The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hagiwara, K.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hagiwara, K.
Right arrow Articles by Nukiwa, T.
The Journal of Immunology, 2003, 170: 1973-1979.
Copyright © 2003 by The American Association of Immunologists

Mouse SWAM1 and SWAM2 Are Antibacterial Proteins Composed of a Single Whey Acidic Protein Motif1

Koichi Hagiwara2,*, Tohru Kikuchi*, Yoshiyuki Endo*, Huqun*, Kazuhiro Usui*, Mitsu Takahashi*, Naoko Shibata*, Takashi Kusakabe{dagger}, Hong Xin*, Sachiko Hoshi*, Makoto Miki*, Nozomu Inooka*, Yutaka Tokue* and Toshihiro Nukiwa*

* Department of Respiratory Oncology and Molecular Medicine, Division of Cancer Control, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, Japan; and {dagger} Department of Pathology, School of Medicine, Fukushima Medical University, Hikarigaoka, Fukushima, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µM. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The 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, 7, 8, 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, 14, 15) and prevents inflammation by protecting tissues against proteases released from neutrophils (16). Its indispensable role in wound healing has been shown using 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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'-AGACAAACCGGAGAAAACACATG-3' and 5'-GCACTTAATCTTTGGTTTCAGGATGG-3' for SWAM1 and 5'-GATATCTCATAAAAATGGCCTCAG-3' and 5'-TTTCTCAGAGTTCTGGAGGTTGG-3' for SWAM2. The nucleotide sequences of both genes were determined by the long distance sequencer method (21, 22, 23).

Plasmids

The entire open reading frames of both SWAM1 and SWAM2 were amplified by PCR using primers S11F (5'-CGAATTCGCTCACCTGCACAGTTTTCTTGGG-3') and S11B (5'-CGTCTAGAGGCTATCCTCGACTGAAACTG-3') for SWAM1 and S21F (5'-CGAATTCTCAGTCTCAGCCTCACAGCAAC-3') and S21B (5'-CGTCTAGACAGTCCTCTGAGGCCATTTTTATGAG-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'-CTCATATGCGACCTGAAATAAAGAAGAAGAACG-3') and S12B (5'-AGAGATCTCTATTCTGGGCTCTCCCATGGATCCAC-3') for SWAM1 and S22F (5'-CTCATATGGGTGGAGTCAAAGGCGAGGA-3') and S22B (5'-GAGGATCCTCACACCTTACTCTGAGGGATCTG-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'.



View larger version (33K):
[in this window]
[in a new window]
  		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 ({blacksquare}, 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-{beta}-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.

 
Northern blot analysis

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 x 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 µg) 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'-GCTCACCTGCACAGTTTTCTTGGG-3') and S13B (5'-TTCTGGGCTCTCCCATGGATCC-3') for SWAM1, and S23F (5'-TCAGTCTCAGCCTCACAGCAAC-3') and S23B (5'-CACCTTACTCTGAGGGATCTG-3') for SWAM2. Probes (50 ng) were labeled with 50 µCi of [{alpha}-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 2x SSC/1% SDS twice and with 0.2x 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.

RT-PCR

Total RNA (1 µg) 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 µl. The primers used for SWAM1 were S14F (5'-CGCATACGGAGGACAGTTCTG-3'; forward) and S14B (5'-GGCTATCCTCGACTGAAACTG-3'; backward), and those for SWAM2 were S24F (5'-TCAGTCTCAGCCTCACAGCAAC-3'; forward) and S24B (5'-CACCTTACTCTGAGGGATCTGTTC-3'; backward). Aliquots (10 µl) 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.

Expression and purification of rSWAM1 and rSWAM2

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-{beta}-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 µg/ml) and DNase I (5 µg/ml) were added, and the sample (lysate) was incubated on ice for 30 min. The lysate was ultracentrifuged at 500,000 x 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-Phe-pNA (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 x 107/ml. The bacterial suspension was diluted to 5 x 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, two-tailed 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 WAP motif of the human elafin as a query sequence gave us two novel EST clones. The 5' and 3' RACE reactions provided the full-length 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 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.



View larger version (61K):
[in this window]
[in a new window]
  		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 ({square}, 5' and 3' untranslated sequences; , signal sequences; {blacksquare}, 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. {blacksquare}, Signal sequences; {square} in which WAP is written, the WAP motifs; in which cem is written, cementoin domains characteristic of elafin (55 ).

 
Tissue-specific expression of SWAM1 and SWAM2 mRNAs and induction of SWAM1 mRNA in lung infected with Streptococcus pneumoniae

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 tissue-specific genes with distinct expression patterns.



View larger version (54K):
[in this window]
[in a new window]
  		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.

 
The expression of elafin and SLPI is induced in inflammation (26, 33, 34). In the elafin gene, NF-{kappa}B is responsible for the inducible expression (35). In the murine SLPI gene, a consensus NF-{kappa}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-{kappa}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.



View larger version (89K):
[in this window]
[in a new window]
  		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 µg/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.

 
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 Müller-Hinton medium for 2 h, and the numbers of surviving cells were counted as CFU. As target bacteria, E. coli JCM 5491 (a Gram-negative rod), S. aureus subspecies aureus JCM2151 (a Gram-positive 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 µM for both strains. It has been reported that human SLPI has an IC90 of 9 µM on E. coli ML-35p (8), and that elafin has an IC90 of 25 µM 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.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. Antibacterial activity of rSWAM1 and rSWAM2. a, Effect of different concentrations of rSWAM1 ({square}), rSWAM2 ({circ}), lysozyme ({blacksquare}), and BSA (•) on E. coli or S. aureus. Log-phase bacteria (5 x 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 * (p < 0.01). b, Effect of rSWAM1 ({square}), rSWAM2 ({circ}), and BSA (•) 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 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.



View larger version (34K):
[in this window]
[in a new window]
  		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.

 
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.



View larger version (34K):
[in this window]
[in a new window]
  		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.

 
We failed in our efforts to isolate the mouse elafin gene, and this 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 {beta}-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.


   Footnotes
 
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. Back

2 Address correspondence and reprint requests to Dr. Koichi Hagiwara, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: hagiwark{at}idac.tohoku.ac.jp Back

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. Back

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). Back

Received for publication March 20, 2002. Accepted for publication December 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mackey, I., F. S. Rosen. 2000. Innate immunity. N. Engl. J. Med. 343:338.[Free Full Text]
  2. Fraser, I. P., H. Koziel, A. Ezekowitz. 1998. The serum mannose-binding protein and the macrophage mannose receptor are pattern recognition molecules that link innate and adaptive immunity. Semin. Immunol. 10:363.[Medline]
  3. Medzhitov, A., P. Preston-Hurlburt, C. A. J. Janeway. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394.[Medline]
  4. Akira, S.. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2:675.[Medline]
  5. Zasloff, M.. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389.[Medline]
  6. Seemuller, U., M. Arnhold, H. Fritz, K. Wiedenmann, W. Machleidt, R. Heinzel, H. Appelhans, H. G. Gassen, F. Lottspeich. 1986. The acid-stable proteinase inhibitor of human mucous secretions (HUSI-I, antileukoprotease): complete amino acid sequence as revealed by protein and cDNA sequencing and structural homology to whey proteins and Red Sea turtle proteinase inhibitor. FEBS Lett. 199:43.[Medline]
  7. Wiedow, O., J.-M. Schroeder, H. Gregory, J. A. Young, E. Christophers. 1990. Elafin: an elastase-specific inhibitor of human skin: purification, characterization, and complete amino acid sequence. J. Biol. Chem. 265:14791.[Abstract/Free Full Text]
  8. Hiemstra, P. S., R. J. Maassen, J. Stolk, R. Heinzel-Wieland, G. J. Steffens, J. H. Dijkman. 1996. Antibacterial activity of antileukoprotease. Infect. Immun. 64:4520.[Abstract]
  9. Simpson, A. J., A. I. Maxwell, J. R. Govan, C. Haslett, J. M. Sallenave. 1999. Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens. FEBS Lett. 452:309.[Medline]
  10. Grutter, M. G., G. Fendrich, R. Huber, W. Bode. 1988. The 2.5 A x-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine {alpha}-chymotrypsin. EMBO J. 7:345.[Medline]
  11. Tsunemi, M., Y. Matsuura, S. Sakakibara, Y. Katsube. 1996. Crystal structure of an elastase-specific inhibitor elafin complexed with porcine pancreatic elastase determined at 1.9 A resolution. Biochemistry 35:11570.[Medline]
  12. Zitnik, R. J., J. Zhang, M. A. Kashem, T. Kohno, D. E. Lyons, C. D. Wright, E. Rosen, I. Goldberg, A. C. Hayday. 1997. The cloning and characterization of a murine secretory leukocyte protease inhibitor cDNA. Biochim. Biophys. Acta 232:687.
  13. Shugars, D. C.. 1999. Endogenous mucosal antiviral factors of the oral cavity. J. Infect. Dis. 179:(Suppl 3):S431.
  14. Wiedow, O., J. Harder, J. Bartels, V. Streit, E. Christophers. 1998. Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes. Biochim. Biophys. Acta 248:904.
  15. Tomee, J. F., P. S. Hiemstra, R. Heinzel-Wieland, H. F. Kauffman. 1997. Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi. J. Infect. Dis. 176:740.[Medline]
  16. Sallenave, J. M.. 2000. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease. Respir. Res. 1:87.[Medline]
  17. Ashcroft, G. S., K. Lei, W. Jin, G. Longenecker, A. B. Kulkarni, T. Greenwell-Wild, H. Hale-Donze, G. McGrady, X. Y. Song, S. M. Wahl. 2000. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nat. Med. 6:1147.[Medline]
  18. Wiedow, O., J. Luademann, B. Utecht. 1991. Elafin is a potent inhibitor of proteinase 3. Biochim. Biophys. Acta 174:6.
  19. Kuijpers, A. L., J. Schalkwijk, H. F. Rulo, J. J. Peperkamp, P. C. van de Kerkhof, E. M. de Jong. 1997. Extremely low levels of epidermal skin-derived antileucoproteinase/elafin in a patient with impetigo herpetiformis. Br. J. Dermatol. 137:123.[Medline]
  20. Simpson, A. J., W. A. Wallace, M. E. Marsden, J. R. Govan, D. J. Porteous, C. Haslett, J. M. Sallenave. 2001. Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection. J. Immunol. 167:1778.[Abstract/Free Full Text]
  21. Hagiwara, K., C. C. Harris. 1996. "Long distance sequencer" method; a novel strategy for large DNA sequencing projects. Nucleic Acids Res. 24:2460.[Free Full Text]
  22. Takenoshita, S., A. Mogi, M. Nagashima, K. Yang, K. Yagi, A. Hanyu, Y. Nagamachi, K. Miyazono, K. Hagiwara. 1998. Characterization of the MADH2/Smad2 gene, a human Mad homolog responsible for transforming growth factor-b and activin signal transduction pathway. Genomics 48:1.[Medline]
  23. Hagiwara, K., M. G. McMenamin, K. Miura, C. C. Harris. 1999. Mutational analysis of the p63/p73L/p51/p40/CUSP/KET gene in human cancer cell lines using intronic primers. Cancer Res. 59:4165.[Abstract/Free Full Text]
  24. Nielsen, H., E. Jacob, J. S. Brunak, G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engin. 10:1.[Abstract/Free Full Text]
  25. Chomczynski, P., N. Nacchi. 1987. Single-step method or RNA isolation by acid guanidium thiocyanate-phenol chloroform extraction. Anal. Biochem. 162:156.[Medline]
  26. Abe, T., Y. Tominaga, T. Kikuchi, A. Watanabe, K. Satoh, Y. Watanabe, T. Nukiwa. 1997. Bacterial pneumonia causes augmented expression of the secretory leukoprotease inhibitor gene in the murine lung. Am. J. Respir. Crit. Care Med. 156:1235.[Abstract/Free Full Text]
  27. Sambrook, J., D. W. Russell, J. Sambrook, D. Russel. 2001. Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  28. St. Croix, B., C. Rago, V. Velculescu, G. Traverso, K. E. Romans, E. Montgomery, A. Lal, G. J. Riggins, C. Lengauer, B. Vogelstein, et al 2000. Genes expressed in human tumor endothelium. Science 289:1197.[Abstract/Free Full Text]
  29. Kramps, J. A., C. H. van Twisk, A. C. van der Linden. 1983. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, a highly specific substrate for granulocyte elastase. Scand. J. Clin. Lab Invest. 43:427.[Medline]
  30. Nakajima, K., J. C. Powers, B. M. Ashe, M. Zimmerman. 1979. Mapping the extended substrate binding site of Cathepsin G and human leukocyte elastase: studies with peptide substrates related to the a1-protease inhibitor reactive site. J. Biol. Chem. 254:4027.[Free Full Text]
  31. Thompson, J. D., D. G. Higgins, T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673.[Abstract/Free Full Text]
  32. Henikoff, S., J. G. Henikoff. 1992. Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89:
  33. Jin, F., C. F. Nathan, D. Radzioch, A. Ding. 1998. Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor. Infect. Immun. 66:2447.[Abstract/Free Full Text]
  34. Sallenave, J. M., J. Shulmann, J. Crossley, M. Jordana, J. Gauldie. 1994. Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes. Am. J. Respir. Cell Mol. Biol. 11:733.[Abstract]
  35. Alkemade, J. A., H. O. Molhuizen, M. Ponec, J. A. Kempenaar, P. L. Zeeuwen, G. J. de Jongh, V.-W. I. van, P. E. van Erp, P. C. van de Kerkhof, J. Schalkwijk. 1994. SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J. Cell Sci. 107:2335.[Abstract]
  36. Kikuchi, T., T. Abe, K. Satoh, K. Narumi, T. Sakai, S. Abe, S. Shindoh, K. Matsushima, T. Nukiwa. 1997. Cis-acting region associated with lung cell-specific expression of the secretory leukoprotease inhibitor gene. Am. J. Respir. Cell. Mol. Biol. 17:361.[Abstract/Free Full Text]
  37. Renesto, P., M. Chignard. 1993. Enhancement of cathepsin G-induced platelet activation by leukocyte elastase: consequence for the neutrophil-mediated platelet activation. Blood 82:139.[Abstract/Free Full Text]
  38. Song, X., L. Zeng, W. Jin, J. Thompson, D. E. Mizel, K. Lei, R. C. Billinghurst, A. R. Poole, S. M. Wahl. 1999. Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis. J. Exp. Med. 190:535.[Abstract/Free Full Text]
  39. Campbell, S. M., J. M. Rosen, L. G. Hennighausen, U. Strech-Jurk, A. E. Sippel. 1984. Comparison of the whey acidic protein genes of the rat and mouse. Nucleic Acids Res. 12:8685.[Abstract/Free Full Text]
  40. Coronel, C. E., J. San Agustin, H. A. Lardy. 1990. Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig. J. Biol. Chem. 265:6854.[Abstract/Free Full Text]
  41. Araki, K., J. Kuroki, O. Ito, M. Kuwada, S. Tachibana. 1989. Novel peptide inhibitor (SPAI) of Na+, K+-ATPase from porcine intestine. Biochim. Biophys. Acta 164:496.
  42. Legouis, R., M. Cohen-Salmon, I. del Castillo, J. Levilliers, L. Capy, J. P. Mornow, C. Petit. 1993. Characterization of the chicken and quail homologues of the human gene responsible for the X-linked Kallmann syndrome. Genomics 17:516.[Medline]
  43. Dear, T. N., I. A. Ramshaw, R. F. Kefford. 1988. Differential expression of a novel gene, WDNM1, in nonmetastatic rat mammary adenocarcinoma cells. Cancer Res. 48:5203.[Abstract/Free Full Text]
  44. Lundwall, A., A. Clauss. 2002. Identification of a novel protease inhibitor gene that is expressed in the prostate. Biochem. Biophys. Res. Commun. 290:452.[Medline]
  45. Kirchhoff, C., I. Habben, R. Ivell, N. Krull. 1991. A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors. Biol. Reprod. 45:350.[Abstract]
  46. Richardson, R. T., P. Sivashanmugam, S. H. Hall, K. G. Hamil, P. A. Moore, S. M. Ruben, F. S. French, M. O’Rand. 2001. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 270:93.[Medline]
  47. Tsunemi, M., Y. Matsuura, S. Sakakibara, Y. Katsube. 1993. Crystallization of a complex between an elastase-specific inhibitor elafin and porcine pancreatic elastase. J. Mol. Biol. 232:310.[Medline]
  48. Hagstrom, J. E., M. P. Fautsch, M. Perdok, A. Vrabel, E. D. Wieben. 1996. Exons lost and found. Unusual evolution of a seminal vesicle transglutaminase substrate. J. Biol. Chem. 271:21114.[Abstract/Free Full Text]
  49. Tamechika, I., M. Itakura, Y. Saruta, M. Furukawa, A. Kato, S. Tachibana, S. Hirose. 1996. Accelerated evolution in inhibitor domains of porcine elafin family members. J. Biol. Chem. 271:7012.[Abstract/Free Full Text]
  50. Bensch, K. W., M. Raida, H. J. Magert, P. Schulz-Knappe, W. G. Forssmann. 1995. hBD-1: a novel {beta}-defensin from human plasma. FEBS Lett. 368:331.[Medline]
  51. Agerberth, B., H. Gunne, J. Odeberg, P. Kogner, H. G. Boman, G. H. Gudmundsson. 1995. FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc. Natl. Acad. Sci. USA 92:195.[Abstract/Free Full Text]
  52. Sabatini, L. M., E. A. Azen. 1989. Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (HIS1 and HIS2). Biochim. Biophys. Acta 160:495.
  53. Krause, A., S. Neitz, H. J. Magert, A. Schulz, W. G. Forssmann, P. Schulz-Knappe, K. Adermann. 2000. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett. 480:147.[Medline]
  54. Schittek, B., R. Hipfel, B. Sauer, J. Bauer, H. Kalbacher, S. Stevanovic, M. Schirle, K. Schroeder, N. Blin, F. Meier, et al 2001. Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat. Immunol. 2:1133.[Medline]
  55. Nara, K., S. Ito, T. Ito, Y. Suzuki, M. A. Ghoneim, S. Tachibana, S. Hirose. 1994. Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin.". J. Biochem. 115:441.[Abstract/Free Full Text]
  56. Deloukas, P., L. H. Matthews, J. Ashurst, J. Burton, J. G. Gilbert, M. Jones, G. Stavrides, J. P. Almeida, A. K. Babbage, C. L. Bagguley, et al 2001. The DNA sequence and comparative analysis of human chromosome 20. Nature 414:865.[Medline]
  57. Kramps, J. A., C. van Twisk, H. Appelhans, B. Meckelein, T. Nikiforov, J. H. Dijkman. 1990. Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain. Biochim. Biophys. Acta 1038:178.[Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
P Sipila, J Jalkanen, I T Huhtaniemi, and M Poutanen
Novel epididymal proteins as targets for the development of post-testicular male contraception
Reproduction, March 1, 2009; 137(3): 379 - 389.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Nishimura, H. Saiga, S. Sato, M. Okuyama, H. Kayama, H. Kuwata, S. Matsumoto, T. Nishida, Y. Sawa, S. Akira, et al.
Potent Antimycobacterial Activity of Mouse Secretory Leukocyte Protease Inhibitor
J. Immunol., March 15, 2008; 180(6): 4032 - 4039.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
A. Bellemare, N. Vernoux, D. Morisset, and Y. Bourbonnais
Human Pre-Elafin Inhibits a Pseudomonas aeruginosa-Secreted Peptidase and Prevents Its Proliferation in Complex Media
Antimicrob. Agents Chemother., February 1, 2008; 52(2): 483 - 490.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
B. Hurle, W. Swanson, NISC Comparative Sequencing Program, and E. D. Green
Comparative sequence analyses reveal rapid and divergent evolutionary changes of the WFDC locus in the primate lineage
Genome Res., March 1, 2007; 17(3): 276 - 286.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
S. Yenugu, R. T. Richardson, P. Sivashanmugam, Z. Wang, M. G. O'Rand, F. S. French, and S. H. Hall
Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif
Biol Reprod, November 1, 2004; 71(5): 1484 - 1490.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
Y. Furutani, A. Kato, R. Kawai, A. Fibriani, S. Kojima, and S. Hirose
Androgen-Dependent Expression, Gene Structure, and Molecular Evolution of Guinea Pig Caltrin II, a WAP-Motif Protein
Biol Reprod, November 1, 2004; 71(5): 1583 - 1590.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
T. I. Brown, R. Mistry, D. D. Collie, S. Tate, and J.-M. Sallenave
Trappin ovine molecule (TOM), the ovine ortholog of elafin, is an acute phase reactant in the lung
Physiol Genomics, September 16, 2004; 19(1): 11 - 21.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
N. Hsia and G. A. Cornwall
DNA Microarray Analysis of Region-Specific Gene Expression in the Mouse Epididymis
Biol Reprod, February 1, 2004; 70(2): 448 - 457.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. M. Torres, H. Y. Wong, M. Desai, S. Moochhala, P. W. Kuchel, and R. M. Kini
Identification of a Novel Family of Proteins in Snake Venoms: PURIFICATION AND STRUCTURAL CHARACTERIZATION OF NAWAPRIN FROM NAJA NIGRICOLLIS SNAKE VENOM
J. Biol. Chem., October 10, 2003; 278(41): 40097 - 40104.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hagiwara, K.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hagiwara, K.
Right arrow Articles by Nukiwa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS