image (14K GIF file)]
-Helix
Fig. 5.
Circular dichroism spectra of CRAMP-1.
CD spectra were recorded at 10-25 µM peptide in 5 mM sodium phosphatase buffer, pH 7.0, in the absence
() or the presence of 15% (- - -) and 45% (
-
)
trifluoroethanol.
[View Larger Version of this Image (21K GIF file)]
Organism and
strain
MIC
CRAMP-1
CRAMP-2
µM
Escherichia coli ATCC 25922
1
1
Escherichia coli ML35
2
2
Escherichia
coli D21
0.5
0.5
Salmonella typhimurium ATCC
14028
8
8
Pseudomonas aeruginosa ATCC
27853
4
4
Serratia marcescens ATCC 8100
4
4
Proteus vulgaris ATCC 13315
>64
>64
Staphylococcus aureus ATCC 25923
32
32
Staphylococcus aureus Cowan I
32
32
Staphylococcus aureus (MRSA)
>64
>64
Staphylococcus aureus (MRSA)
64
64
Staphylococcus epidermidis ATCC 12228
16
16
Streptococcus faecalis ATCC 29212
32
16
Bacillus megaterium Bm11
4
4
Candida
albicans
>64
>64
Cryptococcus
neoformans
16
16
Concentration
E. coli ATCC 25922
S.
aureus ATCC 25923
P. aeruginosa ATCC 27853
µM
%
killed bacteria
CRAMP-1
0.1
18.9
5.8
12
0.3
61 -65.4
32.7 -40.4
42
1.0
73 -99.1
91.2 ->99.99
95.5
10.0
>99.99
->99.99
>99.99 ->99.99
>99.99
CRAMP-2
0.3
81.6
-57.8
93.7 -89.9
48.6
1.0
97.1 -99.5
99.8
->99.99
99.8
10.0
>99.99 ->99.99
>99.99
->99.99
>99.99
The kinetics of E. coli inner membrane permeabilization was
evaluated for both peptides. The reaction rate for permeabilization with CRAMP-2 was faster than for CRAMP-1 (Fig. 6). At a
concentration of 2.5 µM, CRAMP-2 has the capacity to
permeabilize bacteria and thus unmask cytoplasmic -galactosidase
activity at a rate that is 93% of that achieved by sonication.
Steady-state bacterial permeabilization was reached after 4 min.
CRAMP-1 at the same concentration induced about 60% permeabilization
with steady state achieved by 8 min. Despite the potent ability to lyse
bacterial inner membranes, the CRAMP peptides (50 µM) did
not lyse human or sheep erythrocyte membranes (data not shown).
Immunolocalization of CRAMP in Granulocytes
The expression of
CRAMP protein in vivo was evaluated with a polyclonal
antibody prepared against CRAMP-1. Prominent immunostaining was
detectable in myeloid cells present within mouse femoral marrow (Fig.
7A). This staining was specific for CRAMP
since addition of excess synthetic peptide abolished staining (Fig.
7B). In peripheral leukocytes, CRAMP was detected in
abundance in neutrophil granules (Fig. 7C) but was not seen
in monocytes, lymphocytes, or eosinophils. Thus, CRAMP protein is
expressed in vivo in a location similar to that shown for
cathelin-related antimicrobial peptides in other species.
In this study we identify, molecularly clone, and characterize a novel antimicrobial peptide in the mouse. This molecule is a new member of a large family of antimicrobial peptide precursors found in human (13, 28), pig (6, 8, 14, 29, 30), cow (9, 31), rabbit (12), and sheep (10, 11). The homology among the precursors of these antimicrobial peptides lies in a common NH2-terminal region of the unprocessed precursor known as cathelin and has led to use of the term cathelicidins to describe the gene family. The mouse gene described here shares a high degree of homology with the cathelin domain contained within all members of this family; it maps to a region of mouse distal chromosome 9 homologous to the chromosomal location of cathelicidins in pig and human, it is antimicrobial, and it is expressed primarily in neutrophil granules. Therefore, this gene, named Cramp, encodes a new endogenous antimicrobial peptide and identifies a novel host defense molecule in mouse for further study of mechanisms of innate immunity.
The cathelicidins exert their antimicrobial effect based on the structurally diverse COOH-terminal domain of these peptides (7). To identify candidate cathelicidins in mouse, primers were designed against various regions within the highly evolutionarily conserved NH2-terminal cathelin domain of these gene products. This approach has been successful previously in identifying multiple cathelin-related genes in several species (7). Only a single cathelicidin was identified by this approach in mouse despite the use of multiple primers and tissue sources for RNA and large scale screening of more than 500 cathelin-related clones. An additional myeloid gene product, B9, has been described in mouse which contains a cathelin-like domain yet is not known to be antimicrobial (32). The inability to detect this transcript may reflect limitations inherent in a reverse transcriptase-PCR approach such as difficulties in detecting transcripts in low abundance. Thus, although no additional antimicrobial cathelicidin has been described in mice, the current work cannot rule out the presence of multiple cathelin-related antimicrobial peptides such as found in other species.
Identification of a murine cathelin-related gene enabled us to use the mouse for study of tissue-specific and developmental expression. We found that the expression of Cramp transcripts in the mouse is regulated in a tissue-specific and temporal developmental pattern. In the adult, transcript expression was most abundant in marrow. The finding was consistent with our observation that the protein was found abundantly in granulocytes and in marrow cells of myeloid lineage and agrees with the sites of expression of cathelicidins in man and pig (14, 28, 33). However, CRAMP mRNA was also detectable at low levels in mouse testis, the gastrointestinal tract, spleen, heart, lung, and skeletal muscle. It is possible that the presence of CRAMP transcripts in a variety of tissues is due to the presence of small numbers of myeloid precursors in those organs. However, a lack of CRAMP transcripts in liver, kidney, and brain, tissues that are likely to contain significant amounts of blood cell contaminants, argues against this. Furthermore, pig cathelicidins have been found in the gastrointestinal tract, and the human cathelicidin FALL-39 has been found in testis (13). Our findings are therefore supported by the tissue-specific expression of cathelicidins in other species.
Analysis of whole embryo RNA during development demonstrated that CRAMP transcripts are also expressed during embryogenesis. Expression was detected as early as embryonic day 12 in the mouse and increased subsequently. The timing of CRAMP expression coincides with the earliest colonization of the fetal murine liver with hematopoietic stem cells but precedes the establishment of granulocytic lineages (34). Thus, as in adult tissues, CRAMP RNA may be expressed by nonimmune cells. The significance of these observations is 2-fold. First, finding constitutive expression of antimicrobials in a variety of tissues suggests that these tissues may have an innate ability to resist microbial infection before or without stimulating an inflammatory cell influx. Secondly, certain antimicrobial peptides such as PR-39 are known to possess additional biologic activity including the ability to function as stimulators of cell surface syndecan expression (14). Expression of CRAMP in a variety of tissues and during embryogenesis therefore is consistent with functions in addition to antimicrobial activity. Further work to evaluate cell type-specific expression and biological function of CRAMP is needed to address these issues directly.
The 3 region of CRAMP predicts a translation product that is an
effective antimicrobial peptide. Analysis of the processing of the
cathelicidins suggested that CRAMP may be cleaved at multiple sites
(28, 35). Helical wheel projections of two potential cleavage products
predicted that the mature peptides would acquire an
-helical
conformation with an amphipathic arrangement. This structural
prediction is similar to a subset of cathelicidins which includes the
human FALL-37, rabbit CAP 18, and pig PMAP-37, but not the
cathelicidins characterized by proline and arginine-rich or
tryptophan-rich mature peptides. Direct analysis of both synthetically produced CRAMP peptides confirmed this structural prediction and established the antimicrobial function of CRAMP. CD spectral analysis demonstrated that these synthetic peptides can assume an
-helical structure. Antimicrobial assays done against a variety of microbes demonstrated potent activity against Gram-negative bacteria and rapid
permeabilization of the inner membrane of E. coli. These results demonstrate that CRAMP is a potent, membrane-active
antibacterial peptide. Recently, a similar cDNA from the mouse was
described (36). The corresponding synthetic peptide lacked five
NH2-terminal amino acids (GLLRK) used here in CRAMP-1 and
was not antimicrobial. This deletion is likely to be responsible for
the lack of antimicrobial activity because structural analysis done in
other cathelicidin-derived peptides has shown that the
NH2-terminal domain of the mature peptide is critical for
activity (37). Purification of the native peptide from the mouse will
be necessary to establish its native sequence.
In summary, identification of CRAMP and characterization of its expression and antimicrobial activity demonstrate that the murine host defense system includes members of the cathelicidin family. This information permits many powerful experimental tools available in the mouse to be applied to the study of these antimicrobial peptides. Such work may lead to greater insight into the function of these peptides in protecting organisms from infection and influencing cell behaviors.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U43409[GenBank].
We thank Elizabeth Siebert and Martin Naley for expert technical assistance, Renae Bopko for secretarial support, and Dr. Kenneth Huttner for helpful discussions.