SPAG11/isoform HE2C, an atypical anionic beta-defensin-like peptide

A human caput epididymidal cDNA, HE2C, was cloned based on its homology to the known chimpanzee counterpart, suggesting that the encoded beta-defensin-like peptide represented a conserved component of the innate epididymidal epithelial defense system in primates. An approximately 6kDa HE2- related peptide was co-purified together with other HE2 isoforms from human seminal plasma by affinity chromatography. By its antibody reactivity as shown by Western blot analysis, this peptide was distinct from the more abundant HE2 isoforms and was concluded to correspond to HE2C. Similar to other HE2-encoded isoforms, the endogenous HE2C was proteolytically processed from a larger precursor by a furin-like prohormone convertase. This was confirmed by N-terminal sequencing. In order to study the structural and functional properties of HE2C it was recombinantly expressed in insect cells. Post-translational processing also occurred within these cells, yielding the mature processed HE2C peptide. Correct disulfide bonding of the recHE2C peptide was shown by p-aminophenylarsineoxide(PAPAO)-agarose binding assay. Purified recHE2C strongly bound to Escherichia coli DH5alpha and Bacillus subtilis; however, it did not exhibit microbicidal activity when tested in a radial diffusion assay against these bacteria. Different from the previously described beta-defensins, the mature HE2C peptide has an anionic pI and an algebraic net charge of -1. Also, it lacks the amphipathic transitions, which, according to the Shai-Matzusaki-Huang model, are prerequisite for the membranolytic activity of antimicrobial peptides.     

Organization of the human gene encoding the epididymis-specific EP2 protein variants and its relationship to defensin genes

The EP2 gene codes for at least nine message variants that are all specifically expressed in the epididymis. These variants putatively encode small secretory proteins that differ in their N- and C-termini, resulting in proteins that can have little or no sequence similarity to each other. We have isolated and sequenced the human EP2 gene to determine the molecular origin of these variants. The EP2 gene has two promoters, eight exons, and seven introns. Exons 3 and 6 encode protein sequences homologous to beta-defensins, a family of antimicrobial peptides. This sequence homology and the arrangement of promoters and defensin-encoding exons suggest that the EP2 gene originated from two ancestral beta-defensin genes arranged in tandem, each contributing a promoter and two exons encoding a leader sequence and a defensin peptide. The proposed evolutionary relationship between the EP2 gene and defensin genes is supported by the observation that the EP2 gene is located on chromosome 8p23 near the defensin gene cluster and is separated by 100 kilobases or less from DEFB2, the gene for beta-defensin-2. While the EP2 gene transcribes beta-defensin-like message variants, most of the known message variants code for nondefensin proteins or proteins containing only a partial defensin peptide sequence. We suggest that, during its evolution, the EP2 gene has acquired new functions that may be important for sperm maturation and/or storage in the epididymis.     

Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif

The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.     

Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice

Defensins comprise a family of cationic antimicrobial peptides that are characterized by the presence of six conserved cysteine residues. We identified two novel human beta-defensin (hBD) isoforms by mining the public human genomic sequences. The predicted peptides conserve the six-cysteine motif identical with hBD-4, termed hBD-5 and hBD-6. We also evaluated the characteristics of the mouse homologs of hBD-5, hBD-6, and HE2beta1, termed mouse beta-defensin (mBD)-12, mBD-11, and mouse EP2e (mEP2e). The mBD-12 synthetic peptide showed salt-dependent antimicrobial activity. We demonstrate the epididymis-specific expression pattern of hBD-5, hBD-6, mBD-11, mBD-12, and mEP2e. In situ hybridization revealed mBD-11, mBD-12, and mEP2e expression in the columnar epithelium of the caput epididymis, contrasting with the predominant expression of mBD-3 in the capsule or septum of the whole epididymis. In addition, the regional specificity of mBD-11, mBD-12, and mEP2e was somewhat overlapping, but not identical, in the caput epididymis, suggesting that specific regulation may work for each member of the beta-defensin family. Our findings indicated that multiple beta-defensin isoforms specifically and cooperatively contribute to the innate immunity of the urogenital system.     

Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study

Tenecin 1, a peptide consisting of 43 amino acids, exhibits a potent bactericidal activity against various Gram-positive bacteria and shares a common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. Our previous research indicated that an active fragment was successfully extracted from C-terminal beta sheet domain of Tenecin 1, whereas the fragment corresponding to the alpha helical region of the protein had no antibacterial activity. We chose this inactive fragment corresponding to alpha helical region of Tenecin 1 and synthesized derivatives with a different net positive charge by using rational design. Interestingly, we successfully endowed antibacterial activity as well as antifungal activity to the inactive alpha helical fragment by single or double amino acid replacement(s) without an increase of hemolytic activity. The leakage of dye from vesicles induced by the active peptides suggested that these peptides act on the membranes of pathogen as a primary mode of action. Structure-activity relationship study of a series of the active derivatives revealed that amphiphilic structure and high net positive charge were prerequisite factors for the activity and that there was a relationship between the antibacterial activity and the isoelectric point of the active peptides. In this work, we showed an efficient method to endow the antibacterial activity as well as antifungal activity to the inactive fragment derived from a cyclic insect defensin protein and suggested a facile method to screen for active fragments from cyclic host defense peptides.     

Avian antimicrobial peptides: the defense role of beta-defensins

Avian antimicrobial peptides, classified as beta-defensins, have been identified from bloods of chicken, turkey, and ostrich; epithelial cells of chicken and turkey; and king penguin stomach contents. Beta-defensins are a family of antimicrobial peptides characterized by six cysteine residues forming beta-defensin motifs that are also found in bovine, ovine, pig, and human. These peptides are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and yeast. Analysis of evolutionary relationships of vertebrate beta-defensins showed that there might be a common ancestral gene between avian and other mammalian peptides. This ancient gene may have been passed down and evolved from species older than the oldest living birds, forming a beta-defensin-like precursor molecule. This review describes potential applications of these peptides in health care products.     

Effect of vasectomy on gene expression in the epididymis of cynomolgus monkey

Vasectomy has been shown to affect the pattern of mRNA expression of P34H, a human sperm protein added to the acrosomal cap during epididymal transit. It has been reported that vasectomy alters the histology of the reproductive tract in various species as a result of the increased pressure in the epididymis. The aim of this study was to evaluate if other epididymis-specific mRNAs, which are expressed in different patterns along the duct, are altered by vasectomy as well. We analyzed the expression of P31m (a monkey homologue of human P34H) and three different HE-like (HE-l) mRNAs along the epididymis in the cynomolgus monkey (Macaca fascicularis). Sexually mature cynomolgus monkeys were vasectomized unilaterally; then the epididymides were surgically removed at different time points. The ipsilateral normal epididymis was used as a control. Histomorphometric measurements showed that the height of the epididymal epithelial cells started to be affected only at 14 wk postsurgery. However, Northern blot and in situ hybridization analysis showed that the expression pattern of P31m, HE1, and HE5-like mRNA along the epididymis was not affected by vasectomy. Only the HE2-like mRNA predominantly expressed in the normal corpus epididymidis was significantly lowered 14 wk after vasectomy. Thus, ductal obstruction differentially alters mRNA expression along the epididymis of the cynomolgus monkey.     

A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol.

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity

Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate a synthetic antimicrobial peptide cp gene, which was then cloned into the expression vector pPICZalpha-A. The constructed recombinant expression vector pPICZalpha-cp was transformed into Pichia pastoris X-33, in which the synthetic antimicrobial peptide (CP) could be expressed under the control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Results showed that recombinant plasmid is highly stable, and In vitro experiments showed that the recombinant antimicrobial peptide CP is heat and acid-stable, and it has high antibacterial activity against several Gram-positive and -negative bacteria. Only 1 microg of the recombinant antimicrobial peptide CP has an antibacterial activity equivalent to 64 U ampicillin. Thus, this recombinant antimicrobial peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single whey acidic protein motif

Antibacterial proteins are important participants in the innate immunity system. Elafin and SLPI are the whey acidic protein (WAP) motif proteins with both antibacterial activity and antiprotease activity, and their role in innate immunity is under intense investigation. We cloned two novel antibacterial WAP motif proteins from mice, SWAM1 and SWAM2. SWAM1 and SWAM2 are composed of a signal sequence and a single WAP motif that has high homologies with the WAP motifs of elafin and SLPI. SWAM1 is constitutively expressed in kidney and epididymis, and is induced in the pneumonic lung. SWAM2 is constitutively expressed in tongue. SWAM1 and SWAM2 inhibit the growth of both Escherichia coli and Staphylococcus aureus at a IC(90) (concentration that achieves 90% inhibition) of 10 microM. Human genes LOC149709 and huWAP2 are considered to be human SWAM1 and SWAM2, respectively. These and several WAP motif proteins (WAP1, elafin, SLPI, HE4, eppin, C20orf170, LOC164237, and WFDC3) form a gene cluster on human chromosome 20, suggesting that they may be derived from the same ancestral gene by gene duplication. Our results underscore the role of the WAP motif as a skeletal motif to form antibacterial proteins, and warrant the study of antibacterial activity in other WAP motif proteins.     

The structure of human macrophage inflammatory protein-3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding activities with human beta-defensins

Human macrophage inflammatory protein-3alpha (MIP-3alpha; CCL20) is a CC-type chemokine that binds to and activates CC chemokine receptor-6 (CCR6). Although MIP-3alpha does not share the binding site of CCR6 with any other chemokine, human beta-defensin-1 and -2, small cationic antimicrobial peptides, have also been found to bind to and activate CCR6. Conversely, we have found that MIP-3alpha possesses antibacterial activity of greater potency than human beta-defensin-1 and -2 against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, while having no activity against the fungus Candida albicans. There is no clear sequence similarity between beta-defensins and the chemokine MIP-3alpha, beyond an abundance of cationic residues and the presence of disulfide bonds. Nonetheless, there are structural similarities between these three proteins that allow their overlap of chemotactic and antimicrobial activities. In this report, we describe the x-ray crystal structure of human MIP-3alpha refined to a resolution of 1.7 A and compare it with the crystal structures of human beta-defensin-1 and -2. Molecules of MIP-3alpha and the beta-defensins seem to share few structural motifs that are likely associated with their common biological activities.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.     

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.     

Macrophage inflammatory protein-3alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells

Macrophage inflammatory protein (MIP)-3alpha and beta-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3alpha and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3alpha and BD-2 can bind. Real-time PCR showed that MIP-3alpha and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3alpha. MIP-3alpha and BD-2 increased OPN mRNA levels very slightly. MIP-3alpha and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3alpha was lower than that of BD-2. These findings suggest the MIP-3alpha and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.     

Isolation and characterization of four antibacterial peptides from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.