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Characterization and functions of beta defensins in the epididymis

Susan H. Hall¹, Suresh Yenugu², Yashwanth Radhakrishnan¹, Maria Christina W. Avellar³, Peter Petrusz¹,

Frank S. French¹

¹Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill, NC 27599-7505, USA
²Department of Biochemistry and Molecular Biology, Pondicherry Central University, Pondicherry 605014, India
³Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo, SP 04044-020, Brazil

Abstract

The epididymal β-defensins have evolved by repeated gene duplication and divergence to encode a family of proteins that provide direct protection against pathogens and also support the male reproductive tract in its primary function. Male tract defensins also facilitate recovery from pathogen attack. The β-defensins possess ancient conserved sequence and structural features widespread in multi-cellular organisms, suggesting fundamental roles in species survival. Primate SPAG11, the functional fusion of two ancestrally independent β-defensin genes, produces a large family of alternatively spliced transcripts that are expressed according to tissue-specific and species-specific constraints. The complexity of SPAG11 varies in different branches of mammalian evolution. Interactions of human SPAG11D with host proteins indicate involvement in multiple signaling pathways. (Asian J Androl 2007 July; 9: 453_462)

Keywords: defensin; antibacterial; male fertility

Correspondence to: Dr Susan H. Hall, Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599 USA.
Tel: +1-919-966-0728 Fax: +1-919-966-2203
E-mail: shh@med.unc.edu

DOI: 10.1111/j.1745-7262.2007.00298.x

1 Introduction

Defensins emerged from our studies on epididymis-specific proteins in which we were seeking novel male contraceptive targets. Among the candidate targets, the epididymal protease inhibitor Eppin was shown to be a successful reversible male immunocontraceptive in macaques [1]. The first defensin discovered in this program was given the clone name ESC42, and its trefoil-like motif was described [2]. Trefoil proteins are important in host defense; they maintain mucosal integrity and influence defensin and adaptive immunity gene expression [3]. After this motif was recognized as the β-defensin signature, ESC42 was named β-defensin 118 (DEFB118). DEFB118 is a member of a large family of genes clustered primarily on human chromosomes 6, 8 and 20 (Figure 1) [4_11]. Defensins have evolved by repeated gene duplication and divergence, including functional diversification [12]. Except for the 6-cysteine domain, rich in positively charged amino acids, defensins differ considerably in their amino acid sequences and target pathogen specificity [4]. A similar cysteine array is found in some lectins [13] and antibacterial protease inhibitors, including the contraceptive target Eppin [14], and secretory leukocyte protease inhibitor [15] (Figure 2). Ancient guards against pathogen invasion, lectins and protease inhibitors are also important in plant host defense [16].

2 β-defensin primary sequences and functions

Beyond the 6-cysteine signature motif, the simplest β-defensins have little additional sequence (Figure 2) and fall in the molecular weight range of 5_10 kDa. These simple defensins, such as human DEFB1 and DEFB4 (hBD2), are related to defensins in lower animals, including fish [17] and insects [18]. Similar defensins are produced in plants, particularly in the reproductive structures (flowers and seeds) [16]. Male reproductive tract defensins are known only in mammals. These defensins may be as large as 18 kDa (human DEFB129) and often have long N-terminal or C-terminal extensions, generally of unknown function. Reproductive functions are suggested by the sperm surface location of several defensins, including SPAG11 [19, 20], DEFB118 [2] and DEFB126 [21, 22]. Reproductive functions have been reported for rat SPAG11E (Bin1b) [23] and for DEFB126 [21, 22]. Bin1b promotes motility in immature spermatozoa from the caput epididymidis by a mechanism dependent on calcium uptake [23]. The long C-terminal domain of DEFB126, rich in threonine and serine, is highly O-glycosylated. A major component of the sperm glycocalyx [24], DEFB126 is shed during capacitation [22], a loss prerequisite to spermatozoa binding to the zona pellucida [21]. The highly anionic C-terminus of DEFB118 is not thought to have a role in antibacterial action [25], which typically depends on cationic amino acids. The male reproductive tract DEFB123 has a novel function, protection against endotoxemia through restoration of normal tumor necrosis factor-α levels [26].

3 Structures of β-defensins and similar proteins

Structurally, β-defensins typically contain an N-terminal alpha helical domain joined by a disulfide bond to a 2-strand or 3-strand beta sheet stabilized by additional disulfide bridges. The similarity of this fold in human proteins hBD1 [27], SPAG11E [28], in bovine SPAG11C [29] and the human intestinal trefoil protein 3 [30] is shown in Figure 3. The fungal, insect, and plant defensins shown are strikingly similar to a scorpion neurotoxin that shows sequence homology with the male reproductive tract defensins DEFB118 and DEFB126 (identified as GenBank AA335178 and ESP13.2 in [31]). Their cysteine stabilized configuration might represent evidence of broad application of independently evolved structures to common features of host defense challenges [32], or might be evidence of ancient origins of the β-defensins conserving similar domains throughout the animal and plant kingdoms.

4 The SPAG11 gene is a fusion of two β-defensin genes

Unique among the β-defensins, human SPAG11 represents the functional fusion of two ancestrally independent β-defensin genes [33] (Figure 4). Alternatively spliced transcripts are initiated at both promoters. Transcripts initiated at the A promoter may end after exon 3 or may continue past the poly A addition site, presumably a weak termination signal, and continue through the B promoter and the B exons. Species-specific exons are reported for human, monkey and bovine SPAG11 [29, 33_35]. There are fewer bovine mRNA splice variants (only six) than primate variants [29]. Several of the bovine splice sites are in the 3'-untranslated regions, where they may affect mRNA stability. There are three bovine-specific exons. The rat SPAG11 gene is simpler than that in primate and bull and retains the original separate function of the A and B components. There is only one splice site and it is in the A component. No species-specific exons are found in rats [36]. Read-through transcription have to has not been reported for any other pair of defensin genes.

5 SPAG11 proteins

Translation of these alternatively spliced RNAs produces a complex protein family. Immunohistochemical staining has revealed the presence of multiple SPAG11 isoforms in the epithelial cells of the epididymis, showing that these mRNAs are actively translated [20, 29, 36]. Most primate SPAG11 proteins contain the N-terminal common region joined to C-terminal peptides encoded by different combinations of exons (Figure 5). Multiple reading frames are utilized. For human SPAG11A, exon 6 transcripts are translated in one reading frame, in a second reading frame for the D isoform and for the Rhesus macaque J isoform in the third reading frame. Why SPAG11 evolved these special features is not known. Perhaps it is for the same reason that families of alternative splice variants operate where discriminative protein association is crucial in immunity [37, 38], neuronal function [39, 40], hearing [41], olfactory detection [42] and fertility [43]. Families of proteins containing different combinations of peptides can have different but overlapping sets of molecular recognition properties and, therefore, overlapping sets of interacting partners that might be of host and/or pathogen origin. SPAG11 mRNA splicing is regulated by tissue-specific and species-specific mechanisms that have led to the suggestion that different combinations of isoforms more effectively kill the pathogens in different organs [29]. Alternatively, different combinations of isoforms might be required for specific male reproductive functions.

6 SPAG11 sequence conservation in different species

Alignment of amino acid sequences of the defensin-like SPAG11C, and E isoforms using CLUSTALW [44] reveals exon-specific rates of evolutionary divergence (Figure 6). There is strong sequence conservation indicated by the black shading in the defensin regions of SPAG11C and SPAG11E,whereas the N-terminal common region shows broad sequence diversity [29]. This region is sometimes called a propiece. The lysine-arginine cleavage site for a furin-like prohormone convertase has been identified in this propiece in humans [45] and is conserved in all species except horses. All of the SPAG11 sequences found thus far are in mammals.

7 Functions of SPAG11 isoforms

The N-terminal common region has antibacterial activity, although it lacks a defensin motif [46]. Each of the full length human, rhesus and bovine SPAG11 proteins tested as well as the C-terminal peptides of human SPAG11 A, D and G show antibacterial activity against Escherichia coli [28]. In addition, the C-terminal peptide of SPAG11A kills Niesseria, Enterococcus and Staphylococcus [47]. However, the C-terminal peptides of human and rhesus SPAG11C, and rhesus SPAG11K and SPAG11L lack antibacterial activity [46]. SPAG11 isoforms and other defensin-like proteins of the male tract kill E. coli by a membrane disrupting mechanism that has been measured within minutes of contact with the recombinant SPAG11 proteins using fluorescent probes specific for the outer and inner bacterial membranes [14, 25, 28, 48]. SPAG11 and other proteins also inhibit bacterial macromolecular synthesis [46, 48]. Damage to the bacteria can be visualized by scanning electron microscopy [14, 25, 46, 48]. E. coli exposed to different SPAG11 peptides shows a range of responses, including shrinkage, loss of cell contents, especially at the division septa, and knob-like distortions (Figure 7).

The rapid mechanism of β-defensin bacterial killing is illustrated in Figure 8. Defensin proteins might be initially randomly distributed around a bacterium, but rapidly begin to bind the negatively charged bacterial surface. Membrane disruption assays have shown that within 30 s, the outer membrane is damaged and within a few minutes, the inner membrane is also disrupted [25, 48]. Defensins interfere with macromolecular synthesis by destroying the outer and inner membrane barriers and/or by entering the cell [25, 48]. Scanning electron microscopy shows that 30 min of treatment results in the release of cell contents. Bacteria that are unable to seal these pores are not likely to survive.

In the homology model of the SPAG11D defensin domain, conserved residues (light grey) [49] and additional basic residues (dark grey) form a potential protein interaction domain (Figure 9). The possibility that a protein receptor for SPAG11D on sperm might bind this region prompted us to look for interacting partners. In recent studies, using yeast 2 hybrid screening, we identified a number of epididymal proteins that interact with the full length mature human SPAG11D protein in yeast, but not with the amino terminal common region alone (Radhakrishnan et al., unpublished data). Each of these proteins has a role in male fertility that potentially could be modulated by interaction with SPAG11D. Further studies on the interactions of SPAG11 isoforms with epididymis and sperm surface proteins should lead to a better understanding of the full range of male reproductive functions of these antibacterial proteins.

8 Conclusion

The β-defensin proteins are involved in innate immunity and male reproductive functions. Evolutionary conservation of the β-defensin fold in animal and plant kingdoms attests to the broad success of this paradigmatic structure in promoting species survival. Multiple interacting partners of SPAG11D suggest involvement in host signaling pathways.

Acknowledgment

We are grateful for the financial support for this project [CIG-96-06-A] from the Consortium for Industrial Collaboration in Contraceptive Research (CICCR) Program of the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, USA. The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR. This work was also supported by grants from The Andrew W. Mellon Foundation and by National Institutes of Health grants R37-HD04466 and U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and by the Fogarty International Center Training and Research in Population and Health grant D43TW/HD00627.

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