Antimicrobial Peptide

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BIOLOGY OF REPRODUCTION 67, 804–813 (2002)DOI 10.1095/biolreprod.102.004564

Novel Antimicrobial Peptide of Human Epididymal Duct Origin1

Hans Henning von Horsten, Petra Derr, and Christiane Kirchhoff 2

IHF Institute for Hormone and Fertility Research at the University of Hamburg, D-22529 Hamburg, Germany

ABSTRACTHE2, a gene expressed specifically in human epididymis,

gives rise to multiple mRNAs that encode a group of small cat-ionic secretory peptides. Localization of HE2 within the defensingene cluster and prediction that -defensin-like modules existsuggest that these peptides have antimicrobial activity and rep-resent components of the innate epithelial defense system of theepididymal duct. Reverse transcription-polymerase chain reac-tion analysis confirmed the occurrence of eight human HE2-derived transcripts, including minor mRNA variants, that hadpreviously been shown only in animal species. Employing iso-form-specific antibodies against the predicted HE2 products,multiple 4- to 8-kDa peptides were detected in human epidid-ymal epithelium, epididymal fluid, and ejaculate. N-terminal mi-crosequencing has suggested a proteolytic processing of these

peptides by a furin-like proprotein convertase, which cleaves apropiece from the longer precursor peptides. HE2 and HE21,representing major peptide isoforms in the human epididymis,were recombinantly expressed, and their susceptibility to furincleavage was demonstrated in vitro and in vivo. Processed re-combinant peptides and chemosynthetic fragments were includ-ed in antimicrobial tests. In addition to the -defensin-likeHE21 with its expected antibacterial function, HE2 C-termi-nal fragments showed antibacterial activity against Escherichia

coli , although it showed no significant similarity to -defensinsnor to any other known protein family.

epididymis, gene regulation, sperm

INTRODUCTION

Infections of the genitourinary tract are a major cause of morbidity and may contribute to infertility, but possible de-fense mechanisms are largely unknown. Because the lu-minal contents of the testis and epididymis are isolatedfrom adaptive immune responses for obvious reasons (fora review see [1]), it seems compelling that their epitheliahave developed alternative mechanisms to fight ascendinginfections, including local production of antimicrobial pep-tides and proteins. Among the most potent innate immuneeffectors of mammalian epithelia are the -defensins, smallcationic peptides with six cysteines [2]. HE2, a gene ex-pressed specifically in human epididymis [3], is locatedwithin the human defensin gene cluster and predicts -de-fensin-like modules [4], suggesting that it may be part of

the epididymal epithelial defense system. A gene product

1Supported by the German Research Association (DFG contracts Ki 317/5and Ki 317/8) and by a graduate stipend from the Ernst Schering ResearchFoundation to H.H.vH. Results presented in this publication are part of thedoctoral thesis of H.H.vH.2Correspondence: Christiane Kirchhoff, IHF Institute for Hormone and Fer-tility Research, Grandweg 64, D-22529 Hamburg, Germany.FAX: 49 40 56190864; e-mail: [email protected]

Received: 14 February 2002.First decision: 11 March 2002.Accepted: 4 April 2002. 2002 by the Society for the Study of Reproduction, Inc.ISSN: 0006-3363. http://www.biolreprod.org

of the rat epididymal duct, bin1b, has recently been cloned[5] and has been suggested to represent the rat counterpartof the human HE2 gene. Bin1b has a predicted peptide withthe structural characteristics of -defensins. The highly re-stricted expression pattern of the mRNA, which was foundonly in the proximal parts of the rat epididymis, led to theassumption that the encoded peptide was effective againstinvasive pathogenic microorganisms while simultaneouslypreserving spermatozoa from cytotoxic damage during ep-ididymal transport, maturation, and storage [5].

HE2 transcripts had originally been cloned from a hu-man epididymal cDNA library by differential screening [3].The cDNA clones obtained were derived from abundantepithelial transcripts and showed a highly restricted hybrid-ization pattern at levels detectable by Northern blot, sug-gesting that HE2 was produced in large amounts only bythe proximal parts of the human epididymal duct epithelium[3, 6]. It is now obvious that the initially identified HE2mRNA is a member of an entire family of human mRNAvariants [7] that result from alternative mRNA splicing.Differential expression patterns of HE2 variants have beendescribed and suggest that the encoded peptides may exerttheir effects consecutively during epididymal transit [7].Part of the HE2 mRNA variants have predicted cationicpeptides with a pattern of six cysteine residues near theirC-termini [7, 8], which is a hallmark of the -defensin fam-ily of antimicrobial peptides [2]. Moreover, the human ge-nome project localized the HE2 gene on the short arm of chromosome 8, directly adjacent to the -defensin genes

for hBD-2 and hBD-3 (segment 8p22-p23; [4, 9]). The pre-diction of the existence of -defensin-like modules andtheir location within the human defensin gene cluster sug-gest that HE2 might encode novel antimicrobial peptidesof the human male genital tract that are suitable for fightingspecific genital tract infections.

However, very little is known about the production andsecretion of the predicted human peptides. A detailed char-acterization at the peptide level has been hampered becauseHE2 expression is largely restricted to the proximal humanepididymis. The antisera employed previously [3, 7] werenot well suited to detect all major human peptide isoforms.Thus, proof of the existence of the HE2 peptide still doesnot exist. Also, any direct functional evidence of the en-

coded peptides is lacking. Indeed, despite the prediction of -defensin-like modules in rat bin1b and a number of HE2-derived peptides, an antimicrobial activity of these peptideshas not been shown. Moreover, the HE2 sequence is en-tirely different from the defensins and from any otherknown peptide, and a function cannot be inferred. To char-acterize the HE2-derived peptides whose origin is the hu-man epididymal duct, isoform-specific antibodies wereraised and human epididymal tissue, luminal fluid, andejaculates were analyzed by immunohistochemistry, West-ern blotting, immunoprecipitation, and peptide microse-quencing. Chemosynthetic and recombinantly expressedHE2-derived peptides were included in tests of antimicro-bial activity.



80ANTIMICROBIAL PEPTIDE

MATERIALS AND METHODS

Epididymal Tissues, Fluids, and Ejaculates

Human testes and epididymides were obtained from local hospitalswhere patients were undergoing orchidectomy for prostatic carcinoma(courtesy of Drs. Ching-Hei Yeung and Trevor G. Cooper, University of Munster). Two patients had received long-term treatment with GnRH andantiandrogen before surgery. Informed consent was obtained from all pa-tients and, in all cases, investigations were conducted in accordance withthe guidelines of the Helsinki Declaration, as revised in 1983. Epididymal

fluids were collected by cannulation and retrograde perfusion of two ad-ditional organs as described elsewhere [10]. Fluid samples were dispensedto aliquots before storage. Permission was sought from the HansestadtEthics Committee, in Hamburg, Germany, in order to perform studies in-volving human ejaculates from young, healthy volunteers. Rat tissues wereobtained from freshly killed laboratory animals. All tissue and fluid sam-ples were shock-frozen in liquid nitrogen and stored at 80C.

Preparation of Human Sperm Membrane Proteins

A salt wash and 0.3 M lithium 3,5-diiodosalicylate (LIS) extracts of sperm proteins were prepared from liquefied human ejaculates. Seminalplasma was removed by centrifugation at 500 g for 30 min, and spermpellets were resuspended. LIS extracts were prepared as described else-where [11]. To obtain salt wash proteins, sperm pellets were resuspendedin a cold 0.6 M NaCl solution containing 10 mM PMSF. After 10 min of incubation on ice, cold ethanol was added to give a final concentration of

60% (v/v) ethanol. After 10 min on ice, suspensions were centrifuged for10 min at 4000 g. Supernatants were precipitated with four volumes of acetone at 20C overnight. Precipitated proteins were harvested by cen-trifugation for 20 min at 4000 g at 4C. Supernatants were decantedand pellets were dried under vacuum. Protein pellets were resuspended indeionized water, and insoluble proteins were separated by centrifugation.The soluble fraction was employed in SDS-PAGE.

Preparation of Seminal Plasma Proteins

Thirty-five milliliters of liquefied human seminal plasma were pouredinto 200 ml of acetone and stirred at room temperature. To acceleratesuspension formation, up to 5% of n-tributylphosphate was added to themixture. Suspensions were centrifuged at 4000 g for 10 min, and theproteinaceous pellets were dried under vacuum. The powder was resus-pended in sterile deionized water containing a protease inhibitor cocktail(Complete, Boehringer-Roche, Mannheim, Germany). Samples were re-centrifuged at 27 000 g for 30 min, the supernatants were dispensed in500-l aliquots and lyophilized, and the dried samples were stored at80C.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction

RNA from human and rat tissues was extracted into 15–20 volumesof chaotropic solution as described elsewhere [12]. Oligo(dT)-primedcDNA was synthesized from 2–5 g of total RNA according to theGeneScript protocol (Genecraft, Munster, Germany) in the presence of 0.5M betaine (Sigma, Deisenhofen, Germany). Sequences of oligonucleotideprimers were 5-AGACATGAGGCAACGATTGCTCC-3 (the forwardprimer common to HE2, HE21, and HE2C), 5-GGCAGGGAGGTT-CAACGGAC-3 (the forward primer specific to HE2B and HE2E), 5-GGGATCAGAGCAAATGTCACGC-3 (the reverse primer common to

HE2, HE21, HE2B, and HE2E), and 5-CATCAGTTTTAATGTAAA-CAGCAGGCGTC-3 (the reverse primer specific to HE2C as describedby Frohlich et al. [8]). Reactions were carried out using 1 unit of Bio-therme Taq polymerase (Genecraft) per reaction in a touchdown poly-merase chain reaction (PCR) program that dropped from 61C to an an-nealing temperature of 51C. Amplicons were separated and visualized on1% Tris-acetate/EDTA agarose/ethidium bromide gels. Identity of insertswas confirmed by subcloning them into the pGEM-T-Easy plasmid vector(Promega, Mannheim, Germany) and plasmid sequencing by standard pro-cedures.

Chemosynthetic Peptides

Peptides were synthesized according to the amino acid sequences de-duced from the HE2 cDNAs (GenBank accession numbers X67697,AF168616, AF170797, AF168617, AF168618, AF168619, and

AF168620) by standard f-moc solid-phase procedures (courtesy of DMarkus Koppitz, Schering AG, Berlin, Germany). Sequences were as folows: P1, H2N-GELRERAPGQGTNGC-COOH; P2, H2N-KRDLLPPTPPYQVC-COOH; P3, H2N-ISHREARGPSFRICVDFLGPRWARGCSGN-COOH (representing the amino acid sequence of HE22 ([3GenBank accession number X67697); synHE2cycl with a disulfide bondH2N-VISHREARGPSFRICVDFLGPRWARGCSTGN-COOH; P4, H2NCVSNTDEEGKEKPEM-COOH; and P5, H2N-GDVPLGIRNTIC-COOHAll peptides eluted as single peaks upon reverse-phase high-performancliquid chromatography (HPLC). Mass spectroscopy of peptides was kindlperformed by Dr. Jurgen Harder, University of Kiel. Electrospray ionization-mass spectrometry (ESI-MS) analyses (Q-Tof2; Micromass, AlmerNetherlands) revealed a mass of 3346.88 Da for the P3 peptide (3346.7Da calculated for linear synHE2) and 3444.00 Da for the synHE2cy

peptide (the calculated mass of the reduced peptide is 3445.93 Da), whicconfirmed the presence of a disulfide bond.

Antipeptide Antisera

Peptides P1 and P2 were synthesized to contain an artificial C-terminacysteine for subsequent coupling to keyhole-limpet-hemocyanine (KLHSigma, Deisenhofen, Germany) as a carrier; P4 and P5 peptides containean endogenous C-terminal cysteine for coupling. The N-terminal isoleucine of P3 was used for coupling to KLH. Rabbits were immunized witan s.c. injection, and immune sera were obtained after 60, 90, and 12days (Pineda-Antikorper-Service, Berlin, Germany). Monospecific purification of polyclonal antibodies was performed by affinity chromatographafter peptide coupling to epoxy-activated sepharose 4B (Amersham-Phamacia-Biotech, Freiburg, Germany).

Immunohistochemistry

Cryosections of human epididymides were postfixed in 4% parafomaldehyde and used in immunohistochemistry as described elsewher[12]. Immunolocalization of HE2 peptides was achieved by indirect Cyimmunofluorescence employing P3 and P4 antisera as first antibodies anCy2-conjugated anti-rabbit antibodies (1:100; Jackson ImmunoResearcLaboratories, West Grove, PA) as second antibodies. Tissue sections werinvestigated using an epifluorescence microscope (Nikon, Japan) equippewith a 450–490 nm excitation wavelength filter (Omega Optics, Brattleboro, VT).

Immunoprecipitation

All steps were carried out at 4C. Protein samples were titrated to pH7. Twenty microliters of normal rabbit serum or preimmune serum weradded and the samples were incubated for 1 h on a rotating wheel. Thirtmicroliters of water-equilibrated protein A-agarose slurry (BoehringeMannheim) were added, taking care not to compress the beads, and thtubes were again incubated on the rotating wheel for 1 h, followed by brief centrifugation at 7000 g in an Eppendorf centrifuge. Supernatanwere removed into fresh tubes and recentrifuged for 5 min at 14 000 gSeventy microliters of affinity-purified rabbit antibodies were added anthe samples were incubated for 1 h on the rotating wheel. Fifty microliterof equilibrated protein A-agarose were then added, followed by overnighincubation on the rotating wheel. Samples were centrifuged for 30 sec a7000 g, and the supernatants were discarded. Pellets were washed wit1 ml of radioimmunoprecipitation assay buffer (150 mM NaCl, 1.0% v/NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris pH 8.0supplemented with a proteinase inhibitor cocktail (Complete; BoehringeMannheim). Tubes were centrifuged intermittently for 30 sec at 7000

g. Washing was repeated three times. After the final centrifugation stepsupernatants were removed and 30 l of 1 loading buffer (see belowwas added to the pellets. Before electrophoresis (see below) samples werdenatured for 2 min at 97C.

Western Blot Analysis and N-Terminal Peptide Sequencing

Samples were separated on 15% Laemmli gels or on 16.5% Tris-Trcine gels containing 6 M urea [13] and transferred to polyvinylidene dfluoride membranes (Millipore, Eschborn, Germany) in a continuous buffer system us ing a semidry blott er (Phase, Lubeck, Germany). Moleculweight standards employed in Laemmli gels were Rainbow low-range molecular weight markers (Amersham-Pharmacia); in Tris-Tricine gels, molecular weight standards (BioRad, Hercules, CA) were used. The proteitransfer procedure was modified as described in [14] to efficiently bl



806 VON HORSTEN ET AL.

cationic peptide isoforms. Immunodetection of proteins was carried out bystandard procedures as described in [15], employing the CL-HRP substratesystem (Pierce Chemical Company, Rockford, IL) at a dilution of 1:10and exposure to x-ray-film (Kodak, Rochester, NY). Specificity of anti-body binding was confirmed by comparing it with the correspondingpreimmune serum and by competition with the peptide as described in[10]. Corresponding peptide bands were out from Coomassie-stained par-allel blots and subjected to N-terminal peptide sequencing (TopLab-GmbH, Munchen, Germany).

Recombinant Protein Expression in Escherichia coli

An HE21 cDNA fragment [3] lacking the intrinsic signal peptidecoding region was subcloned into the pMAL-c2x vector (New EnglandBiolabs, Schwalbach/Ts, Germany) after addition by PCR amplification of a 3- HindIII restriction site. Oligonucleotide primers were as follows:sense oligomer, 5-TCTCAAGCCAGACATGTG-3 ; antisense oligomer,5-TACTAAAGCTTCTAATTCCCAGTGGAAC-3. 3- HindIII-digested5-blunt-end fragments were ligated into the XmnI/ HindIII-digested vector.The recombinant plasmid was amplified in E. coli DH5 (Life Technol-ogies, Rockville, MD) and verified by sequencing. The cytosolic protease-deficient E. coli strain ER2508 (New England Biolabs) was transformedand induced for protein expression according to suggestions of the supplier(New England Biolabs). Bacteria were harvested by centrifugation and thecells were lysed. Supernatants were diluted and loaded onto an amyloseresin column by gravity flow. The column was washed and the recombi-

nant protein was competitively eluted. Elutes containing recombinantHE21-maltose-binding protein (MBP) fusion were dialyzed against de-ionized water and lyophilized.

Recombinant Protein Expression in Insect Cells

Complementary DNA fragments without intrinsic signal peptide-cod-ing regions were subcloned into the pMelBacB transfer vector (Invitrogen,Leek, Netherlands). BamHI and HindIII restriction sites were added byPCR amplification for insertion. The sense oligomer for the HE21 variantwas 5-CAACGGATCCAGACATGAGGCAACGATTGCTC-3; the anti-sense oligomer was 5-CAACAAGCTTAGATCCCAGATCTGCCATCC-3. Recombinant virions were selected by plaque assay and propagated inSf9 cells. Virions were used to infect High Five insect cells at a multi-plicity of three plaque-forming units per cell, and recombinant peptideswere recovered from the culture medium after 96 h of infection. Super-natants containing the HE21 peptides were adsorbed to an Oasis MAX

mixed-mode anion exchange column (Waters-GmbH, Eschborn, Germa-ny). Fifty microliters of culture supernatant were loaded onto a methanol-conditioned 500 mg Oasis MAX column. The column was washed with5 ml of 5% methanol in 20 mM Tris-HCl pH 8.0, followed by 5 ml of 100% methanol. Seven milliliters of 50% (v/v) 100 mM acetic acid/ace-tone were used to elute the recombinant peptide. Elutes were heated to56C for 30 min to remove excess acetone and lyophilized.

Protease Digestion of Recombinant HE21

Recombinant MBP-HE21 from E. coli was digested using 1.5 unitsof recombinant truncated human furin (New England Biolabs) per micro-gram of fusion protein in 100 mM Hepes pH 7.5 at 25C, 0.5% Triton-X-100, 2 mM CaCl2, and 1 mM -mercaptoethanol reaction buffer for 6 hat 30C. For the double digest with furin and Factor Xa (New EnglandBiolabs), the digest mix was chilled and incubated overnight at room tem-perature using the same activity of recombinant furin as above in combi-

nation with 1 g of factor Xa per 50 g of MBP-HE21. The digestswere extracted with StrataClean resin (Stratagene) [16] in order to obtainsalt-free samples for subsequent Western blotting.

In Vitro Antibacterial Assays

Gel overlay assays were performed essentially as described in [17, 18]. E. coli DH5 were grown to log-phase and harvested by centrifugation at4000 g for 3 min. The pellet was washed once in 10 mM sodiumphosphate buffer pH 7.2 and resuspended as described. A volume con-taining 4 106 bacterial colony forming units (CFUs) was added to 10ml of 42C sodium phosphate buffer that contained 1% low electroendos-mosis, low sulfate agarose (Metaphor; Biozym, Hamburg, Germany). Bac-teria-containing agar was poured to a depth of 2 mm into a Petri dish.Continuous acid urea (CAU)-PAGE of protein samples to be tested forantimicrobial activity was performed as described by Lehrer et al. [18].

Parallel control gels were blotted and analyzed as described [19]. Theantibiotic dye, crystal violet, served as a positive control. After electro-phoresis, the gel was washed for 15–25 min in 10 mM sodium phosphatebuffer pH 7.2, and the buffer was changed every 5 min. The pH wascontrolled using pH strips in the 0 –14 range (Macherey & Nagel, Duren,Germany). As the gel reached pH 7 it was removed and placed on top of the prepared E. coli agar plates. Plates were incubated for 3 h at 37 C.CAU-PAGE gels were then removed and the bacteria-containing agar wasoverlayed with Luria-Bertani (LB) agar (Difco, Detroit, MI). The platewas incubated overnight at 37C. Zones of clearing were readily apparentafter this time. To perform the CFU assay as described by Harder et al.[20], 3 103 E. coli DH5 (Life Technologies) was incubated with thelinear synHE2 and the synHE2cycl peptide at increasing concentrationsin 100 l of 10 mM sodium phosphate buffer (pH 7.4) containing 10%(v/v) LB broth. While the linear HE21 was dissolved in sterile water, amixture of 99.5% dimethyl sulfoxide/0.5% trifluoracetic acid was em-ployed primarily to dissolve the cyclic peptide. The primary stock solu-tions were then diluted to concentrations that were suitable for antimicro-bial assays.

RESULTS

RT-PCR Analysis of Human Epididymal HE2mRNA Variants

Previous analyses have described six human HE2mRNA variants ([3, 7]; GenBank accession numbers

X67697, AF168616, AF170797, AF168617, AF168618,AF168619, and AF168620). Three additional variants havebeen described for Pan troglodytes by Frohlich et al. [8](GenBank accession numbers AF263552, AF263553, andAF263555). Based on these sequences, HE2-specific prim-ers were chosen to span at least two exons to be able todistinguish the amplification of contaminating DNA andunspliced precursors (Fig. 1). Amplicons were subclonedand their identity was verified by sequencing (data notshown). In addition to HE2 and HE21, three minor PCRproducts, HE2B, HE2C, and HE2E were identified (Fig. 1),which were homologous to the EP2B, EP2C, and EP2Evariants of chimpanzee [8]. Besides HE21, the HE2C andHE2E mRNA variants also predicted -defensin-like pep-tides. A quantitative comparison of the HE2 mRNA vari-ants was difficult to perform from the standard PCR runs,however, analysis of cDNAs from two different patient tis-sues at nonsaturating conditions suggested that HE2 andHE21 transcripts represented major splice variants (Fig.1). HE2 was also found at low levels in testicular cDNA,however, the minor transcripts were not detected. Compar-ing the staining intensities of amplicons derived from con-taminating genomic DNA, amplification of HE2C frag-ments appeared to be less efficient (Fig. 1), possibly notreflecting true relative levels of the epididymal HE2CmRNA variant.

Characterization of HE2-Related Peptides in HumanEpididymal Tissue and Fluid

A panel of monospecific antibodies against five che-mosynthetic peptides (see Materials and Methods) wasraised to identify HE2-related antigens in human epididy-mal tissue and in flushed epididymal secretions. P3 and P4antibodies specifically directed against HE2 /HE2B andHE21/HE2E, respectively (Fig. 2), were chosen to detectHE2-related antigens in cryosections of human epididymis.From our previous in situ transcript hybridization results[6], maximum levels of HE2 expression were expected inthe distal caput and proximal corpus regions. Indirect Cy2immunofluorescence showed that both antibodies reactedspecifically with epithelial antigens in this region (Fig. 3).In tissues from two patients with no known history of an-




FIG. 1. Detection by RT-PCR analysis of HE2, HE21, and three minoHE2-derived mRNAs, HE2B, HE2C, and HE2E (named by Frohlich et a[8]) in human epididymal cDNA. a) Separation of amplicons obtained bRT-PCR on ethidium-bromide stained agarose gel. The specificity of thprimer pairs employed is indicated above each lane; identity of ampliconwas confirmed by sequencing. Two different patient tissues are represented by two sets of human epididymal cDNA data. M indicates a 100base pair (bp) ladder. Lanes 1–3, RT-PCR of human testicular cDNA; 1 primers detect HE2 (404 bp amplicon). Lanes 4–6 show human epididymal cDNA from patient 1 and lanes 7–9 show human epididyma

cDNA from patient 2; in both tissues, HE2 (404 bp) and HE2 1 (32bp) are predominant (lanes 4 and 7). HE2B (269 bp) and HE2E (193 bpare separated in lanes 5 and 8; HE2C (354 bp) appears in lanes 6 and 9Lanes 10–12 are negative controls for each pair of primers. b) Structuof the human HE2 gene locus (modified after Jia et al. [9]) as proven herby RT-PCR, containing eight exons and two promoters, plus a diagrammatic representation of the nine human epididymal HE2 mRNA varianidentified to date. Open reading frames are highlighted by gray shadin-defensin domains by dark shading. Primers (positions marked by arows) were chosen to span at least two exons to discriminate ampliconoriginating from contaminating genomic DNA by their increased siz(larger than the approximately 400-bp HE21 fragment).

tiandrogen treatment, P3- and P4-reactive antigens were lo-calized in the apical part of the duct epithelium; a weak labeling of luminal contents was observed in some of theductal cross-sections. Epididymides from two other patientswho had received long-term antiandrogen treatment showeda deviating morphology in that the duct lumen was largelyabsent. They also showed a deviating pattern of HE2-re-lated antigen expression (Fig. 3). Whereas high levels of P3-reactive antigen appeared to be associated with the api-

cal stereocilia of the duct epithelium, P4 immunoreactivitywas weak in these tissues, persisting only in the basal partsof the epithelium (Fig. 3).

Luminal fluid flushed from two human epididymideswas analyzed by Western blotting by employing antibodiesP1–P4 (see Materials and Methods). The P1 antiserum di-rected against a sequence common to six predicted HE2peptide isoforms (Fig. 2) detected no antigens in the ex-pected molecular weight range (not shown). The P2 anti-body, directed against another peptide sequence commonto all HE2 isoforms except HE2B and HE2E (Fig. 2), re-acted with multiple peptides in the range of approximately5 to 8 kDa (Fig. 4). This was in agreement with the massespredicted for HE21 and HE22, as well as for HE21 andHE22 (Fig. 2). The most prominent products separatedeither as a broad band or as a doublet of approximately 8kDa. The P3 antibody, specific for HE21/2 and HE2B(calculated masses of 8.5 and 3.7 kDa, respectively; Fig. 2)detected an antigen of approximately 8 kDa as well as asmaller band, the higher apparent mass being in agreementwith that of the HE2 form. Unexpectedly, the P4 antibody,specific for HE21 and HE2E (Fig. 2), revealed two closebands of approximately 8 kDa as well (Fig. 4). This ap-parent mass corresponded to the calculated mass of HE2E(7.2 kDa), but not HE21 (12.2 kDa; Fig. 2). Specificityof antibody binding was confirmed by competition experi-ments employing the corresponding chemosynthetic oligo-peptides and custom peptide sequencing of the immunore-active protein bands in the range of 8 kDa (see below).

From our RT-PCR analysis (Fig. 1) the HE21 isoform,rather than HE2E, was assumed to represent the predomi-nant P4-reactive -defensin-like isoform. None of the an-tisera, however, reacted with peptides in the molecular massrange of 12 kDa as predicted from the HE21 cDNA se-quence after cleavage of the signal peptide ([7]; Fig. 2). Toexplain this apparent discrepancy, a peptide band was cutin the range of 8 kDa from a parallel blot and prepared forN-terminal amino acid sequencing. The sequences obtainedrevealed a mixture of HE2-related peptides with differentN-termini. These were either RHV[N]HSA, correspondingto the N-terminus of the majority of HE2 isoforms aftersignal peptide cleavage, or DLLPPRT, corresponding to aninternal peptide sequence common to most HE2 isoforms

and representing a proprotein convertase cleavage consen-sus. From this result, limited endoproteolysis of proximalpromoter-derived HE2 peptide isoforms, including HE21,was suggested, resulting in processed peptides of approxi-mately 8 kDa. Edman sequencing further suggested that theasparagine at position 4 of the RHV[N]HSA sequence wasat least partially occupied by N -linked glycans, possiblyrelated to the failure of P1 antibody reactivity on humanepididymal fluid (see above). The internal DLLPPRT motif is part of the P2 epitope and is common to all peptideisoforms predicted by transcripts from the proximal HE2promoter, including HE2 and HE21 (see Fig. 1 and Fig.2), however, excluding HE2B and HE2E. A truncated N-terminus starting at this motif would result from cleavage

of the peptide isoforms between arginine35 and aspartate3

by a furin-like endoprotease.To specifically reveal the presence of HE2 in humaepididymal fluid and to determine the N-terminal sequencof the secreted product, immunoprecipitation of proteisamples was performed by employing the P3 antibody. Separation of the precipitated proteins was performed on SDgels, the proteins were blotted, and a band in the range o8 kDa was cut out. The major N-terminal amino acid sequence obtained was again DLLP, corresponding to the Nterminus of a proteolytically processed HE2 peptide. Other HE2-related N-termini, for example, predicted from thHE2E cDNA sequence, were not found. Still, the occurrence in human epididymal fluid of blocked N-termini ostill-undetected N-termini of the minor HE2 peptide iso




FIG. 2. Diagrammatic representation of nine human HE2 peptide isoforms as pre-dicted from the cloned cDNAs, and thelocation of peptide sequences chosen foroligopeptide synthesis and antibody pro-duction (P1–P5). HE21 and HE22 pep-tides differ only by amino acid exchangesas marked by three dotted lines (comparewith Osterhoff et al. [3]). Signal peptidaseand proprotein convertase cleavage sitesare represented by arrows (↓). Signal pep-tides are shown as blocks with a whitebackground; mature peptides are shown aslight gray shaded blocks; the location of peptide sequences P1–P5 is shown as darkgray shaded blocks; numbers indicate cor-responding amino acids in HE2-peptides;and -defensin-like modules are shown asblocks with a dark background. The tableshows molecular mass and isoelectricpoint values as calculated from the pep-tide sequences of all isoforms.

FIG. 3. Localization of HE2-related peptides in the human epididymalduct epithelium. Indirect Cy2 immunofluorescence was performed in 8-m duct cryosections of human distal caput/proximal corpus epididymis.A and C) Immunofluorescence employing P3 antibody (1:200; HE2 / HE2B-specific) as a primary antibody; E) corresponding preimmune serum(1:200); B and D) indirect Cy2 immunofluorescence employing P4 anti-body (1:200; HE21/HE2E-specific) as a primary antibody; F) correspond-ing preimmune serum (1:200). Panels A and B are tissue sections from apatient with no known history of antiandrogen treatment; the apical cy-toplasm and the apical stereocilia were strongly immunopositive. PanelsC and D are tissue sections from a patient after long-term antiandrogentreatment; the duct lumen has collapsed. Lu, Duct lumen; Ep, duct epi-thelium. Employing the HE2 /HE2B-specific P3 antibody, apical stereo-cilia were still strongly immunopositive, whereas the HE21/HE2E-spe-cific P4 antibody reacted only with the basal part of the duct epithelium.Bar 50 m.

forms (i.e., HE2B and the bin1b-homologous HE2E [Fig.2]) is not excluded.

Detection of HE2-Related Peptides in Human Ejaculate

LIS extracts and salt wash membrane protein prepara-tions of human ejaculated sperm were analyzed by em-ploying P3 and P4 antibodies (Fig. 2). With the P3 anti-serum, only inconsistent labeling in the range of approxi-mately 8 kDa was observed (data not shown), corroboratingprevious observations that a Western blot detection of HE2 on human ejaculated sperm was difficult to perform[7]. However, using the P4 antiserum directed against iso-

forms HE2

1, HE2E, or both, a faint band of approxi-mately 8 kDa was detected in LIS extracts, and a prominentband of a similar apparent mass was detected in salt washpreparations of cationic sperm proteins (Fig. 5a).

The presence of HE2-related antigens in human seminalplasma was confirmed by immunoprecipitation employingisoform-specific antibodies. Proteins were precipitated bythe P2 antiserum and subsequently analyzed by Westernblotting using a combination of P2, P3, P4, and P5 antisera(Fig. 5b). The P3 antiserum was negative (data not shown).A prominent 8-kDa band was detected by the other threeantisera, probably representing the processed form of HE21 (Fig. 2). The P2 antiserum detected additional mi-nor peptides of 6 kDa (Fig. 5b). Because these smallerpeptides reacted with neither P4 nor P5 antiserum (Fig. 2),

they were assumed to represent alternative, smaller HE2peptide isoforms rather than degradation products of HE21.

Recombinant Expression and Processing of HE2and HE21

An HE21 cDNA fragment [3] and an HE21 cDNAfragment, both encoding the complete propeptides ([7];Figs. 1 and 2) were chosen for recombinant protein pro-duction. HE21 was expressed in E. coli as a maltose-bind-ing protein (MBP) fusion. The cysteine-rich, -defensin-like HE21, on the other hand, was expressed in insect cellsto allow correct peptide folding and formation of disulfide

bonds. The recHE21/MBP fusion protein from E. coli wasaffinity-purified and processed by protease treatment in vi-tro. Factor Xa was used to cleave the fusion protein so thatno vector-derived residues were attached. To obtain a pep-tide containing the DLLPP motif at its N-terminus, recom-binant human furin was employed, either alone or in com-bination with factor Xa. Protease treatment of recHE21 invitro and subsequent Western blot analysis confirmed thatthe peptide was indeed a substrate for furin-like proproteinconvertases (Fig. 6a). A structural model for the mature,processed HE2 peptide was proposed (Fig. 7A), startingwith the DLLPP motif and containing an intramoleculardisulfide bond.

The -defensin-like HE21 peptide (Fig. 7B) was pro-duced in High Five cells and enriched from the cell culturesupernatants by anion exchange chromatography. Recom-binantly expressed proteins were analyzed by employingP1, P2, P4, and P5 antisera (Fig. 2). Although multiplebands were detected with all antisera, including P1, the fast-est migrating (approximately 8 kDa) band reacted with allantisera except P1 (Fig. 6b). Subsequent N-terminal se-quencing of the corresponding peptide band cut from par-allel blots revealed the DLLPP motif. From this result weconcluded that the 8-kDa band contained the processedHE21 propeptide, and that processing of the prepropeptide







FIG. 4. Detection of secreted HE2-related peptides in human epididymalfluid. a) Western blot analysis after standard Laemmli PAGE of flushedepididymal fluid (ef). Lane 1, 4 l of ef reacted with P2 antibody aftercompetition with P2 peptide; lane 2, 4 l of ef reacted with P2 antibody(1:250); lane 3, 8 l of ef reacted with P2 antibody; lane 4, 8 l of ef reacted with pre-P4; lane 5, 8 l of ef with P4 antiserum (1:250). b)Western blot analysis after Tris-Tricine PAGE of flushed ef. Lane 1, 8 lof ef reacted with P3 antiserum (1:250); lane 2, 8 l of ef reacted with

P4 antiserum (1:250).

FIG. 5. Detection of HE2-related peptides in a human ejaculate. a)Western blot analysis of human sperm membrane protein preparationsemploying the P4 antibody. Lane 1, 8 g of sperm membrane proteinsfrom LIS extracts; lane 2, 80 g of sperm membrane proteins from LISextracts reacted with P4 antiserum (1:250); lane 3, 10 g of sperm saltwash proteins with P4 antiserum; lane 4, 40 g of sperm salt wash pro-teins with P4 antiserum. b) Immunoprecipitation of HE2-related peptidesfrom human seminal plasma. Lane 1, preclearing and detection with P2-antiserum, 1:250; lane 2, immunoprecipitation with P2 antiserum anddetection with P2 antiserum; lane 3, immunoprecipitation with P2 anti-serum and detection with P4 antiserum; lane 4, immunoprecipitationwithP2 antiserum and detection with P5 antiserum.

FIG. 6. Proteolytic processing of recombinantly expressed HE2 in vitroand in vivo. a) Furin peptidase cleavage of E. coli -expressed MBP-HE21fusion protein and subsequent Western blot analysis with P3 antiserum(1:500). Lane 1, Factor Xa/furin-double digest of 50 g of MBP-HE21;lane 2, furin digest of 50 g of MBP-HE21. Furin cleavage of HE21was more effective in the double digest. b) Recombinantly expressedHE21 as recovered from approximately 200-l supernatants of trans-fected High Five insect cell cultures. Proteins were characterized by West-ern blot analysis with isoform-specific antibodies. Lane 1, detection withP1 antiserum (1:250); lane 2, detection with P4 antiserum (1:250); lane3, marker proteins.

also occurred in vivo by a furin-like endoprotease presentin insect cells [21]. By optimizing the multiplicity of in-fection, most of the recombinant protein products corre-sponded to the fully processed form (data not shown).

In Vitro Antibacterial Activity of HE2-Derived Peptides

Recombinant HE21 protein was enriched from insectcell culture supernatants as processed form and run on acid-

urea gels. Gels were subsequently included in the bacterialgel overlay assay [17, 18] against E. coli DH5. In the agaroverlying the recHE21 lane, a bacteria-free, clear zonewas observed. Parallel acid-urea gels were processed forWestern blot analysis. Employing the P4 antiserum specif-ically directed against the HE21 peptide, an immunore-active band with a migration pattern corresponding to thatexhibiting antibacterial activity was identified (Fig. 8).

Proteolytic processing, disulfide bond formation, and

subsequent HPLC-purification of the E. coli-derivedrecHE2 peptide proved difficult at a preparative scale. Toprovide the larger amounts of pure peptide necessary for invitro antibacterial assays, HE22 was obtained as linearand cyclic chemosynthetic C-terminal peptide fragments,the latter containing an intramolecular disulfide bond (Fig.7A). Because there is yet no information on the minimallength of an in vivo, bioactive HE2 peptide, the C-ter-minal part of the sequence common to the HE2 and HE2Bpeptide isoforms (compare Figs. 2 and 7) was chosen foroligopeptide synthesis. The linear peptide was readily dis-solved in water; however, the cyclic form was difficult todissolve.

On acid-urea PAGE gels, the linear and cyclicsynHE22 peptides showed different migration patterns(Fig. 9A). The linear peptide, despite its elution as a singleHPLC peak and its homogenous mass as determined byESI-MS, showed several bands, which is indicative of olig-omer formation. The cyclic peptide, equally pure as deter-mined by HPLC and ESI-MS, showed similar bands in thelower part of the gel, however, a large proportion of mol-ecules migrated as aggregates in the upper part of the gels.In the acid-urea PAGE bacterial gel overlay assay, zonesof clearing corresponded to the peptide oligomers presentin the lower part of the gels, whereas the aggregates in theupper part showed no killing of E. coli (Fig. 9A). Quanti-tative CFU assays were performed by employing the HPLCpeak-purified, synthetic HE22 peptides at increasing con-




FIG. 7. Diagrammatic representation of the major human epididymal HE2 peptideisoforms. A) Proposed processing andstructure of the processed HE2 peptidecompared with the synthetic HE2 peptidfragments employed in the antibacterialassays. Signal peptide is indicated as ablock with a white background; thecleaved prodomain is shown as light grayshaded blocks; the processed peptide isshown as blocks with a dark background.Ciphers refer to the numbers of amino ac-ids starting with methionine 1 (1–25, sig-nal peptide; 25–60, prolonged propeptide61–103, mature HE2; 73–103, syntheticpeptide). Amino acid differences betweenthe two allelic forms are indicated; plusand minus symbols mark charged aminoacids; the proposed loop structure is stabilized by a cysteine (black bar) and an ionic (black dots) bond. B) Deduced aminoacid sequences of processed HE21 andHE2C have been aligned with the knownhuman -defensins (hBD1–hBD4) and the-defensin consensus. Dashes have beeninserted to improve alignment.

FIG. 8. Antibacterial activity of recombinant HE21 protein expressein High Five insect cells. A) CAU-PAGE separation and Coomassie staining of a supernatant from untransfected cultures (lanes are referred to a) and of HE21 peptide enriched from the supernatant of a transfectecell culture (lanes are referred to as 1). B) Subsequent bacterial gel ovelay assay of the same gel against E. coli DH5. Locations of substancewith antimicrobial activity in the gel are visualized by bacteria-free, cleazones in the agar. They are accentuated by arrows. C) Western blot anaysis of recombinantly expressed HE21 run on a parallel acid-urea geemploying the P4 antibody (1:250).

centrations. The results suggested that the minimal concen-trations of both peptides necessary to kill 90% of E. coliwere 60 g/ml at a sodium concentration of 20 mM (Fig.9B). This minimal inhibitory peptide concentration may,however, be an underestimate because of the tendency of the peptides to form high molecular weight aggregates thatwere biologically inactive (Fig. 9A).

DISCUSSION

Immunohistochemistry, Western blot analyses employ-ing isoform specific antibodies, and peptide microsequenc-ing confirmed for the first time the occurrence of multiplehuman HE2-encoded peptides in the human male genitaltract, including the HE2 isoform. We have shown thatHE2 and HE21 represent the major isoforms producedby the epididymal epithelium, processed at the peptide lev-el, and secreted apically into the duct lumen. These resultsare different from those in other mammalian species [5, 7,8], but they are congruent with previous results on mRNAlevels in humans [3, 7]. Minor HE2-derived peptides were

also detected, but they cannot yet unequivocally be as-signed to minor mRNA variants. The presence of HE2B,HE2C, and HE2E transcripts in human epididymal RNAhad not been shown before; rather, the existence of corre-sponding exons in the human HE2 gene had been inferredby extrapolation from the homologous chimpanzee tran-scripts [8].

Epididymis-restricted expression and sperm bindingproperties of HE2-derived peptides [3, 7] had suggestedthat the HE2 gene may be involved in the process of post-testicular sperm maturation. More recent results suggestthat it is related to the -defensin gene family [4, 5, 9].Indeed, in three human variants (i.e., HE21, HE2C, andHE2E), the six-cysteine motif reminiscent of the -defen-

sins is predicted (Fig. 7B). To corroborate this assumptionwe have shown that recombinant HE21 containing a defensin-like motif has antibacterial activity. A weak similarity to the defensin propeptide consensus also reside




FIG. 9. Antibacterial activity of synthetic HE2 peptide fragments. A) CAU-PAGE separation and Coomassie staining of C-terminal peptides with andwithout an intramolecular cysteine bond. Because this separation system did not contain SDS, the polypeptide standard (BioRad) served only as alandmark. M indicates the polypeptide standard. Lane 1, approximately 50 g of synHE22cycl; lane 2, 10 g of linear synHE2;2; lane 3, crystal violetwas included as a positive control. B) Bacterial gel overlay of parallel acid-urea gel containing the same amounts of synthetic HE2 peptide fragments.Antimicrobial activity is visualized by the bacteria-free, clear zones. C) Quantification of antibacterial activity by CFU assay of linear synHE22 ()and synHE22cycl () against E. coli DH5. Bacteria were incubated for 3 h at 37C in 100 l of 10 mM sodium phosphate buffer pH 7.2, containing10% LB broth with increasing concentrations of peptide (20 mM total sodium concentration). Antibacterial activity was measured by plating serial

dilutions of each incubation mixture and counting of the colony forming units the following day. The limit of detection (1 colony per plate) correspondedto 1 102 CFU/ml.

within the propeptide sequence predicted from HE2mRNA. However, its processed isoform, besides beingstrongly cationic, showed no other reminiscence to any of the known classes of peptide antibiotics. Still, our resultsshow that HE2, like the -defensin-like HE21, has an-tibacterial activity, indicating that the human gene producesantimicrobial peptides with a sequence that is entirely dif-ferent from the known defensins. Thus, we may have de-tected a novel class of antibacterial peptides that had notbeen observed before in other species or tissues, and whichmay be involved in defending the epithelia of the male

genital tract in humans.Complementary DNA cloning of bin1b, which was pro-

posed as representing the rat HE2 homologue [5], and iden-tification of a homologous mouse EST clone (GenBank ac-cession number AK020333), allows the generation of de-ficient animals by gene targeting. Targeted gene mutationwill certainly help to promote a better understanding of thefunctional role of the -defensin-like HE2 isoforms in vivo.However, HE2E, which shows closest similarity to bin1b[5], appeared to represent only a minor product in the hu-man epididymis. Other variants, including a counterpartHE2, have not yet been found in rodents, and it is possiblethat they do not exist. Still, alternative mRNA splicing isa potent mechanism of expression regulation, and the hu-

man HE2 isoforms, which are constitutively expressed onlyas minor forms, may be induced during infection or inflam-mation.

Our results suggest that in addition to extensive pro-cessing at the mRNA level, proteolytic processing of prox-imal promoter-driven HE2 peptides occurs by a ubiquitousfurin-like serine protease, by cleaving a glycosylated 35-amino acid propiece from the precursor peptides. Activa-tion by endoproteolytic cleavage has been shown only forthe -defensins, which are synthesized as longer prepro-defensins [22]. A similar posttranslational processing hasnot previously been shown for -defensins, but may applyto all HE2 peptides that contain the corresponding propro-tein convertase cleavage consensus. Limited endoproteo-

lysis at sites marked by this consensus sequence is a wide-spread process by which biologically active peptides areproduced within the secretory pathway of eukaryotic cells(for a review see [23]). HE2 processing was observed inthe same way in human epididymis and in insect cell cul-tures, and could play a role in regulating cellular transportand peptide function.

Additional putative cleavage sites for other endoproteas-es are present in some of the HE2 isoforms as well. Region-specific expression in the epididymis of various proteasesand their presence in epididymal fluid has been recently

demonstrated [24]. Matrilysin, a matrix metalloprotease im-plicated in -defensin activation [25] is secreted luminallyby the epididymis, seminal vesicles, and prostate [26]. Itmay also cleave prosegments of HE2 peptides, possibly fur-ther increasing the number of potentially bioactive HE2-derived peptides.

Regulation of defensins occurs via signal transductionpathways that are common to other immune responses (fora review see [27]. However, bin1b expression in rat epidid-ymis [28] and HE2 gene expression in nonhuman primates[7, 8] seems to depend on androgens. Therefore, it was anunexpected finding that HE2-related peptides were still pre-sent in the epididymides of two patients after long-termantiandrogen treatment. No differences were observed at

the mRNA level by Northern blot analysis (data notshown), and it is possible that these patients were not com-pletely androgen-deprived. Still, the duct morphology inboth tissues exposed to antiandrogens was dramatically al-tered compared with untreated tissues. Thus, our observa-tion is interesting in that the -defensin-like peptides (i.e.,HE21/HE2E), were located only basally in androgen-de-prived ducts. Location of the defensin-unrelated peptides(i.e., HE2 /HE2B), on the other hand, was comparable tothat in tissues of untreated patients.

The human male genital tract is a recognized site of antimicrobial peptide secretion [29], and it also expressesthe -defensins hBD-1 [30] and hBD-4 [31]. It may stilluse a more tissue-specific repertoire of antimicrobial pep-




tides; the most compelling evidence for this is the in vitroantibacterial activity of HE2 and HE21 peptides shownhere. The antimicrobial activity of HE2, which shows nosimilarity to the known classes of peptide antibiotics, wasnevertheless surprising. However, the functions and require-ments of the epididymis during transport, maturation, andpreservation of spermatozoa are different from those of oth-er epithelia. Thus, the in vivo functions of HE2-encodedpeptides may be more complex. Specifically, their presence

in the ejaculate and on the sperm surface point to additionalfunctions other than epithelial defense.

Other antimicrobial peptides also revealed additionalfunctions. Besides their antimicrobial activity, a chemoat-tractant role for cells of the adaptive immune system hasbeen described [32]. Enteric defensins form anion-conduc-tive pores in phospholipid bilayers and stimulate chloridesecretion [33]. Defensins are cytotoxic to both bacterial andnormal eukaryotic cells (for a review see [2]). Moreover,CAP37 has been reported to immobilize sperm [34]. Somecases of male idiopathic infertility thus may be caused byendogenous peptide antibiotics, which may bear a contra-ceptive potential.

ACKNOWLEDGMENTSWe are indebted to Drs. Ching-Hei Yeung and Trevor G. Cooper, In-

stitute of Reproductive Medicine, Munster, for t heir generous help in pro-viding human epididymal tissue and fluid. We thank Drs. Richard Ivelland Caroline Osterhoff, IHF, Hamburg, and Drs. Jens-Michael Schroderand Jurgen Harder, University of Kiel, for helpful discussions. Dr. F. Lei-denberger, IHF, Hamburg, provided excellent facilities and continued in-terest and support. Dr. Marcus Koppitz, Schering AG, Berlin, synthesizedmost peptides employed in this study.

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