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J. Biol. Chem., Vol. 278, Issue 31, 28540-28546, August 1, 2003
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Processing of Seminal Plasma hCAP-18 to ALL-38 by Gastricsin
A NOVEL MECHANISM OF GENERATING ANTIMICROBIAL PEPTIDES IN VAGINA*
From the
Granulocyte Research Laboratory, the
Department of Hematology, the ||Department of
Clinical Biochemistry, Fertility
Clinic, Copenhagen University Hospital, Rigshospitalet, DK-2100 Copenhagen,
Denmark, the ¶Department of Seafood Research,
Danish Institute for Fisheries Research, DK-2800 Lyngby, Denmark, the
**Department of Laboratory Medicine, Malmö
University Hospital, SE-205 02 Malmö, Sweden, and the
Department of Pulmonology, Leiden
University Medical Center, 2300 RC Leiden, The Netherlands
Received for publication, February 14, 2003 , and in revised form, May 16, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Antimicrobial peptides are important effector molecules of the innate immune system from insects to humans (1, 2). The peptides are active against a broad spectrum of Gram-positive and Gram-negative bacteria as well as some fungi and enveloped viruses. These peptides may play a role in the regulation of the normal microflora (3). In mammals, the antimicrobial peptides are of major importance for the antimicrobial efficacy of professional phagocytes such as neutrophils and macrophages, but the peptides are also expressed in epithelial cells (4). In mammals, there are two large families of antimicrobial peptides, defensins and cathelicidins. Defensins are divided into the
-defensins found in neutrophils,
macrophages, and Paneth cells in the small intestine, and the
-defensins, which are found widespread in epithelial cells.
Cathelicidins are found mainly in neutrophils
(5). Members of this protein
family share a highly conserved N terminus of 12 kDa, named cathelin after a
protein isolated from porcine neutrophils
(6). Antimicrobial peptides are synthesized as preproproteins and (with the exception of defensins in neutrophils) stored as inactive proproteins (7). In order to become biologically active, the peptides must be liberated from the proproteins by proteolytic cleavage.
The proteolytic generation of antimicrobial peptides is very important for
the clearance of bacteria at sites of infection. In mice, -defensins
from Paneth cells in the small intestine are generated by matrilysin-mediated
cleavage of prodefensins, and matrilysin knock-out mice have increased
susceptibility to intestinal infections
(8). Inhibition of activation
of the porcine neutrophil cathelicidins by elastase impairs clearance of
bacteria from wounds in vivo
(9).
The only human cathelicidin, hCAP-18, is a major protein of the specific granules of human neutrophils (10). It is processed to the antimicrobial peptide LL-37 by extracellular cleavage by proteinase 3 from azurophil granules following exocytosis (11). LL-37 has broad spectrum antimicrobial activity toward Gram-negative and Gram-positive bacteria (12, 13). Furthermore, LL-37 binds and neutralizes the effects of lipopolysaccharide (14) and is a chemoattractant toward neutrophils, monocytes, and T-lymphocytes (15).
hCAP-18 is also present in epithelial cells (i.e. in various squamous epithelia (16), in the lungs (17, 18), in sweat glands (19), in salivary glands (20), and in keratinocytes during inflammatory disorders (21)). The strongest epithelial expression of hCAP-18 is found in the epididymis with a subsequent high concentration in seminal plasma (22, 23).
How the epithelium-derived hCAP-18 is processed to generate an active antimicrobial peptide is not known. Because of the high concentration of hCAP-18 in seminal plasma, we chose this as a model to study the processing and antimicrobial activation of hCAP-18 expressed in epithelial cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials
Vaginal fluid and freshly ejaculated semen was collected from healthy volunteers at an outpatient fertility clinic. The semen was allowed to liquefy for 1 h at room temperature followed by centrifugation at 800 x g for 5 min, and the supernatant was collected. Both the seminal plasma and the vaginal fluid were then stored at –20 °C until further use. After thawing, the seminal plasma and vaginal fluid were used immediately for cleavage experiments. Semen was installed in the vagina of women undergoing insemination and collected after 4 h. Postcoital vaginal fluid was collected 10 h following sexual intercourse and stored at –20 °C until further analysis.
Polyclonal anti-hCAP-18 antibodies were previously described (24). A monoclonal antibody against LL-37 was generated by immunization of mice with glutaraldehyde-cross-linked synthetic LL-37 using conventional hybridoma technology.1 Polyclonal rabbit antibodies against progastricsin (pepsinogen C) were purchased from U.S. Biological (Swampscott, MA).
Synthetic LL-37 and ALL-38 were purchased from Schafer-N (Copenhagen, Denmark). The peptides were synthesized using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy, purified by reverse phase chromatography, and analyzed by HPLC coupled to a Shimadzu LCMS-QP8000 spectrometer. The purity of the peptides was greater than 95%. Gastricsin, purified as described, was generously provided by Pal B. Szecsi (25).
Cleavage Experiments
The pH of samples of seminal plasma was adjusted to pH 4 by the addition of
0.5 M sodium acetate and incubated at 37 °C for 6 h. Following
incubation, the pH was neutralized by the addition of Tris buffer, and the
samples were boiled in Laemmli sample buffer.
SDS-PAGE and Immunoblotting
SDS-PAGE (26) and
immunoblottting (27) were
performed with Mini-Protean 3 cells and Mini Trans-Blot Electrophoretic
Transfer cells according to the instructions given by the manufacturer
(Bio-Rad). For immunoblotting, the polyvinylidene difluoride
(PVDF)2 membranes
(Millipore Corp., Bedford, MA) were blocked for 1 h with 5% skimmed milk in
phosphate-buffered saline after the transfer of proteins from the 14%
SDS-polyacrylamide gels. For detection of hCAP-18 or fragments derived
thereof, the PVDF membranes were incubated overnight with primary antibodies.
The following day, the membranes were incubated for 2 h with horseradish
peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and
visualized by diaminobenzidine/metal concentrate and stable substrate buffer
(Pierce).
Purification and Identification of hCAP-18 Fragments Generated by
Cleavage in Seminal Plasma
Fragments of hCAP-18, generated by cleavage of hCAP-18 in seminal plasma at
pH 4, were affinity-purified on an anti-hCAP-18 antibody column.
To identify the cathelin part, the eluted material was dialyzed against 25
mM Tris (pH 8.5) and subjected to anion exchange chromatography on
a MonoQ column using Äkta-FPLC (Amersham Biosciences). Bound material was
eluted with a gradient of NaCl from 25 mM to 1 M in 50
mM Tris, pH 8.0. One peak containing proteins of 
14 kDa was
eluted at 0.2 M NaCl. The protein was repurified and desalted by
reverse phase HPLC employing a Vydac C4 column (2.1 x 150 mm)
equilibrated with 10% solvent B and eluted with a 1%/min gradient from solvent
A to solvent B (solvent A: 0.1% trifluoroacetic acid; solvent B: 0.1%
trifluoroacetic acid in acetonitrile). The purified protein was reduced and
derivatized with iodoacetamide, as described by Matsudaira
(28), followed by HPLC
purification as described above. The derivatized cathelin was dissolved in 50
µl of 50 mM sodium phosphate (pH 8.0) and digested with 0.5
µg of endoproteinase Asp-N (Roche Molecular Biochemicals) overnight at 37
°C. Then 50 µl of 5% formic acid was added, and the reaction mixture
was loaded onto a C18 ZipTip (Millipore), washed with 0.5% formic acid, and
eluted with 10 µl of 0.5% formic acid in 50% acetonitrile. An aliquot of 2
µl of the purified peptide mixture was introduced into a Q-Tof-2 tandem
mass spectrometer (Micromass, Manchester, UK) using the nanospray interface
and analyzed in MS mode as well as in MSMS mode for selected fragments to
confirm their structure.
Amino Acid Sequence Analysis
Amino acid sequence was analyzed on the PVDF-blotted protein in a 494 A
Procise Protein Sequencer (PerkinElmer Life Sciences) using the blot cartridge
and PVDF cycles. All reagents and solvents were supplied by PerkinElmer Life
Sciences.
Immunoprecipitation
Antibodies against gastricsin and normal rabbit immunoglobulins were
incubated with Protein A-Sepharose (Amersham Biosciences) for 30 min at room
temperature in phosphate-buffered saline (pH 7) with 0.5 M NaCl.
The Sepharose particles were subsequently washed seven times in
phosphate-buffered saline with 0.5 M NaCl to remove unbound
antibodies followed by incubation with seminal plasma at 4 °C for 4 h. 0.5
M NaCl were added to the seminal plasma before immunoprecipitation
to prevent nonspecific absorption to the Sepharose particles. The Sepharose
particles were pelleted by centrifugation. The supernatants were aspirated and
immediately used for cleavage experiments.
Purification of hCAP-18 from Seminal Plasma and Neutrophils for
Cleavage Experiments
Seminal plasma was applied to an affinity chromatography column with
anti-hCAP-18 antibodies immobilized on CNBr-activated Sepharose (Amersham
Biosciences) as described by the manufacturer. The column was washed
extensively, and the bound protein was eluted with 0.2 M glycine
HCl (pH 2.5).
hCAP-18 was purified from specific granules of human neutrophils by affinity chromatography and by cation exchange chromatography as previously described (11).
Antibacterial Activity of ALL-38 and LL-37
Growth Inhibition—The bacterial strains of Escherichia
coli (ATCC 25922 and strain 1655 (genome-sequenced K12 strain));
Staphylococcus aureus (ATCC 25923); Bacillus megaterium
(Bm11), and Pseudomonas aeruginosa PAO1 were tested for purity on
trypticase soy agar (Oxoid CM131) and inoculated in M9 salts
(29) with glucose and casamino
acids (M9GC) overnight at 37 °C. LL-37 and ALL-38 were dissolved in M9CG
at 100 µg/ml and sterile-filtered, and further 10-fold dilutions were
prepared in sterile M9CG. The peptide containing M9GC was pipetted into
96-microwell plates in volumes of 200 µl. All concentrations were tested in
duplicate. The plate included M9GC with no peptide as growth control and
noninoculated M9GC for check of sterile conditions. Experiments were conducted
with M9GC adjusted to pH 7 and M9GC adjusted to pH 5 (using HCl) to determine
whether increased antibacterial activity was observed at the lower pH.
The wells were inoculated with 20 µl of a 10-fold dilution of Bacillus and Staphylococcus and 20 µl of a 100-fold dilution of the two E. coli strains and of P. aeruginosa. This corresponded to the addition of 2 x 105 cfu to each well. Absorbance at 450 nm was read, and the plate was incubated at 37 °C. Absorbance was read after 1, 2, 3, 6, 8, and 10 days. At day 10, 10-fold dilution series were prepared from all wells with peptides where bacterial growth had not occurred (S. aureus and B. megaterium). Colony counts were made by surface plating on trypticase soy agar.
It has previously been noted that the composition of the growth medium is important when testing the antibacterial activity of LL-37 (12). Accordingly, we used a defined medium M9GC. In this medium, we repeatedly found a growth delay of S. aureus, which was not found in nutrient-rich media like brain heart infusion. This could be due to the autoinhibition, which has been described for other Gram-positive bacteria (30, 31).
Bactericidal Activity against Log Phase Cells—E. coli MG1655
was precultured in M9GC and inoculated at 100 cfu/ml in M9GC and
incubated at 37 °C with aeration. When A450 nm reached
0.1, the culture was diluted 1000-fold in medium and mixed (400-µl volume)
with sterile-filtered solutions of LL-37 and ALL-38 (125-µl volumes)
dissolved in distilled water. Concentrations of peptides tested were 0, 3, 6,
12, 25, 50, and 100 µg/ml. The bacteria-peptide mixtures were incubated at
37 °C, and samples were taken immediately after mixing for colony counts
and after 2, 6, and 24 h.
Bactericidal Activity against Stationary Phase Cells—S. aureus was cultured in brain heart infusion (Oxoid CM225), and E. coli was cultured in M9GC overnight at 37 °C. The cultures were harvested at 3,500 x g for 3 min and resuspended in either PIPES (pH 7; Sigma catalog no. P6757) or HOMOPIPES (pH 4). Absorbance at 450 nm was adjusted to 0.2. The bacterial suspensions were mixed (equal volumes) with peptide ALL-38, resulting in final concentrations of 50 or 5 or 0.5 µg/ml at both pH values. Stock solutions of peptide were prepared in either PIPES or HOMOPIPES. Bacterial levels were determined by surface plating 0, 3, and 24 h after mixing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cleavage of hCAP-18 in Seminal Plasma at Low pH— hCAP-18 is present in seminal plasma, appearing as a double band around 18 kDa as observed by immunoblotting (22). Incubation of seminal plasma at neutral pH for 6 h at 37 °C did not change the observed molecular forms of hCAP-18 (Fig. 1, A and B, lane a). However, following incubation at pH 4, bands were observed around 14 kDa together with a low molecular weight band at 5–6 kDa when immonoblotting was performed with polyclonal anti-hCAP-18 antibodies (Fig. 1A, lane b), but only the 5–6-kDa band was observed when blotting was done with the monoclonal anti-LL-37 antibody (Fig. 1B, lane b). Thus, cleavage of endogenous hCAP-18 in seminal plasma at low pH generates a fragment at the size of LL-37, which reacts with the monoclonal anti-LL-37 antibody. The bands at
14 kDa detected with the polyclonal
anti-hCAP-18 antibodies were not detected by the monoclonal anti-LL-37
antibody. These fragments represent the cathelin part of hCAP-18 as further
validated below. This corresponds to the cleavage of hCAP-18 found in
exocytosed material from neutrophils, where LL-37 is cleaved from the cathelin
part (11).
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The double bands of hCAP-18 observed previously in seminal plasma (22, 32) must be due to either posttranslational modifications or to proteolytic processing occurring during sampling of the ejaculate. To exclude an artifactual proteolysis occurring during sampling, endogenous hCAP-18 was removed, and the ability of seminal plasma to process purified hCAP-18 from neutrophil-specific granules was examined. Fig. 2 shows that purified hCAP-18 is processed to generate a low molecular weight band at low pH, which is recognized by the monoclonal anti-LL-37 antibody (Fig. 2B, lane b) and an additional single band of 14 kDa, which can be observed when immunoblotting is performed with the polyclonal anti-hCAP-18 antibodies (Fig. 2A, lane b). This argues that the endogenous epididymal-derived hCAP-18 and exogenous neutrophil-derived hCAP-18 are processed in a similar way in seminal plasma and that the double band appearance of epididymal-derived hCAP-18 is due to a possible posttranslational modification and not to proteolysis occurring during sampling.
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Identification of hCAP-18 Fragments Generated in Seminal Plasma at Low pH—To further identify the hCAP-18 fragments generated in seminal plasma at pH 4, these fragments were affinity-purified using an anti-hCAP-18 antibody column.
An aliquot of the eluate from the antibody column was concentrated by precipitation with trichloroacetic acid and was subjected to SDS-PAGE and blotted to a PVDF membrane. The low molecular mass fragment of 5–6 kDa was analyzed by N-terminal amino acid sequencing of the first 10 residues. These were identified as ALLGDFFRKS, consistent with the 38-amino acid peptide ALL-38.
To purify the peptides of 14 kDa seen by immunoblotting with polyclonal
anti-hCAP-18 antibodies, the remains of the eluate from the antibody column
were subjected to anion exchange chromatography. The 14-kDa peptides eluted at
0.2 M NaCl. As expected, these peptides were not recognized by
the monoclonal anti LL-37 antibody but only by the polyclonal anti-hCAP-18
antibodies (Fig. 1, A and
B, lane c). The N terminus of hCAP-18 (and of
the cathelin part) is blocked for protein sequence analysis
(33). In order to further
identify the purified 14-kDa peptides, fragments hereof were generated by
cleavage with endoproteinase Asp-N and identified by mass spectrometry. All of
the generated fragments corresponded to segments in the cathelin part of
hCAP-18 (Fig. 3). The most
C-terminal fragment identified was the fragment 98–102 DNKRF
(Table I), which is the
expected C terminus of the cathelin part of hCAP-18 if this is cleaved to
liberate ALL-38.
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The immunoblotting with the monoclonal anti-LL-37 antibody and the identity of purified fragments generated in seminal plasma demonstrated that hCAP-18 was cleaved between the cathelin part of the molecule and its C-terminal peptide ALL-38 in seminal plasma.
Antimicrobial Activity of LL-37 and ALL-38 —To verify the assumed antimicrobial activity of ALL-38, both synthetic LL-37 and ALL-38 were generated. The antimicrobial activity of LL-37 and ALL-38 was tested against strains of E. coli, P. aeruginosa, S. aureus, and B. megaterium (Fig. 4), where no difference was found between the two peptides. Both E. coli strains were inhibited by 100 µg/ml for 2 days, after which normal growth occurred. The growth of P. aeruginosa was not affected by the presence of the peptides (data not shown). The Gram-positive strains were more sensitive, being inhibited by 100 and 10 µg/ml for 10 days.
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, 100 µg/ml; 
, 10 µg/ml; 
, 1 µg/ml;
, 0.1 µg/ml; 
, 0 µg/ml (control). The absorbance was
measured at 450 nm at days 0, 2, 4, 6, and 10 to monitor bacterial growth.At day 10, 10-fold dilution series were made from samples with peptides where no growth had occurred (B. megaterium and S. aureus) (i.e. from wells where the absorbance remained as the sterile control). Colony counts were made by surface plating on trypticase soy agar. No growth was detected from wells inoculated with B. megaterium, whereas colonies were detected from S. aureus-containing wells where growth had not occurred. These findings indicate that the peptides had a bactericidal effect toward B. megaterium and a bacteriostatic effect toward the staphylococci. The same result was found for both LL-37 and ALL-38. The results also indicate that the peptides were stable over time, since the bacteriostatic effect was retained for more than a week.
Exponentially growing E. coli cells were then exposed to the peptides in different concentrations. Initial concentrations of cells were approximately 106 cfu/ml. Immediately after mixing, the peptides caused a reduction in culturable count of approximately 1 log unit (Table II). At the sampling after 2 h, both LL-37 and ALL-38 caused a dramatic reduction of viable cells to or below 104 cfu/ml at 12–100 µg/ml. After 6 h, cell densities in the three highest concentrations were approximately 102 to 103 cfu/ml. Bacterial densities remained low in the highest concentrations of the two peptides 24 h after mixing, whereas growth resumed in the lower concentrations. Again, the antibacterial activity of ALL-38 was equal to that of LL-37.
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Since no difference was seen between the two peptides, only ALL-38 was used to test bacteriolytic activity on nongrowing cell suspensions. No bacteriolytic activity was seen when cells suspended in buffers were exposed to the peptide, independent of pH (Table III). Overall, our findings indicate that the peptides primarily are active against growing bacterial cells.
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Identification of the Protease Responsible for Cleavage of hCAP-18 in Seminal Plasma—To identify the protease responsible for the generation of the antimicrobial peptide ALL-38, protease inhibitors were added to seminal plasma before incubation at pH 4. The cleavage of hCAP-18 was not inhibited by phenylmethylsulfonyl fluoride or aprotinin (general inhibitors of serine proteases), EDTA (inhibitor of metalloproteases), chymostatin (chymotrypsin-like protease inhibitor), or E-64 (cysteine protease inhibitor) (Fig. 5, lanes b–f). The cleavage of hCAP-18 was, however, totally abolished by pepstatin A (an aspartic protease inhibitor) (Fig. 5, lane g). Low pH activates the proform of the aspartic protease gastricsin (pepsin C) in seminal plasma (34), and the addition of the specific pepsin inhibitor, pepsinostreptin, totally abolished the cleavage of hCAP-18 (Fig. 5, lane h).
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To further validate that gastricsin was responsible for the cleavage of hCAP-18, gastricsin was immunoprecipitated from seminal plasma. Following precipitation with preimmune rabbit antibodies, cleavage was still observed at low pH (Fig. 6, lane b). However, following immunoprecipitation with antigastricsin antibodies, cleavage of hCAP-18 was abolished (Fig. 6, lane a). The antibodies used for immunoprecipitation of gastricsin reacted with purified gastricsin (data not shown).
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Cleavage Experiments with Purified Gastricsin—To further
verify the gastricsin-mediated cleavage of hCAP-18 in seminal plasma, hCAP-18,
purified from both neutrophils and seminal plasma, was incubated with
gastricsin at pH 4. Gastricsin cleaved hCAP-18 from both sources
(Fig. 7, A and
B, lanes b and d). The cleavage of
seminal plasma-derived hCAP-18 gave rise to two bands at 14 kDa, and the
neutrophil-derived hCAP-18 gave rise to one band at 14 kDa as visualized by
immunoblotting with polyclonal antibodies, in addition to the 5–6-kDa
band. This low molecular weight band and the unprocessed hCAP 18 were detected
by the monoclonal anti-LL-37 antibody, whereas the bands at 14 kDa were not.
When hCAP-18 was incubated with gastricsin at neutral pH, no cleavage was
observed (Fig. 7A,
lane e). The cleavage of hCAP-18 by gastricsin thus resembles that in
seminal plasma at low pH.
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In Vivo Processing of hCAP-18 following Sexual Intercourse—To validate that the processing of hCAP-18 takes place in vivo, samples of vaginal fluid, seminal plasma collected from vagina 4 h after installation, and a postcoital sample were analyzed. Very little hCAP-18 is present in normal vaginal fluid (Fig. 8, A and B, lane b). When samples of seminal plasma inseminated in vagina were collected after 4 h, the pH of the samples was neutral, and no processing of hCAP-18 had occurred (data not shown). A vaginal sample obtained 10 h after sexual intercourse had a pH value below 5. Immunoblotting of this with polyclonal anti-hCAP-18 antibodies demonstrated both a double band at 18 kDa corresponding to unprocessed hCAP-18 and 14 kDa corresponding to the cathelin part of processed hCAP-18 (Fig. 8, A and B, lane c). When immunoblotting was performed with the monoclonal anti-LL-37 antibody, only the double band at 18 kDa (Fig. 8B, lane c) was visualized, not the 14 kDa in agreement with the interpretation that this latter is the cathelin part of hCAP-18. The double bands at both 18 and 14 kDa observed in the postcoital vaginal sample, furthermore, demonstrate that the observed hCAP-18 is derived from seminal plasma, since processed hCAP-18 from neutrophils would only have one band at 18 kDa and one at 14 kDa. We were, however, not able to detect any band at 5–6 kDa in the postcoital sample by immunoblotting either with the monoclonal or with the polyclonal antibody.
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To rule out that the observed in vivo processing of hCAP-18 was due to a vaginal fluid-derived protease, vaginal fluid was then mixed with seminal plasma in a ratio of 10:1. Because of the buffer capacity of the seminal plasma, the pH remained neutral, and no cleavage of hCAP-18 was observed following prolonged incubation (Fig. 8, A and B, lane d). When the vaginal fluid was incubated with seminal plasma and the pH was adjusted to 4, cleavage was observed similar to the cleavage of hCAP-18 in seminal plasma at low pH (Fig. 8, A and B, lanes e and f). The cleavage of hCAP-18 in seminal plasma mixed with vaginal fluid at low pH was also inhibited by pepsinostreptin as in "pure" seminal plasma at low pH, demonstrating that also in the presence of vaginal fluid, hCAP-18 was processed by gastricsin at low pH (Fig. 8, A and B, lanes g and h). It is, furthermore, noteworthy that the mature ALL-38 was not degraded in the presence of vaginal fluid at low pH.
These findings are in accordance with previous in vivo studies of the activation of gastricsin in the vagina, where gastricsin was activated only several hours after either sexual intercourse or insemination in the vagina and paralleled a drop in the vaginal pH to acidic levels (35). The observed postcoital processing of hCAP-18 in vivo was therefore similar to that observed in seminal plasma at low pH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We have found that the promicrobicidal protein, hCAP-18, which is present in seminal plasma at high concentrations (22), is processed by gastricsin to generate the antimicrobial peptide ALL-38 at a low pH similar to the pH in the vagina. A similar processing was found in the vagina after sexual intercourse. Whereas we easily detected the holoprotein and the cathelin fragments of hCAP-18 in the postcoital sample, we were unable to detect the C-terminal antimicrobial domain. This is not likely due to proteolytic degradation of this peptide. Our data show that processing of hCAP-18 in vagina only takes place after several hours preceded by a drop in the pH. In the cleavage experiments with seminal plasma, where the pH instantaneously was adjusted to 4, we found no visible degradation of ALL-38 after 6 h although hCAP-18 in this setting was nearly completely processed. Furthermore, the proteolytic activity toward hCAP-18 was only found in seminal plasma, and the presence of vaginal fluid at low pH did not cause degradation of ALL-38. A likely explanation for our inability to recover the ALL-38 peptide could be that this very cationic and hydrophobic peptide binds to substances in the vagina (e.g. polyanions) that were not collected during the postcoital sampling.
It has previously been found that gastricsin degrades many seminal plasma proteins at low pH in vitro (36). This role of gastricsin was subsequently confirmed by in vivo studies (35), where it was demonstrated that the gastricsin in seminal plasma is activated at the low pH in the vagina 2–7 h postcoitus and that gastricsin activity is present more than 24 h thereafter (35). Thus, gastricsin-mediated cleavage of seminal plasma proteins is a well documented in vivo phenomenon. In contrast to seminal plasma, vaginal fluid has a very low protein content (37), and no protease activity has been detected.
Following incubation of seminal plasma at pH 4 for 6 h, most of the hCAP-18 was cleaved by gastricsin into two parts: the cathelin part and ALL-38. The cleavage site between ALL-38 and the cathelin part was found both by determination of the N terminus of the C-terminal antimicrobial peptide and of the C terminus of the cathelin part. Both corresponded to a cleavage between the phenylalanyl and the alanyl residue, whereas in exocytosed material from neutrophils, cleavage is observed between the alanyl and leucyl residue (38, 39).
Thus, in seminal plasma, the cleavage of hCAP-18 leads to generation of ALL-38, in contrast to neutrophils, where cleavage of hCAP-18 leads to generation of LL-37. Peptides comprising the 33, 35, 37, and 39 C-terminal residues of hCAP-18 all possess antimicrobial activity (38). The 37-amino acid peptide (LL-37) was found to be the most potent of these hCAP-18-derived antimicrobial peptides (38). The antimicrobial activity of ALL-38 has not previously been determined. We found it to be equal to that of LL-37. Both LL-37 and ALL-38 were inhibitory (bacteriostatic or bacteriocidal) when tested on actively growing cells, and a killing effect was exerted against Bacillus and E. coli. In contrast, no antimicrobial effect was found on nongrowing bacterial cells suspended in buffer.
Other human promicrobicidal proteins are cleaved to generate antimicrobial
peptides of different size (e.g. human -defensin 1 and human
-defensin-5). Human -defensin 1 has been purified from human
urine with different N-terminal truncations
(40). However, these peptides
were not equally antimicrobially active
(40), and how the different
forms of human -defensin 1 are generated from the proprotein is not
known. Human -defensin-5 has been isolated from ileal tissue in
different forms, all of which are presumably generated by the same enzyme,
trypsin (41).
The concentration of hCAP-18 in seminal plasma is around 85 µg/ml with a range between 40 and 140 µg/ml (22). This corresponds to a concentration of ALL-38 of 24 µg/ml with a range between 12 and 40 µg/ml. This is within the concentration range where we found antimicrobial activity of ALL-38.
Although processing of epithelium-derived hCAP-18 in seminal plasma and of neutrophil-derived hCAP-18 are mediated by different proteases, there are important similarities in these processes. In neutrophils, the expression and localization of hCAP-18 and the processing enzyme, proteinase 3, are separated although they are present in the same cells. Proteinase 3 is localized in azurophil granules (42), and hCAP-18 is localized in specific granules (10). hCAP-18 and proteinase 3 are localized together extracellularly following exocytosis, and only then is LL-37 generated (11). This strict control of generation of LL-37 is probably important because of the cytotoxic effects of this peptide (43). The hCAP-18 in seminal plasma is derived from epithelial cells in epididymis (22, 23), whereas gastricsin is derived from the prostatic gland and seminal vesicles (44). Thus, hCAP-18 and gastricsin only co-localize following ejaculation. Furthermore, significant processing requires activation of gastricsin by the low pH of vagina, which indicates that premature processing of the hCAP-18 stored in epididymis is prevented.
In summary, we found that the human cathelicidin, hCAP-18, is processed in seminal plasma to generate a novel antimicrobial peptide ALL-38 by gastricsin and that this process takes place following intercourse. The enzymatic activation of an antimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a hitherto undiscovered mechanism to prevent infection following sexual intercourse.
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FOOTNOTES |
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* This work was supported by grants from the Danish Medical Research Council, the A. P. Møller and Chastine Mærsk McKinney Møller Foundation, and the Copenhagen Hospital Corporation. 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.
To whom correspondence should be addressed: Host Defense Research Laboratory,
Dept. of Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte
Ave., 52-164 CHS, Los Angeles, CA 90095-1690. Tel.: 310-825-7499; Fax:
310-206-8766; E-mail:
sorensen{at}ucla.edu.
1 G. S. Tjabringa, J. Aarbiou, D. K. Ninaber, J.-W. Drijfhout, O. E.
Sørensen, N. Borregaard, K. F. Rabe, and P. S. Hiemstra, submitted for
publication.
2 The abbreviations used are: PVDF, polyvinylidene difluoride; PIPES,
piperazine-N,N'-bis-2-ethanesulfonic acid; HOMOPIPES,
homopiperazin-N,N'-bis-2-(ethansulfonsyre); HPLC, high pressure
liquid chromatography; MS, mass spectrometry; MSMS, tandem MS; cfu,
colony-forming units.
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ACKNOWLEDGMENTS |
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The expert technical assistance of Hanne Kidmose, Charlotte Horn, Jette Melchiorsen, and Allan Kastrup is greatly appreciated. We thank Charlotte Horn, Malene D. Bjerregaard, Lene Udby, and Mikkel Faurschou for critical review of the manuscript. We thank Jannie Jacobsen and the staff at the Department of Growth and Reproduction at Rigshospitalet for outstanding assistance providing samples of seminal plasma and Pal B. Szecsi for helpful and entertaining discussions.
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