1 Department of Laboratory Medicine, Divisions of Clinical Chemistry and 2 Medical Microbiology, Lund University, University Hospital MAS, SE- 205 02 Malmö, Sweden and 3 Granulocyte Research Laboratory, Department of Hematology, University Hospital, DK-2100 Copenhagen, Denmark
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Abstract |
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Key words: cathelicidin/hCAP-18/LL-37/prostasome/semen
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Introduction |
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We have recently shown that hCAP-18 is expressed in the male reproductive system (Malm et al., 2000). High levels were found in seminal plasma and hCAP-18 was also associated with sperm. High expression of the hCAP-18 gene was demonstrated in the epithelium of epididymis.
In this study, hCAP-18 was purified from seminal plasma and its identity confirmed by N-terminal amino acid sequencing. We also show that hCAP-18 appears in two major peaks after gel filtration of seminal plasma. hCAP-18 in the high molecular peak represents hCAP-18 bound to prostasomes, small membrane-bound vesicles derived from prostate epithelial cells.
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Materials and methods |
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Isolation of hCAP-18 from seminal plasma and N-terminal amino acid sequencing
An immunoglobulin fraction of rabbit anti-hCAP-18 antiserum (Sorensen et al., 1997b) was coupled to Affi 10 gel (BioRad, Hercules, CA, USA), 7 mg antibody/ml gel. A fresh sample of seminal plasma (4 ml) diluted 1:3 in equilibration buffer [50 mmol/l TrisHCl, 0.5 mol/l NaCl, 0.1% Triton-X 100 (v/v), pH 7.5] was applied to the affinity chromatography column. The bound protein was eluted by lowering the pH [0.1 mol/l glycine, 0.5 mol/l NaCl, 0.1% Triton-X 100 (v/v), pH 2.2]. The fractions were analysed by Western blotting using rabbit anti-hCAP-18 antibody. Fractions containing hCAP-18 were pooled and concentrated using a Centricon 3 (Millipore, Bedford, MS, USA) centrifugal filter. The protein present in the pooled and concentrated fractions was cleaved by pyroglutamate aminopeptidase (EC 3.4.19.3; Sigma, St Louis, MO, USA) to remove pyroglutamate before Edman degradation. Thirty units of the enzyme were incubated with 2.7 µg hCAP-18 in 100 mmol/l Na2HPO4, pH 8.0, 10 mmol/l EDTA, 5 mmol/l dithiothreitol (ICN Biomedicals Inc., Aurora, OH, USA) and 5% (v/v) glycerol for 16 h at 50°C. After cleavage, the sample was run on sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis (PAGE) (see below) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The 18 kDa doublet band was cut out and N-terminal amino acid sequencing performed (Procise 494 Protein Sequencer; Applied Biosystems).
Analysis for detection of glycosylation of hCAP-18
The purified hCAP-18 was investigated by using the DIG Glycan/Protein Double Labeling Kit (Biochemica Boehringer Mannheim, Mannheim, Germany). In brief, the purified hCAP-18 was subjected to electrophoresis on a SDSPAGE (Laemmli, 1970) and the protein was then transferred onto a nitrocellulose membrane (Towbin et al., 1979
). Staining of the blotted protein was performed according to the manufacturers instructions.
Gel filtration of seminal plasma and immunoblots
Seminal plasma was diluted in 1 volume of phosphate-buffered saline (PBS). A 200 µl sample was applied to a Superose 12 column (Pharmacia, Uppsala, Sweden). The gel filtration was run under non-denaturing conditions. Fractions of 0.5 ml were collected and analysed with an hCAP-18 ELISA (Sorensen et al., 1997b). To investigate a possible co-localization of prostasomes and hCAP18, an antibody against the prostasome marker dipeptidyl peptidase IV (CD26) (Schrimpf et al., 1999
) was used for immunoblotting (Dakopatts, Glostrup, Denmark). A total of 6 µl of each fraction was dotted onto PVDF membranes (Millipore). After blocking in skimmed milk [3% (w/v) in 10 mmol/l TrisHCl, pH 8.0, 150 mmol/l NaCl, 0.05% Tween 20, (TBST)] the membranes were washed and CD26 visualized using monoclonal mouse anti-human CD26 antibody (2 µg/ml; Dakopatts) and porcine anti-mouse IgG conjugated with alkaline phosphatase (Promega Corp., Madison, WI, USA) as a secondary antibody. Bound antibodies were visualized by nitrobluetetrazolium/5-bromo-4-chloro-3-indolylphosphate (Sigma) in developing buffer (100 mmol/l TrisHCl, 100 mmol/l NaCl, 5 mmol/l MgCl2, pH 9.5).
ELISA
A previously described (Sorensen et al., 1997b) sandwich ELISA was used to quantify hCAP-18 in the fractions after gel filtration.
Flow cytometry
Prostasomes were investigated for the presence of surface-associated hCAP-18 by flow cytometry (FACScan; Becton Dickinson, San José, CA, USA). The freshly prepared prostasomes were washed extensively with PBS (50 mmol/l, pH 7.5) containing 0.1% bovine serum albumin and thereafter incubated with the primary antibody. Rabbit anti-hCAP-18 antibody (0.2 µg) was used against an amount of prostasomes corresponding to 90 µl human semen. After repeated washings, bound hCAP-18 antibodies were detected by a secondary fluorescein isothiocyanate-labelled anti-rabbit IgG antibody (Dakopatts). The prostasomes were neither fixed nor permeabilized before analysis in the flow cytometer. As a negative control, the primary antibody was replaced with an irrelevant isotype-matched factor V antibody (kindly provided by Professor Björn Dahlbäck, Malmö, Sweden).
SDSPAGE and Western blot
SDSPAGE (Laemmli, 1970) and immunoblotting (Towbin et al., 1979
) were performed with BioRad systems according to the manufacturers instructions. Samples of 1.65x106 sperm, prostasomes corresponding to the amount in 16 µl semen and 5 µl of 1:10 diluted ultracentrifuged seminal plasma were analysed on a 15% SDSpolyacrylamide gel under reducing conditions. After protein transfer, the PVDF membranes (Millipore) were blocked for 30 min with 3% (w/v) skimmed milk in TBST. To detect hCAP-18, the membranes were incubated for 60 min with rabbit anti-human hCAP-18 antibody (2 µg/ml). After washing three times in TBST, incubation for 30 min with porcine anti-rabbit IgG conjugated with alkaline phosphatase (Promega) followed. The following washing steps and the visualization of bands on the membranes were performed in the same manner as described for the immunoblot experiment.
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Results |
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Discussion |
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Prostasomes are complex membrane-bound corpuscular organelles produced by prostate epithelial cells. These organelles are expelled with the prostate secretions at ejaculation and can be purified from seminal plasma (Ronquist et al., 1978a,b
). Prostasomes have a diameter of
150 nm (Ronquist et al., 1990
) and are claimed to have several functions. For example, prostasomes possess immunosuppressive capacity (Kelly et al., 1991
; Skibinski et al., 1992
) and after fusion with sperm (Arienti et al., 1997
), they enhance sperm motility (Stegmayr and Ronquist, 1982
). The biochemical background behind these effects is still unknown. Apart from hCAP-18, some other seminal plasma proteins have been found in association with prostasomes, e.g. tissue factor (Fernandez et al., 1997
), the complement-regulatory protein membrane cofactor protein (CD46) (Simpson and Holmes, 1994
; Kitamura et al., 1995
) and membrane attack complex inhibitory protein CD59 (Rooney et al., 1993
). The finding that hCAP-18 is associated with prostasome membranes is analogous to the finding that hCAP-18 is bound to the lipid part of lipoproteins in blood plasma (Sorensen et al., 1999
). Essentially all hCAP-18 in blood plasma is bound to lipoproteins. Prostasome-associated hCAP-18 accounts for
70% of all hCAP-18 in seminal plasma. The association of hCAP-18 with lipoproteins has been characterized as a non-covalent, hydrophobic interaction, with the cationic C-terminal end of LL-37 associated with the lipoprotein (Sorensen et al., 1999
). It is therefore reasonable to believe that hCAP-18 in seminal plasma is associated with the prostasome membrane in the same way. This is also supported by the fact that LL-37 interacts with lipid bilayers (Turner et al., 1998
).
The antimicrobial activity seen in prostasomes (Carlsson et al., 2000) could be due to hCAP-18, possibly in interaction with other antimicrobial peptides in seminal plasma such as the C terminal fragment of chromogranin B, secretolytin (Strub et al., 1995
; Stridsberg et al., 1996
). Expression of the CAMP gene and the large amount of hCAP-18 found in the epididymis (Malm et al., 2000
) indicate its importance, probably in local defence against potentially invasive pathogenic micro-organisms. hCAP-18 has also been described intracellularly in the acrosome of human sperm (Hammami-Hamza et al., 2001
), and this hCAP-18 is assumed to originate from the epithelial cells of testis (Hammami-Hamza et al., 2001
). It is, however, possible that hCAP-18 attached to sperm (Malm et al., 2000
) originates from prostasomes which have fused with sperm. The binding of hCAP-18 to prostasomes instead of directly to sperm could be a way of protecting sperm cells from the cytotoxic effects of LL-37. LL-37 could be cytotoxically active against prokaryotes and not eukaryotic cells due to the absence of cholesterol in the plasma membrane of prokaryotes. The plasma membrane of sperm possesses only a low cholesterol content (Mack et al., 1986
) compared with prostasome membranes (Arvidson et al., 1989
). When prostasomes, with associated hCAP-18, are fused with sperm, the sperm membranes will be enriched in cholesterol and the sperm could subsequently become resistant to the toxicity of LL-37.
Several studies have shown that human semen has antimicrobial activity. The antimicrobial capacity has been ascribed to different components of semen, for example lysozyme (Mardh and Colleen, 1974), spermine (Williams-Ashman and Lockwood, 1970
) and the high zinc concentration (Mardh and Colleen, 1975
). A possible reason for the relatively weak antimicrobial effect of seminal fluid is that hCAP-18 is present predominantly in its inactive proform with the C-terminal part, LL-37, still bound to the cathelin region. Activation may take place in the uterus. About 45 min after the sperm have entered the uterus, there is an extensive invasion of granulocytes from the female. These granulocytes release high concentrations of proteases, such as elastase and proteinase 3, which kill most of the sperm cells (Phillips and Mahler, 1977
). Both elastase (data not shown) and proteinase 3 (Sorensen et al., 2001
) have been shown to activate hCAP-18. The neutrophil granulocytes also contribute LL-37, since hCAP-18 is released from its specific granules in parallel with the release of proteinase 3 from the azurophilic granules (Sorensen et al., 2001
). The physiological importance of semen-mediated and prostasome-mediated antimicrobial effects is easily envisaged. Both Gram-positive and Gram-negative bacteria and also some fungal species are found in the environment outside the cervix. We suggest that seminal plasma hCAP-18 is important for the protection of sperm cells in this unfriendly milieu. Free, uncomplexed hCAP-18 does not pass the cervix barrier and consequently does not enter the uterus. In contrast, hCAP-18 bound to sperm, probably transferred from prostasomes, may have its main function in the uterus or at conception in the oviduct. hCAP-18 may thus contribute to the sterile environment during fertilization.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 1, 2002; resubmitted on April 19, 2002; accepted on May 9, 2002.