Mouse SWAM1 and SWAM2 Are
Antibacterial Proteins Composed of a Single
Whey Acidic Protein Motif
This information is current as
of June 16, 2017.
Koichi Hagiwara, Tohru Kikuchi, Yoshiyuki Endo, Huqun,
Kazuhiro Usui, Mitsu Takahashi, Naoko Shibata, Takashi
Kusakabe, Hong Xin, Sachiko Hoshi, Makoto Miki, Nozomu
Inooka, Yutaka Tokue and Toshihiro Nukiwa
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Copyright © 2003 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2003; 170:1973-1979; ;
doi: 10.4049/jimmunol.170.4.1973
http://www.jimmunol.org/content/170/4/1973
The Journal of Immunology
Mouse SWAM1 and SWAM2 Are Antibacterial Proteins
Composed of a Single Whey Acidic Protein Motif1
Koichi Hagiwara,2* Tohru Kikuchi,* Yoshiyuki Endo,* Huqun,* Kazuhiro Usui,*
Mitsu Takahashi,* Naoko Shibata,* Takashi Kusakabe,† Hong Xin,* Sachiko Hoshi,*
Makoto Miki,* Nozomu Inooka,* Yutaka Tokue,* and Toshihiro Nukiwa*
T
he innate immune system forms the first line of host defense against invading pathogens. Participants in this system include the phagocytes (polymorphonuclear leukocytes and macrophages), complements, and recently identified
groups of proteins that directly recognize the bacterial components
(pattern-recognition receptors) (1), such as mannose-binding lectin
(2), and the Toll-like receptors (3, 4). Antibacterial peptides are
considered to be some of the earliest participants in the system (5).
Both animals and plants are equipped with numbers of antibacterial peptides to protect them from a wide range of microbes.
SLPI (6) and Elafin (7) are proteins with antibacterial activity
and antiprotease activities (6 –9). They belong to a group of proteins that have a whey acidic protein (WAP)3 (1) motif, hereafter
called the WAP motif protein. The WAP motif (InterPro code
IPR002221) is a 50-aa protein motif with eight cysteine residues at
defined positions. They form four intracellular disulfide bonds creating a tightly packed structure (10, 11). Both human and mouse
have the SLPI gene (6, 12). SLPI protein provides protection
against a variety of pathogens (13–15) and prevents inflammation
by protecting tissues against proteases released from neutrophils
(16). Its indispensable role in wound healing has been shown using
*Department of Respiratory Oncology and Molecular Medicine, Division of Cancer
Control, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi, Aoba-ku, Sendai, Japan; and †Department of Pathology, School of Medicine,
Fukushima Medical University, Hikarigaoka, Fukushima, Japan
Received for publication March 20, 2002. Accepted for publication December
11, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by the grant-in-aid for scientific research from the
Ministry of Education, Science, Sports, Culture, and Technology of Japan.
2
Address correspondence and reprint requests to Dr. Koichi Hagiwara, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. E-mail: [email protected]
3
Abbreviations used in this paper: WAP, whey acidic protein; AMV, avian myeloblastosis virus; BAC, bacterial artificial chromosome; SLPI, secretory leukoprotease
inhibitor; Ni-NTA, nickel-nitrilotriacetic acid.
Copyright © 2003 by The American Association of Immunologists, Inc.
knockout mice (17). Human elafin has an antiprotease spectrum
different from that of SLPI (18) and plays a protective role in
various diseases (19, 20). Mouse elafin has not yet been cloned and
despite the use of several methods we have been unsuccessful in
our attempts. However, during our cloning attempts we did identify two novel WAP motif proteins, SWAM1 and SWAM2. Here
we report the cDNA and genomic cloning of both genes, their expression patterns, and their protein functions. Both are antibacterial
proteins probably derived from the same ancestral gene as that of
SLPI and elafin. Our results emphasize that the WAP motif is an
important structural unit in the generation of antibacterial proteins.
Materials and Methods
SWAM1, SWAM2 cDNA, and genomic cloning4
The GenBank mouse EST database was searched by the tblastn program
using the WAP motif of human elafin as a query sequence. This search
provided partial sequences for two cDNAs that we named SWAM1 and
SWAM2. The full-length sequences were obtained by repeated 5⬘RACE
reactions using the Marathon-ready mouse placental cDNA (Clontech, Palo
Alto, CA) until most of the 5⬘ ends of the RACE clones clustered within
a 10-bp region. Mouse genomic clones for SWAM1 (clone 44J10) and
SWAM2 (clone 177A1) were isolated using the mouse (129/SvJ strain)
genomic BAC library PCR screening system, release 1 (Incyte Genomics).
The PCR primer pairs used were 5⬘-AGACAAACCGGAGAAAACAC
ATG-3⬘ and 5⬘-GCACTTAATCTTTGGTTTCAGGATGG-3⬘ for SWAM1
and 5⬘-GATATCTCATAAAAATGGCCTCAG-3⬘ and 5⬘-TTTCTCAGAGT
TCTGGAGGTTGG-3⬘ for SWAM2. The nucleotide sequences of both genes
were determined by the long distance sequencer method (21–23).
Plasmids
The entire open reading frames of both SWAM1 and SWAM2 were amplified by PCR using primers S11F (5⬘-CGAATTCGCTCACCTGCA
CAGTTTTCTTGGG-3⬘) and S11B (5⬘-CGTCTAGAGGCTATCCTCGA
4
The nucleotide sequences of SWAM1 and SWAM2 cDNAs have been deposited in
GenBank under GenBank accession AF276974 (SWAM1) and AF276975 (SWAM2).
The nucleotide sequences of SWAM1 and SWAM2 genes have been deposited in
GenBank under GenBank accession AF482008-AF482009 (SWAM1) and AF482010
(SWAM2).
0022-1767/03/$02.00
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Antibacterial proteins are important participants in the innate immunity system. Elafin and SLPI are the whey acidic protein
(WAP) motif proteins with both antibacterial activity and antiprotease activity, and their role in innate immunity is under intense
investigation. We cloned two novel antibacterial WAP motif proteins from mice, SWAM1 and SWAM2. SWAM1 and SWAM2
are composed of a signal sequence and a single WAP motif that has high homologies with the WAP motifs of elafin and SLPI.
SWAM1 is constitutively expressed in kidney and epididymis, and is induced in the pneumonic lung. SWAM2 is constitutively
expressed in tongue. SWAM1 and SWAM2 inhibit the growth of both Escherichia coli and Staphylococcus aureus at a IC90
(concentration that achieves 90% inhibition) of 10 ␮M. Human genes LOC149709 and huWAP2 are considered to be human
SWAM1 and SWAM2, respectively. These and several WAP motif proteins (WAP1, elafin, SLPI, HE4, eppin, C20orf170,
LOC164237, and WFDC3) form a gene cluster on human chromosome 20, suggesting that they may be derived from the same
ancestral gene by gene duplication. Our results underscore the role of the WAP motif as a skeletal motif to form antibacterial
proteins, and warrant the study of antibacterial activity in other WAP motif proteins. The Journal of Immunology, 2003, 170:
1973–1979.
1974
MOUSE SWAM1 AND SWAM2 ARE ANTIBACTERIAL PROTEINS
CTGAAACTG-3⬘) for SWAM1 and S21F (5⬘-CGAATTCTCAGTCTC
AGCCTCACAGCAAC-3⬘) and S21B (5⬘-CGTCTAGACAGTCCTCT
GAGGCCATTTTTATGAG-3⬘) for SWAM2. Each amplified PCR fragment was inserted into pCI-neo vector (Promega, Madison, WI) to make
either pSWAM1 or pSWAM2. pET-SWAM1 and pET-SWAM2 that express the polyhistidine-tagged (His-tagged) mature proteins of SWAM1
and SWAM2 (rSWAM1 and rSWAM2) in Escherichia coli were constructed as follows. Signal peptides of SWAM1 and SWAM2 proteins were
predicted by the computer program SingalIP (24). The nucleotide fragments encoding either the mature SWAM1 or SWAM2 proteins (i.e., proteins without a signal peptide) were amplified from pSWAM1 or pSWAM2
using PCR primer pairs S12F (5⬘-CTCATATGCGACCTGAAATAAA
GAAGAAGAACG-3⬘) and S12B (5⬘-AGAGATCTCTATTCTGGGC
TCTCCCATGGATCCAC-3⬘) for SWAM1 and S22F (5⬘-CTCATATG
GGTGGAGTCAAAGGCGAGGA-3⬘) and S22B (5⬘-GAGGATCCT
CACACCTTACTCTGAGGGATCTG-3⬘) for SWAM2. The amplified
PCR fragments were inserted into the pET-15b vector in-frame with the
His-tag sequence (see Fig. 4a). pET-SLPI expressing the His-tagged, mature
mouse SLPI protein (rSLPI) was constructed in the same way using PCR
primer pairs 5⬘-CTCATATGGGCAAAAATGATGCTATCAAAATCGG-3⬘
and 5⬘-GAGGATCCTCACATCGGGGGCAGGCAGACTTTC-3⬘.
Northern blot analysis
RT-PCR
Total RNA (1 ␮g) from the individual tissues of C57BL/6J mice was
reverse transcribed by AMV reverse transcriptase (Takara Shuzo, Otsu,
Japan) from backward primer. After adding forward primer, 20, 30, or 40
cycles of PCR were performed using Taq DNA polymerase (Takara Shuzo)
in a reaction volume of 100 ␮l. The primers used for SWAM1 were S14F
(5⬘-CGCATACGGAGGACAGTTCTG-3⬘; forward) and S14B (5⬘-GGC
TATCCTCGACTGAAACTG-3⬘; backward), and those for SWAM2 were
S24F (5⬘-TCAGTCTCAGCCTCACAGCAAC-3⬘; forward) and S24B (5⬘CACCTTACTCTGAGGGATCTGTTC-3⬘; backward). Aliquots (10 ␮l)
were run on 1% agarose gels, and the gels were photographed.
In situ hybridization
In situ hybridization was performed as previously described (28) with some
modifications. Full-length SWAM1 and SWAM2 cDNA were amplified by
PCR using S12F and S12B(T7) for SWAM1 and S22F and S22B(T7) for
SWAM2. S12B(T7) and S22B(T7) are S12B and S22B primers tagged
with T7 RNA polymerase promoter. The digoxigenin-labeled antisense
RNA probes were generated by in vitro transcribing each PCR fragment
using T7 RNA polymerase (Roche, Indianapolis, IN). Thin sections of
paraffin-embedded (kidney and epididymis) or frozen (tongue) tissues were
hybridized with RNA probe (200 ng/ml) in RNA in situ hybridization
solution (Dako Cytomation, Glostrop, Denmark) at 37°C overnight. The
signal was amplified and detected by serially treating the sections with
HRP-conjugated
rabbit
anti-digoxigenin
Ab,
biotin-tyramide
(PerkinElmer, Norwalk, CT), streptavidin-HRP, biotin-tyramide, alkaline
phosphatase-conjugated rabbit anti-biotin, and Fast Red TR/Naphthol
AS-MX (Sigma-Aldrich, St. Louis, MO). The sections were counterstained
with hematoxylin and observed under a microscope.
E. coli strain BL21(DE3)/pLysS transformed by either pET-SWAM1 or
pET-SWAM2 was grown in 750 ml of NYZM medium (BD Biosciences,
Mountain View, CA) at 37°C until an OD of 1 was reached. Isopropyl-␤D-thiogalactoside was added to a final concentration of 1 mM to induce
rSWAM1 and rSWAM2, and cultivation was continued at 30°C for 3 h.
The bacterial cells were harvested, and the cell pellet was suspended in 10
ml of lysis buffer (500 mM NaCl, 20 mM sodium phosphate (pH 7.8), and
1% Triton X). After lysing the cells by freeze-thawing, RNase A (10 ␮g/
ml) and DNase I (5 ␮g/ml) were added, and the sample (lysate) was incubated on ice for 30 min. The lysate was ultracentrifuged at 500,000 ⫻ g
at 4°C for 30 min to separate the pellet and the supernatant. Because
rSWAM1 and rSWAM2 were found in both pellet and supernatant fractions, we purified the proteins separately from each fraction. For purification from the pellet, the pellet was washed by two cycles of suspension (in
lysis buffer) and centrifugation, and dissolved in Gdn buffer (6 M guanidine
HCl, 500 mM NaCl, 20 mM sodium phosphate (pH 7.8), and 1% Triton X).
The solution was loaded onto an nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, Chatsworth, CA), washed with Gdn buffer containing 30 mM imidazole, and eluted by Gdn buffer containing 300 mM imidazole. For purification from the supernatant, the supernatant was loaded
onto an Ni-NTA agarose column, washed with lysis buffer containing 30
mM imidazole, and eluted with lysis buffer containing 300 mM imidazole.
The rSWAM1 was dialyzed against 500 mM NaCl and 20 mM sodium
phosphate (pH 5.6), and the rSWAM2 was dialyzed against 500 mM NaCl
and 20 mM sodium phosphate (pH 8.2). The positive control for the protease inhibition assay, rSLPI, was purified from the supernatant of
BL21(DE3)/pLysS containing pET-SLPI as described above, except that
the column was washed with lysis buffer without imidazole. The rSLPI was
dialyzed against 500 mM NaCl and 20 mM sodium phosphate (pH 7.4).
Protease inhibition assay
Elastase activity was measured as the amidolytic effect of human neutrophil elastase (15 nM; Athens Research & Technology, Athens, GA) on
pyroGlu-Pro-Val-pNA (1 mM; Chromogenix, Molndal, Sweden) (29) and
on MeOSuc-Ala-Ala-Pro-Val-pNA (0.4 mM; Calbiochem, La Jolla, CA)
(30). Cathepsin G activity was measured as the amidolytic effect of human
neutrophil cathepsin G (30 nM; Calbiochem) on Suc-Ala-Ala-Pro-PhepNA (0.4 mM; Calbiochem) and on MeOSuc-Ala-Ala-Pro-Met-pNA (0.4
mM; Calbiochem) (30). In all assays, elastase or cathepsin G was incubated
with different concentrations of rSWAP1, rSWAP2, or rSLPI (a positive
control) at 37°C for 30 min. Then the substrate for each enzyme was added
and incubated at 37°C for 1 h, and the residual enzyme activity was measured by the change in the absorbance at 405 nm.
Antibacterial assay
The bacterial strains E. coli JCM 5491 and S. aureus subspecies aureus
JCM2151 were obtained from the Japan Collection of Microorganisms
(Wako, Japan). Clones of E. coli and S. aureus isolated from clinical samples (clinical isolates) were obtained from Tohoku University Hospital.
The antibacterial assay was performed as previously described (8) with
some modifications. Bacteria were cultured in Müller-Hinton medium (BD
Biosciences) to a midlogarithmic phase. A620 was measured by a spectrophotometer, and the number of bacterial cells was calculated on the basis
of A620 0.2 ⫽ 5 ⫻ 107/ml. The bacterial suspension was diluted to 5 ⫻
104/ml with Müller-Hinton medium containing different concentrations of
rSWAP1, rSWAP2, lysozyme, or BSA. After incubation at 37°C for 2 h
with vigorous shaking, the number of CFU was determined by plating
serial dilutions. CFU without sample proteins (control) was normalized to
100%. The logarithm of the normalized CFU was used for the statistical
analysis. Significant differences were tested using Student’s unpaired, twotailed t test.
Data analysis
Multiple protein sequence alignment was performed by MacVector (Oxford Molecular) using CLUSTAL W algorithm (31) with the BLOSUM
protein substitution matrix (32). Searches of the GenBank databases were
performed using blast (blastn, blastp, and tblastn; http://www.ncbi.nlm.
nih.gov/blast/). Signal peptides were predicted by SignalIP (http://www.
cbs.dtu.dk/services/SignalP/).
Results
Cloning of the mouse SWAM1 and SWAM2 genes
In our attempts to clone mouse elafin gene, a search of the GenBank mouse EST database using the tblasn program with the entire
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Total RNA was prepared using the acid-guanidium phenol-chloroform
method (25, 26). Normal tissue RNA was isolated from the individual
tissues of C57BL/6J mice. Total RNA from mouse inflammatory lung tissue was prepared by administering 8 ⫻ 107 CFU of Streptococcus pneumoniae strain FP1284 intranasally to male ICR strain mice (4 wk). After 0,
10, 24, or 48 h, the mice were killed by exsanguination through a carotid
artery, and the lungs were removed. Total RNA (10 ␮g) was run on a 1.2%
formaldehyde gel and transferred to a Hybond N⫹ membrane (-Pharmacia
Biotech, Arlington Heights, IL). Probes of SWAM1 and SWAM2 containing the entire coding region of each gene were prepared by amplifying 10
pg of pSWAM1 or pSWAM2 using PCR primer pairs S13F (5⬘-GCTCAC
CTGCACAGTTTTCTTGGG-3⬘) and S13B (5⬘-TTCTGGGCTCTCCCA
TGGATCC-3⬘) for SWAM1, and S23F (5⬘-TCAGTCTCAGCCTCACAG
CAAC-3⬘) and S23B (5⬘-CACCTTACTCTGAGGGATCTG-3⬘) for SWAM2.
Probes (50 ng) were labeled with 50 ␮Ci of [␣-32P]dCTP (DuPont, Wilmington, DE) using the Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech), purified using the ProbeQuant G-50 Micro Column (Amersham Pharmacia Biotech), and hybridized in ULTRAhyb solution
(Ambion, Austin, TX) at 68°C overnight. Filters were washed at 68°C with
2⫻ SSC/1% SDS twice and with 0.2⫻ SSC/1% SDS once (27), and then
exposed to Kodak XA-R film (Eastman Kodak, Rochester, NY) at ⫺70°C
for 2 to 14 days depending on signal intensity.
Expression and purification of rSWAM1 and rSWAM2
The Journal of Immunology
1975
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FIGURE 1. Isolation of the SWAM1 and SWAM2 cDNA and genomic clones. a, Nucleotide sequences of SWAM1 and SWAM2 cDNAs. The open
reading frames and the translated amino acid sequences are written in bold text. The signal sequences are shadowed. The positions of the translational stop
codons are marked by asterisks. Polyadenylation signals (AATAAA) are underlined. An in-frame, upstream stop codon (TGA) for SWAM1 cDNA is boxed.
b, Genomic organization of the SWAM1 and SWAM2 genes. Boxes show exons (䡺, 5⬘ and 3⬘ untranslated sequences; u, signal sequences; f, mature
proteins). The positions of the restriction enzyme sites are indicated (B, BamHI; E, EcoRV; H, HindIII; Ps, PstI; Pv, PvuII; S, StuI; X, XbaI). The length
of the intron 1 of the SWAM1 gene was not determined. c, Comparison of the WAP motifs. SLPI NH2, the N-terminally located WAP motif; SLPI COOH,
the C-terminally located WAP motif (see d). Identical amino acids are written in bold, boxed, and shadowed. Similar amino acids are written in bold and
boxed. The consensus amino acid sequence for the WAP motif is shown above the protein sequences. d, Domain structures. f, Signal sequences; 䡺 in
which WAP is written, the WAP motifs; u in which cem is written, cementoin domains characteristic of elafin (55).
WAP motif of the human elafin as a query sequence gave us two
novel EST clones. The 5⬘ and 3⬘ RACE reactions provided the fulllength cDNA sequences (Fig. 1a). Both genes are composed of a
signal sequence followed by a single WAP motif. We named them as
SWAM1 (single WAP motif protein 1) and SWAM2. Screening of
the mouse BAC genomic library gave us genomic clones of SWAM1
(clone 44J10) and SWAM2 (clone 177A1). SWAM1 and SWAM2
resemble each other in the spatial placements of exons in the genome
1976
MOUSE SWAM1 AND SWAM2 ARE ANTIBACTERIAL PROTEINS
and in the assignment of protein domains to each exon, suggesting
that both genes may derive from a common ancestral gene (Fig. 1b).
The WAP motifs of SWAM1 and SWAM2 have homologies with
those of several proteins (Fig. 1c), although each protein has a different domain structure (Fig. 1d). Elafin and SLPI have inhibitory
activity against neutrophil elastase and cathepsin G as well as an antibacterial activity in their WAP motifs, suggesting that SWAM1 and
SWAM2 might have both antiprotease and antibacterial activities.
Tissue-specific expression of SWAM1 and SWAM2 mRNAs and
induction of SWAM1 mRNA in lung infected with Streptococcus
pneumoniae
Purification of rSWAM1 and rSWAM2
Under our culture conditions, rSWAM1 and rSWAM2 proteins
move into both the soluble fraction (the supernatant) and the insoluble fraction (the pellet) of the bacterial lysate. The purity of
both proteins was much better when purified from the pellet (Fig.
4). We used rSWAM1 and rSWAM2 prepared from the pellet in
this study unless otherwise stated. Two milligrams of purified
rSWAM1 and rSWAM2 protein was isolated from 5 g wet weight
of E. coli recovered from 750 ml of culture.
Protease inhibition assay
To investigate whether rSWAM1 and rSWAM2 are protease inhibitors, either elastase or cathepsin G was incubated with
rSWAM1, rSWAM2, or rSLPI (a positive control for the activity),
and the remaining protease activity was measured. We observed no
antiprotease activity in rSWAM1 or rSWAM2 (data not shown).
Proteins purified from the supernatant also gave negative results.
On the other hand, rSLPI inhibited both elastase and cathepsin G
with an IC50 (concentration that achieves 50% inhibition) of 300
nM, a value consistent with previous reports (37, 38). Because the
presence of His-tag or expression in E. coli may have inactivated
the proteins, we could not conclude absolutely that rSWAM1 and
rSWAM2 are not protease inhibitors, but it is unlikely, since
rSLPI, which was similarly designed and purified, still showed
activity.
Antibacterial activity of rSWAM1 and rSWAM2
To investigate the antibacterial activity of rSWAM1 and
rSWAM2, bacteria was incubated with rSWAM1, rSWAM2, lysozyme (positive control), or BSA (negative control) in MüllerHinton medium for 2 h, and the numbers of surviving cells were
FIGURE 2. Tissue-specific expression of SWAM1 and SWAM2. a,
Northern hybridization and RT-PCR. Numbers of PCR cycles are written
on the left. Positions of the specific bands are marked on the right.
Ethidium bromide staining (18S ribosomal RNA) shows the equal loading
and good quality of RNA. kB, kilobases. b, In situ hybridization. Cells
expressing SWAM1 or SWAM2 are stained red by Fast Red, and the cell
nuclei are stained purple by hematoxylin. In kidney, SWAM1-positive
cells are mainly found in proximal tubules. G, glomerulus; P, proximal
tubule; D, distal tubule. In epididymis, the smooth muscle cells that surround the epithelium of the ductus epididymis express SWAM1. Sm,
smooth muscle layer; S, spermatozoa; E, epithelium. In tongue, cells in the
lamina propria under the stratified squamous epithelium express SWAM2.
Lp, lamina propria; P, filiform papillus; Sq, stratified squamous epithelium;
M, striated muscle.
FIGURE 3. Induction of the SWAM1 expression in the pneumonic
lung. Induction of SWAM1 expression in the pneumonic lung established
by the intranasal administration of S. pneumoniae strain FP1284 in male
ICR mice. Total lung RNA (10 ␮g/lane) isolated 0, 10, 24, or 48 h after the
inoculation was loaded. Hybridized filters were exposed for 14 days.
Ethidium bromide staining (18S ribosomal RNA) shows the equal loading
of RNA.
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The tissue-specific expression of SWAM1 and SWAM2 was studied by Northern hybridization and RT-PCR (Fig. 2a). In Northern
hybridization, SWAM1 expression was detected in kidney and epididymis, SWAM2 expression was in tongue. RT-PCR confirmed
these results and also detected weak expression in several other tissues. In situ hybridization demonstrated which cells express each gene
(Fig. 2b). Like other antibacterial proteins (see http://www.bbcm.
univ.trieste.it/⬃tossi/antimic.html), SWAM1 and SWAM2 are tissuespecific genes with distinct expression patterns.
The expression of elafin and SLPI is induced in inflammation
(26, 33, 34). In the elafin gene, NF-␬B is responsible for the inducible expression (35). In the murine SLPI gene, a consensus
NF-␬B binding sequence is present 300 bp upstream of the transcription start site and may be responsible for the inducible expression (36). We studied SWAM1 and SWAM2 expression in
inflammation using a mouse pneumonia model. SWAM1 expression was induced after the intranasal administration of S. pneumoniae, while SWAM2 expression was not (Fig. 3). The NF-␬B
binding sequence is not found in the 5⬘-flanking sequence of the
SWAM1 gene (Fig. 1b). The mechanism of this inducible expression needs to be studied.
The Journal of Immunology
the amino acid sequences of SWAM1 and SWAM2 found
LOC149709 and huWAP2 (44), respectively. Highly conserved
amino acid sequences strongly suggest that they are human
SWAM1 and SWAM2 (Fig. 6a). LOC149709, huWAP2, elafin,
SLPI, HE4 (45), eppin (46), and four other ill-characterized WAP
motif genes form a WAP gene cluster in a 700-kb region on the
long arm of chromosome 20 (Fig. 6b). They may be descendents
of a common ancestral gene produced by gene duplication and thus
may share some biological functions, although WAP proteins have
a variety of functions, as stated above.
Because SLPI is one of the most-studied WAP motif proteins
and has an antiprotease activity, many of the WAP proteins have
been proposed to be antiproteases without any experimental evidence. The active center for the antiprotease activity of SLPI is
called the primary contact region (11, 47). Another WAP motif
protein with an antiprotease activity, elafin, has a homologous
amino acid sequence in the corresponding position (Fig. 7). On the
other hand, other WAP motif proteins in the cluster do not have
homology with this region and may not have antiprotease activity.
Our results suggest that they may have antibacterial activity. Positioning of cationic amino acids and hydrophobic amino acids in
a three-dimensional structure is considered important for this activity (5). Appropriate positioning makes the protein amphipathic,
helping it to intrude into the outer membrane of bacteria. Therefore, the prediction of antibacterial activity from amino acid sequences is difficult. The activity of the protein needs to be tested
individually.
We failed in our efforts to isolate the mouse elafin gene, and this
counted as CFU. As target bacteria, E. coli JCM 5491 (a Gramnegative rod), S. aureus subspecies aureus JCM2151 (a Grampositive coccus), and six clinical isolates (E. coli, three clones; S.
aureus, three clones) were used. Both rSWAM1 and rSWAM2
showed significant antibacterial activity on E. coli JCM 5491 and
S. aureus subspecies aureus JCM2151 (Fig. 5a). Comparable results were obtained from the clinical isolates (Fig. 5b). This indicates that rSWAM1 and rSWAM2 have antibacterial activity, and
that their IC90 (the concentration that achieves 90% reduction of
CFU) is ⬃10 ␮M for both strains. It has been reported that human
SLPI has an IC90 of 9 ␮M on E. coli ML-35p (8), and that elafin
has an IC90 of 25 ␮M on S. aureus (9). In addition, it is known that
many antibacterial proteins operate at micromolar concentrations
(5). The IC90 values obtained from rSWAM1 and rSWAM2 are
comparable with those of other antibacterial proteins.
Discussion
In this study we cloned two novel mouse WAP motif proteins,
SWAM1 and SWAM2. We investigated tissue-specific expression.
We found that SWAM1 expression is induced in inflammation of
the lung. Using recombinant proteins, SWAM1 and SWAM2 were
shown to have an antibacterial activity.
Except for the conserved cysteine residues, amino acids in the
WAP motif are quite divergent, and proteins with a WAP motif
have a variety of functions. Examples are WAP in milk that has an
unknown function (39), caltrin-like protein II that is an inhibitor of
calcium transport in the guinea pig (40), SPAI-1 that is a sodium/
potassium ATPase inhibitor in the pig (41), KAL1 that is the gene
responsible for the Kallman syndrome (42), and WDNM1 that has
been reported to be involved in cancer metastasis (43).
Blast search in the GenBank nr (nonredundant) database using
FIGURE 5. Antibacterial activity of rSWAM1 and rSWAM2. a, Effect
of different concentrations of rSWAM1 (䡺), rSWAM2 (E), lysozyme (f),
and BSA (F) on E. coli or S. aureus. Log-phase bacteria (5 ⫻ 104/ml) were
incubated with the indicated concentrations of protein in Müller-Hinton
medium at 37°C for 2 h, and the number of CFU was determined by plating
serial dilutions. CFU obtained by medium without sample protein was
normalized to 100%. Incubations were performed in triplicate. Significant
differences in the means compared with control (Müller-Hinton medium
only) are shown by # (p ⬍ 0.05) and ⴱ (p ⬍ 0.01). b, Effect of rSWAM1
(䡺), rSWAM2 (E), and BSA (F) on E. coli and S. aureus clinical isolates.
Experiments were performed as described in a. Results for each isolate
were shown by solid lines, dotted lines, or broken lines.
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FIGURE 4. Expression and purification of rSWAM1 and rSWAM2. a,
Schematic presentation of pET-SWAM1 and pET-SWAM2. DNA fragments encoding the mature SWAM1 or SWAM2 proteins were inserted
into pET-15b vector in-frame with a polyhistidine tag (f, His-tag) that
allows purification of the protein using the Ni-NTA agarose column. The
T7 RNA polymerase promoter (T7) expresses the downstream gene in E.
coli BL21(DE3)/pLysS in the presence of 1 mM isopropyl-␤-D-thiogalactoside. b, Purification of rSWAM1 and rSWAM2. Both proteins were purified from the insoluble fraction (the pellet) of the bacterial lysate. Bacterial lysates and purified proteins were run on a NuPAGE gel (Invitrogen).
Lane 1, Bacterial lysate; lane 2, purified protein. The calculated m.w. are
shown in parentheses.
1977
1978
MOUSE SWAM1 AND SWAM2 ARE ANTIBACTERIAL PROTEINS
may be because the mouse elafin gene does not exist. In the guinea
pig, the GP1G gene (a homologue of the human semenogelin II
gene) has been reported to superimpose on an elafin gene-like
sequence (48). The GP1G gene has a sequence homologous to the
human elafin gene in its intron 1, exon 2, and exon 3. As shown in
Fig. 6, the semenogelin genes are located next to the elafin gene. It is
possible that in rodents GP1G overrode the abutting elafin gene and
inactivated it. On the other hand, in porcine the elafin gene family has
undergone an accelerated evolution developing three copies of closely
related genes (49). These observations suggest that the elafin gene
may be under strong selective evolutionary pressure.
Multicellular organisms, such as plants, insect, or animals, produce a variety of antibacterial peptides to protect themselves from
attacks by microbes. Both mice and humans have developed a
number of antibacterial proteins such as ␤-defensins (50), LL-37
(51), histatins (52), LEAP-1 (53), and dermcidin (54), all of which
are considered to derive from different groups of genes. This diversity may reflect the need to develop a set of antibacterial peptides suitable for the living environment of each animal (5). In this
study we report two novel WAP motif proteins with antibacterial
activity. Our results suggest that the WAP motif may be an important skeletal unit in the formation of antibacterial protein in
mice and possibly in humans. The functions of many of the WAP
motif proteins remain untested. Studies of their roles in innate
immunity is warranted and may provide significant insight into the
development of immune system.
FIGURE 7. Comparison of the amino acid sequences of WAP motifs in the cluster. Two WAP motifs that have antiprotease activity (upper panel) and
those for which the antiprotease activity function has not been confirmed were aligned. Consensus cysteine and proline residues that characterize WAP motif
are shadowed. SLPI has two WAP motifs in the molecule, and that in the C terminus (SLPI-COOH) has an antiprotease activity (57), while that in the N
terminus has an antibacterial activity (8). HE4 and WFDC3 have two and three WAP motifs, respectively, and individual motifs were presented with a suffix
(⫺1, ⫺2, and ⫺3). The amino acid sequences of elafin and SLPI-COOH around the primary contact region (11, 47), including the scissile peptide bond
(10, 47), are well conserved, while those of other WAP motifs are different.
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FIGURE 6. WAP motif protein cluster on the long arm of human chromosome 20 (20q). a, Search for the human homologue of SWAM1 and SWAM2.
Two human genes, LOC149709 and huWAP2, showed high homologies with SWAM1 and SWAM2 and thus are considered their human homologues.
Identical amino acids are shown by bold type in gray boxes. Similar amino acids are shown in bold type in open boxes. b, A cluster of genes of the WAP
motif proteins on the long arm of human chromosome 20 (Chr20q). In a 700-kb region of Chr20q, the WAP motif proteins WAP1 (GenBank accession
no. XM086673), huWAP2 (NM080869; human SWAM2 homologue), elafin (Z18538), LOC149709 (XM086637; human SWAM1 homologue), SLPI
(AF114471), HE4 (X63187), eppin (NM020398), C20orf170 (XM029885), LOC164237 (XM092693), and WFDC3 (XM173052) cluster from the centromeric side to the telomeric side in this order (56). The locations of the genes of the WAP motif proteins are shown above the horizontal line, and the
locations of other genes are shown by filled boxes below the line. Directions of the centromere (Chr20 cen) and the telomere of the long arm of the
chromosome (Chr20q tel) are indicated. The distance from the telomere of the short arm is shown as kilobases (e.g., 43,500k ⫽ 43,500,000 bp). The
genomic region syntenic to what is depicted here is on chromosome 2 in mouse.
The Journal of Immunology
1979
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