INTRODUCTION
Serine protease inhibitors are a superfamily of proteins that were first identified as a set of proteins able to inhibit proteases; they play key roles in a variety of physiological and cellular functions and are associated with the vertebrate blood coagulation cascade, complement activation, inflammation, programmed cell death, cell development, and fibrinolysis (Marshall, Reference Marshall1993; Carrell et al. Reference Carrell, Stein, Fermi and Wardell1994; Huber and Carrell, Reference Huber and Carrell1989; Huntington et al. Reference Huntington, Read and Carrell2000; Gettins, Reference Gettins2002). The acronym ‘serpin’ was originally coined because many serpins acted by inhibiting chymotrypsin-like serine proteases (serine protease inhibitors) (Huntington et al. Reference Huntington, Read and Carrell2000). Serpins range in size from 350–400 amino acids with corresponding molecular weights of 40–60 kDa (van Gent et al., Reference van Gent, Sharp, Morgan and Kalsheker2003), and they fall within two basic categories, namely inhibitory and non-inhibitory.
Serpins are thought to have evolved through gene duplication and divergence events, giving rise to a large number of serpin genes within an organism, each encoding a protein with a unique reactive region and physiological function(s) (Hunt and Dayhoff, Reference Hunt and Dayhoff1980). This broad family of proteins was initially identified through similarities between the primary structure of 3 human proteins; anti-thrombin, α 1-protease inhibitor and chicken egg white albumin (Hunt and Dayhoff, Reference Hunt and Dayhoff1980). Over 1000 serpins have now been described in viruses, bacteria, archaea, fungi, plants, eukaryotes and include 36 human proteins; they represent the largest and most diverse family of protease inhibitors (Rawlings et al. Reference Rawlings, Tolle and Barrett2004). Many additional serpins are likely to be identified as more sequenced genomes become available. All serpins so far described have been classified into one of 16 clades, designated A through P, with an additional 10 unclassified ‘orphan’ sequences, all based on phylogenetic relationships (Irving et al. Reference Irving, Pike, Lesk and Whisstock2000). This review discusses serpin structure and function generally, and then details those serpins described to date for helminth parasites, emphasizing their possible biological functions, including their roles in the host-parasite interplay.
SERPIN STRUCTURE AND FUNCTION
Serpin structure
The structural archetype of the serpin superfamily is the main human blood plasma anti-proteolytic inhibitor α 1-antitrypsin (Axelsson and Laurell, Reference Axelsson and Laurell1965). All members of the serpin superfamily have a single common core domain consisting of 3 β-sheets and 8–9 α-helices, and this is responsible for the highly unusual structural and functional properties of these proteins (Gettins, Reference Gettins2002; van Gent et al. Reference van Gent, Sharp, Morgan and Kalsheker2003; Silverman et al. Reference Silverman, Whisstock, Bottomley, Huntington, Kaiserman, Luke, Pak, Reichhart and Bird2010; Whisstock et al. Reference Whisstock, Silverman, Bird, Bottomley, Kaiserman, Luke, Pak, Reichhart and Huntington2010). Serpins are also characterized by the presence of a single protein motif called the reactive centre loop (RCL) (Fig. 1). The RCL (Fig. 1) contains a scissile bond between the P1 and P1′ residues, which is recognized and subsequently cleaved by a target protease. The P1 residue acts as the ‘bait’ amino acid presented in the reactive centre which is thought to mimic the normal substrate of the target enzyme. All amino acids towards the N-terminal side of the scissile bond are labelled in order P1, P2, P3 etc, while those on the C-terminal side are labelled P1′, P2′, P3′ etc (Lawrence et al., Reference Lawrence, Olson, Palaniappan and Ginsburg1994). The selectivity of a serpin for a particular protease is determined absolutely by this RCL (Elliott et al. Reference Elliott, Lomas, Carrell and Abrahams1996; Irving et al. Reference Irving, Pike, Lesk and Whisstock2000). The functionality of serpin family members as either inhibitory or non-inhibitory also depends on the structure of the RCL, which is generally composed of approximately 20 amino acids near the C-terminus and is the fastest evolving region within the serpin nucleotide sequence (Graur and Li, Reference Graur and Li1988). Inhibitory serpins are generally recognized by a consensus pattern in their sequences in the hinge region P17 P16, P15, P14, P12-P9 (Hopkins et al. Reference Hopkins, Carrell and Stone1993). P15 is usually glycine, P14 threonine or serine and positions P12-P9 are occupied by residues with short side-chains such as alanine, glycine or serine (Irving et al. Reference Irving, Pike, Lesk and Whisstock2000). These consensus residues are thought to permit efficient and rapid insertion of the RCL into the A β-sheet whereas, the corresponding regions of non-inhibitory serpins deviate from the consensus.
Fig. 1. Conformational states of serpins as differentiated by the reactive centre loop (RCL) structures (shown in magenta). (a) Native α1AT (adapted from Elliot et al. (Reference Elliott, Pei, Dafforn and Lomas2000)); (b) cleaved α1AT (adapted from Engh et al. (Reference Engh, Lobermann, Schneider, Wiegand, Huber and Laurell1989)); (c) latent anti-thrombin (d) the δ conformation of a variant of α 1-antichymotrypsin. Part of the F-helix is unwound and inserted into the bottom of the A β sheet (orange) (adapted from Gooptu et al. (Reference Gooptu, Hazes, Chang, Dafforn, Carrell, Read and Lomas2000)); (e) polymer of cleaved antitrypsin. In all parts of Fig. 1, the A β sheet is in red, the B β sheet in green and the C β sheet in yellow. The α helices are represented by cylinders coloured blue while the important breach, shutter, gate and hinge regions are shown by the broken circles. Adapted from Irving et al. (Reference Irving, Pike, Lesk and Whisstock2000) with permission from Elsevier.
Five conformational states have been structurally characterized for serpins, differing primarily in their RCL structure (Fig. 1). These conformational states are referred to as native, cleaved, latent, δ and polymeric (van Gent et al. Reference van Gent, Sharp, Morgan and Kalsheker2003). For inhibitory serpins, the native state is characterized by an exposed RCL that is accessible for interaction with a target protease. The transition from native to cleaved state is referred to as ‘stressed to relaxed’ (S–>R) because the cleaved state is generally associated with increased stability (Carrell and Owen, Reference Carrell and Owen1985). This S–>R transition is integral for serpin inhibitory function. The latent state is characterized by the insertion of an uncleaved RCL into the β-sheet-A, and was first described from the crystal structure of Plasminogen Activator Inhibitor-1 (PAI-1) (Mottonen et al. Reference Mottonen, Strand, Symersky, Sweet, Danley, Geoghegan, Gerard and Goldsmith1992). This latent state has also been described in human anti-thrombin (Carrell et al. Reference Carrell, Stein, Fermi and Wardell1994), α 1-antitrypsin (Lomas et al. Reference Lomas, Elliott, Chang, Wardell and Carrell1995) and α 1-antichymotrypsin (Gooptu et al. Reference Gooptu, Hazes, Chang, Dafforn, Carrell, Read and Lomas2000). The δ state represents an intermediate structural conformation between the native and latent states resulting from the oxidation of reactive centre residues as demonstrated with the crystal structure of δ-antichymotrypsin (Gooptu et al. Reference Gooptu, Hazes, Chang, Dafforn, Carrell, Read and Lomas2000). Polymeric forms occur as a result of mutant serpins, aggregating together to form stable polymers. In humans, the aggregation of these mutant serpins in the organs where they are produced results in various human pathologies such as thrombosis, emphysema, cirrhosis and mental disorders (Gils and Declerck, Reference Gils and Declerck1998).
Serpin activity and stoichiometry of inhibition (SI)
As mentioned earlier, the structure of the RCL is a critical feature for serine protease inhibitors to undergo the conformational change necessary for inhibitory activity. Upon recognition and cleavage of the scissile bond between the PI and PI' residues by the target protease, the RCL forms an additional strand which inserts into the β-sheet A, effectively trapping the protease. This mechanism of inhibition involves the formation of a very stable complex between the cleaved inhibitor and the protease, similar in some respects to an enzyme-ligand complex (Irving et al. Reference Irving, Pike, Lesk and Whisstock2000). This tight association results in significant conformational changes in the serpin molecule, including the permanent loss of 37% of the structure and overall distortion of the protease (Huntington et al. Reference Huntington, Read and Carrell2000; Irving et al. Reference Irving, Pike, Lesk and Whisstock2000; van Gent et al. Reference van Gent, Sharp, Morgan and Kalsheker2003). Huntington et al. (Reference Huntington, Read and Carrell2000) showed that this permanent loss of the protease structure is a direct consequence of the limited length of the serpin RCL, which causes the ‘plucking away’ of the protease ester-linked serine from its catalytic partners, hence the name ‘suicide’ substrate inhibitors. The regions within the serpin molecule that are important in controlling and modulating its conformation change include the hinge, the breach, the shutter and the gate (Fig. 1a and 2). The basic mechanism of serpin inhibition is also known as the branched pathway suicide inhibition mechanism (Gettins, Reference Gettins2002). In this system, the protease recognizes and attacks the scissile bond of the reactive centre loop of the serpin thereby cleaving the bond.
Fig. 2. Important domains in serpin conformations. Several regions are important in controlling and modulating serpin conformational changes. The Reactive Centre Loop is involved in protease recognition and conformational transformation as strand 4A after inhibition. The P15–P9 portion of the RCL is called the hinge region. The point of initial insertion of the RCL which is the breach region, located at the top of the A β-sheet. Near the center of A β-sheet is the shutter domain. The breach and shutter are 2 major regions that assist sheet opening and accept the conserved hinge of the RCL when it inserts. The gate region is composed of s3C and s4C strands which has been primarily observed by studies of the transition latency. The image was drawn in chimera using the PDB file of native antitrypsin conformation. Adapted from Khan et al. (Reference Khan, Singh, Azhar, Naseem, Rashid, Kabir and Jairajpuri2011) with permission from the Journal of Amino Acids.
There are 5 steps involved in the serpin inhibitory mechanism which are: (i) formation of an initial non-covalent Michaelis complex with the target protease; (ii) attack of the active-site serine on the peptide bond by the protease resulting in a tetrahedral intermediate (Peterson et al. Reference Peterson, Gordon and Gettins2000); (iii) cleavage of the peptide bond of the serpin to give a covalent acyl ester intermediate with the release of the first product, the free amino group of the peptide bond; (iv) formation of the second tetrahedral intermediate through attack of water; and then (v) departure of the second product (Gettins, Reference Gettins2002). In non-serpin inhibitors, the only step involved in inhibition is the initial recognition, with the specificity and stability of the complex being dependent on the nature and extent of interactions between the two proteins (Gettins, Reference Gettins2002). In contrast, the formation of the initial non-covalent complex between the inhibitory serpins and their target proteases influences the specificity and the rate of reaction since serpin inhibition goes beyond the formation of this non-covalent Michaelis complex.
In a 2D-Nuclear Magnetic Resonance (2D-NMR) study of the complex between S195A trypsin and α 1-PI-Pittsburgh (P1 Met – Arg), Peterson et al. (Reference Peterson, Gordon and Gettins2000) found that, despite the extreme sensitivity of the serpin to conformational changes, as demonstrated by significant shifts of all alanine resonances, the conformation of the serpin body in the complex was still identical to that of the native serpin and no loop insertion of any RCL residue occurred into the β-sheet-A. This observation was later confirmed by Ye et al. (Reference Ye, Cech, Belmares, Bergstrom, Tong, Corey, Kanost and Goldsmith2001), who elucidated the X-ray crystal structure of the complex formed between the protease S195A trypsin and a different serpin (Serpin 1 K). The study by Ye et al. (Reference Ye, Cech, Belmares, Bergstrom, Tong, Corey, Kanost and Goldsmith2001) clearly showed there was no insertion of the RCL into the β-sheet-A and that there was considerable structural similarity between the body of the serpin in the complex and the native serpin.
The major molecular or functional consequence of the first serpin-serine protease inhibition pathway is the continuation of the proteolysis reaction and subsequent release of the cleaved form of the serpin. The second pathway involves the trapping of the acyl intermediate by disrupting the effectiveness of the protease to complete the proteolytic reaction as a result of the conformational change within the serpin and consequent distortion of the protease active site. Regions in the serine protease inhibitor that are crucial for controlling and modulating the conformational change are shown in Fig. 2.
HELMINTH SERPINS AND SMAPINS
A summary of the serpins and small serine protease inhibitors (smapins) identified in parasitic nematodes, trematodes and cestodes is provided in Tables 1 and 2. A description of their individual characteristics now follows.
Table 1. Characteristics of serpins from parasitic helminths
Table 2. Characteristics of nematode smapins
PARASITIC NEMATODE SERPINS AND SMAPINS
Nematodes occupy a relatively low place in invertebrate evolution (Aguinaldo et al. Reference Aguinaldo, Turbeville, Linford, Rivera, Garey, Raff and Lake1997) but many of their parasitic representatives are of significant veterinary and medical importance, particularly as over two billion people are infected in tropical countries (Michael et al. Reference Michael, Bundy and Grenfell1996; Zang et al. Reference Zang, Yazdanbakhsh, Jiang, Kanost and Maizels1999; de Silva et al. Reference de Silva, Brooker, Hotez, Montresor, Engels and Savioli2003; Meeusen et al. Reference Meeusen, Balic and Bowles2005). Useful reviews of parasitic nematode serpins are available (Zang and Maizels, Reference Zang and Maizels2001; Knox, Reference Knox2007). Nematode serpins have limited sequence homology to their mammalian counterparts, although the key amino acid residues required for tertiary structure and functionality are well conserved, with the commonality of hypervariability restricted to the RCL (Zang and Maizels, Reference Zang and Maizels2001). This hypervariability is thought to result from the unusually high rates of non-synonymous substitutions occurring within the reactive site loops (Hill and Hastie, Reference Hill and Hastie1987; Goodwin et al. Reference Goodwin, Baumann and Berger1996). Through the analyses of nucleotide sequences, Zang and Maizels (Reference Zang and Maizels2001) were able to clarify many evolutionary aspects of nematode serpins, specifically by comparing the genomic sequences of 8 genes encoding Caenorhabditis elegans serpins and a novel Brugia malayi serpin gene. The intron map of the 3′ end of the genes showed varied patterns with no single conserved position between the two organisms, and not even within C. elegans itself, suggesting that the extreme divergence in the position of introns may be indicative of the functional constraint for the C-terminus of the protein.
Two distinct nematode serpin families have been identified by this gene sequence database mining (Zang and Maizels, Reference Zang and Maizels2001). One family exhibits particular homology to mammalian serpins in terms of predicted structure based on nucleotide sequence, and a second encodes a totally different novel group of small proteins of less than 100 amino acids. Members of the latter family have been termed smapins (small serine protease inhibitors) and appear unique to parasitic nematodes, with no relatives from free-living nematodes or any other taxa evident (Zang and Maizels, Reference Zang and Maizels2001). Smapins have been identified in Ascaris suum (Grasberger et al. Reference Grasberger, Clore and Gronenborn1994), Anisakis simplex (Kobayashi et al. 2007a), Onchocerca volvulus (Ford et al. Reference Ford, Guiliano, Oksov, Debnath, Liu, Williams, Blaxter and Lustigman2005), Trichuris suis (Rhoads et al. Reference Rhoads, Fetterer and Hill2000a,Reference Rhoads, Fetterer, Hill and Urbanb)), Ancylostoma caninum (Duggan et al. Reference Duggan, Dyson and Wright1999). The major distinguishing feature of the smapin family is the presence of 10 cysteine residues that form 5 disulphide bonds at the protein core. Structural studies of smapins from A. suum (Grasberger et al. Reference Grasberger, Clore and Gronenborn1994) and A. caninum (Duggan et al. Reference Duggan, Dyson and Wright1999), using nuclear magnetic resonance (NMR), identified 2 anti-parallel strands of β-sheets with the remainder of the tertiary structure consisting of extended loops and turns.
Serpins from Brugia malayi
Brugia malayi is one of the causative agents of human lymphatic filariasis. Its life cycle involves mosquitoes and humans and a correspondingly complex set of interactions with the human host immune system (Maizels et al. Reference Maizels, Bundy, Selkirk, Smith and Anderson1993). Only 2 serpin genes, designated Bm-SPN-1 (Yenbutr and Scott, Reference Yenbutr and Scott1995) and Bm-SPN-2 (Zang et al. Reference Zang, Yazdanbakhsh, Jiang, Kanost and Maizels1999), have been characterised so far from B. malayi. Yenbutr and Scott (Reference Yenbutr and Scott1995) employed reverse transcription PCR-based technology, which exploited the presence of a conserved 22-nucleotide spliced sequence present at the 5′ end of a proportion of nematode transcripts, and cloned a PCR product that encoded the first described nematode serine protease inhibitor. Sequence analysis showed that the PCR product was 1287 bp long and the estimated molecular weight of the predicted protein was 44 kDa. Further reverse transcription PCR analysis showed that the protein – termed Bm-SPN-1- was expressed in all life stages of the parasite. These authors also demonstrated that Bm-SPN-1 was immunogenic in gerbils and that it was strongly recognized by sera from immunized animals suggesting that Bm-SPN-1 may play a role in the survival of the parasite during the early phase of its development in the vertebrate host.
In order to identify prominent antigens from blood-borne B. malayi microfilariae (mf) larvae that might be recognized by host T lymphocytes, Zang et al. (Reference Zang, Yazdanbakhsh, Jiang, Kanost and Maizels1999) identified a mf fraction containing proteins of 35-55 kDa in size that proved highly potent at inducing antigen-specific T-cell proliferation and cytokine production. Immunoscreening of an mf cDNA library isolated a clone encoding a native serpin protein termed Bm-SPN-2 with a molecular mass of 47·5 kDa. The expression of Bm-SPN-2 was highly stage-specific, being expressed only in the mf as one of the most abundant proteins of this life-cycle stage (Zang et al. Reference Zang, Yazdanbakhsh, Jiang, Kanost and Maizels1999). Bm-SPN-2 was tested for its ability to inhibit a panel of mammalian serine proteases with differing substrate specificity and functions, but only neutrophil serine protease, elastase and cathepsin G were inhibited in a dose-dependent and highly specific manner. This high specificity of inhibition was confirmed by the fact that Bm-SPN-2 showed no cross reactivity with bovine pancreatic α-chymotrypsin or porcine pancreatic elastase in a dose-specific manner, 2 enzymes with similar substrate specificity to neutrophil cathepsin G and elastase, respectively. It is noteworthy that neutrophil-derived cathepsin G is known to be an important chemoattractant for monocytes (Chertov et al. Reference Chertov, Ueda, Xu, Tani, Murphy, Wang, Howard, Sayers and Oppenheim1997). Mice infected with B. malayi mf mounted a strong, but short-lived Bm-SPN-2-specific Th1 response with significant increases in IFN-γ production (Zang et al. Reference Zang, Yazdanbakhsh, Jiang, Kanost and Maizels1999, Reference Zang, Atmadja, Gray, Allen, Gray, Lawrence, Yazdanbakhsh and Maizels2000). Filariasis patients elicited a potent Th2 immune response to Bm-SPN-2 in both IgG1 and IgG4 antibody subclasses (Zang et al. Reference Zang, Atmadja, Gray, Allen, Gray, Lawrence, Yazdanbakhsh and Maizels2000). Overall, these studies suggested that Bm-SPN-2 functions by neutralizing the immunostimulatory properties of the host cathepsin G, thereby contributing to the longevity and pathogenicity of mf in the mammalian bloodstream.
However, the complete picture regarding the function of Bm-SPN-2 in vivo has yet to be determined, as a subsequent study by Stanley and Stein (Reference Stanley and Stein2003) failed to repeat the earlier results of Zang et al. (Reference Zang, Yazdanbakhsh, Jiang, Kanost and Maizels1999). This group cloned the Bm-SPN-2 gene from a different mf cDNA library, expressed the Bm-SPN-2 protein in E. coli, and characterized its structural and functional properties (Stanley and Stein, Reference Stanley and Stein2003). Sequence alignment, circular dichroism spectroscopy, and susceptibility to cleavage by proteases suggested that the Bm-SPN-2 shared the tertiary structure typical of the serpin family, including an accessible reactive centre loop (Irving et al. Reference Irving, Pike, Lesk and Whisstock2000). However, the protein had no effect on the activity of neutrophil elastase or cathepsin G, did not form SDS-stable complexes with these proteases, and did not undergo the characteristic stressed to relaxed transition required for protease inhibition by serpins. These authors concluded that Bm-SPN-2 was a new non-inhibitory serpin, in keeping with its sequence.
Smapins from Onchocerca volvulus
Onchocerca volvulus is another filarial nematode parasite of humans causing onchocerciasis. Ford and colleagues (2005) adopted a transcriptomics approach to identify novel proteins from O. volvulus involved in the parasite moulting process. Analysis of the datasets derived from expression sequence tags (ESTs) of cDNA libraries constructed from the infective third-stage larva (L3) and molting L3 (mL3) of O. volvulus identified novel cysteine proteases involved in the moulting process (Hashmi et al. Reference Hashmi, Britton, Liu, Guiliano, Oksov and Lustigman2002; Guiliano et al. Reference Guiliano, Hong, McKerrow, Blaxter, Oksov, Liu, Ghedin and Lustigman2004). In addition to these cysteine proteases, these authors also identified a novel family of small molecular weight serine protease inhibitor (Ov-SPI-1 and Ov-SPI-2) with structural similarity to smapins already identified in A. suum (Peanasky et al. Reference Peanasky, Bentz, Paulson, Graham and Babin1984; Martzen et al. Reference Martzen, Geise, Hogan and Peanasky1985, Reference Martzen, Geise and Peanasky1986) and hookworm (Stassens et al. Reference Stassens, Bergum, Gansemans, Jespers, Laroche, Huang, Maki, Messens, Lauwereys, Cappello, Hotez, Lasters and Vlasuk1996). The expression profile for Ov-spi-1 and Ov-spi-2 genes demonstrated that both genes were expressed in all life stages of the parasite with expression increasing during moulting larval stages and reproducing adult worms. Immunolocalization of the native Ov-SPI proteins carried out with specific antibodies raised against rOv-SPI-1 showed that Ov-SPI-1 and -2 were endogenous proteins found within the body channels, multivesicular bodies and in the basal layer of the cuticle of the L3 larva. Protease inhibition assays carried out showed that Ov-SPI-1 reduced the enzymatic activity of a panel of serine proteases including elastase, chymotrypsin, trypsin and cathepsin G. However, although the specific endogenous target enzyme of the Ov-SPI-1 was not identified, the authors suggested that Ov-blisterase, a subtilisin-like serine protease (Poole et al. Reference Poole, Jin and McReynolds2003), could be the potential target of the Ov-SPI proteins since Ov-blisterase was shown to co-localize with Ov-SPI proteins to the same regions of the curticle during moulting of the O. volvulus L3 s.
Indirect evidence for involvement of the Ov-SPIs in immune regulation was reported by Ford et al. (Reference Ford, Guiliano, Oksov, Debnath, Liu, Williams, Blaxter and Lustigman2005) who showed that these proteins are antigenic and strongly recognized by persons previously exposed to O. volvulus, suggesting that Ov-SPIs are released from the parasite during the early stages of the parasite establishment in the host. The mechanism(s) involved in the possible release of these endogenous Ov-SPIs remains unknown.
Serpins from Haemonchus contortus
Haemonchus contortus is an important parasitic nematode of veterinary importance that affects the gastrointestinal tract of ruminant animals, especially sheep, goats and cattle, in various regions of the world (Meeusen et al. Reference Meeusen, Balic and Bowles2005). In a recent study, a serpin from H. contortus termed Hc-Serpin was identified and its biological activities described. The rHc-Serpin inhibited trypsin activity effectively and prolonged the coagulation time of rabbit blood in vivo. Thermostability assays indicated that the rHc-Serpin was thermally inert, maintaining its proteolytic activity even at temperatures above 75°C. Immunohistochemistry, using rat anti-rHc-Serpin antibodies, showed that native Hc-Serpin was localized exclusively to the epithelial cells of the gastrointestinal tract in adult worms (Yi et al. Reference Yi, Xu, Yan and Li2010). Analysis of its deduced amino acid sequence showed the serpin was devoid of a typical signal peptide cleavage site at its N-terminal end, suggesting an intra-cellular location. However, the rHc-Serpin was recognized by serum from goats naturally infected with H. contortus indicating exposure to the host immune system.
Three possible explanations were provided by Yi et al. (Reference Yi, Xu, Yan and Li2010) the authors for the recognition of the Hc-Serpin protein by serum from naturally infected hosts. First, the protein may be exposed to the immune system on the death of the adult nematodes. Second, the larvae (L3, L4, and L5) of H. contortus are killed by the host immune response, and the killing process exposes many internal cytoplasmic components that are expressed by all life stages of the parasite. Third, some intracellular proteins from the L4/L5 larvae and adults of H. contortus may be excreted through undefined pathways and be recognized by the host immune system. It was further suggested that the internalization of the Hc-serpin by host tissues is an active process and that the targets of the serpin are the host proteases rather than endogenous parasite proteases (Yi et al. Reference Yi, Xu, Yan and Li2010). The release of intra-cellular proteins in vitro is thought to depend on the presence of secretory vesicles (Zhang et al. Reference Zhang, Park, Wang, Shah, Liu, Murmann, Wang, Peter and Ashton-Rickardt2006; Merckelbach and Ruppel, Reference Merckelbach and Ruppel2007). However, the precise mechanism as to how the Hc-Serpin is shed into host tissue is still unknown, and the role it plays in the host-parasite interplay warrants further research.
A serpin from Trichinella spiralis
Trichinella spiralis infects the skeletal muscles of a wide variety of vertebrate hosts including pigs, rats, horses, wild animals and humans causing zoonotic trichinellosis (Nagano et al. Reference Nagano, Wu, Nakada, Matsuo and Takahashi2001; Mitreva et al. Reference Mitreva, Jasmer, Zarlenga, Wang, Abubucker, Martin, Taylor, Yin, Fulton, Minx, Yang, Warren, Fulton, Bhonagiri, Zhang, Hallsworth-Pepin, Clifton, McCarter, Appleton, Mardis and Wilson2011). Nagano et al. (Reference Nagano, Wu, Nakada, Matsuo and Takahashi2001) isolated a cDNA clone – Ts11-1 – from a cDNA library constructed from the muscle larvae of T. spiralis that encoded a recombinant protein with protease inhibitory activity. The 42 kDa recombinant protein encoded by the Ts11-1 clone was cloned, expressed in a prokaryotic system and purified. Multiple sequence alignment of the predicted amino acid sequence of the Ts11-1 clone with serpins from Caenorhabditis elegans serpin and B. malayi (Bmserp) indicated that Ts11-1 was a serpin because of its sequence homology to these proteins at the putative reactive region. This conclusion was further strengthened by the inhibition of trypsin activity in vitro when co-incubated with recombinant Ts11-1. Nagano et al. (Reference Nagano, Wu, Nakada, Matsuo and Takahashi2001) showed that Ts11-1 was expressed only in the early developmental stage of the muscle larvae of T. spiralis, but further studies are required to more fully understand its biochemical and biological functions.
A serpin from Trichostrongylus vitrinus
Trichostrongylus spp. are the cause of ovine parasitic gastroenteritis. MacLennan et al. (Reference MacLennan, McLean and Knox2005) isolated a novel serpin (TvSERP) cDNA from Trichostrongylus vitrinus following the screening of a cDNA library prepared from adult worms with rabbit antisera to adult excretory/secretory products. Sequence analysis of the predicted protein sequence for TvSERP indicated the absence of a signal sequence but the presence of 4 N-linked glycosylation sites. A phylogenetic comparison of the TvSERP sequence with serpins from other invertebrates and vertebrates showed that the protein was most closely related to C. elegans serpins. Immunoblot analysis showed that TvSERP was expressed in all life-cycle stages of the parasite and that it formed complexes with other T. vitrinus proteins suggesting a functional role in regulating its endogenous proteases. The serpin was recognized by antibodies in the serum and lymph of lambs immunized with recombinant TvSERP. Protease inhibition assays showed that TvSERP inhibited not only serine proteases of T. vitrinus origin but also those produced by the host, including those of potential importance for host anti-parasite immune responses such as mast cell proteases (Miller, Reference Miller1984). Although these data did not prove a specific biological function for TvSERP, they did indicate possible roles in the regulation of T. vitrinus serine proteases as well as in modulation of the host immune response by inhibiting the activity of serine proteases released from host inflammatory cells (MacLennan et al. Reference MacLennan, McLean and Knox2005). Additional studies are necessary to more fully understand the complete biological role of TvSERP and its possible function in worm survival in the host intestine.
Smapins from Ascaris spp
Two early studies showed that the activities of host proteases such as trypsin and chymotrypsin, disappeared from the micro-environment of live Ascaris suum with a functioning gastrointestinal system (Juhasz and Nemeth, Reference Juhasz and Nemeth1979; Hogan, Reference Hogan1980). Both studies revealed that the only proteases removed from the environment were those for which the parasite had developed inhibitors, although the mechanism involved with the disappeance of the proteases was not determined. A subsequent immunolabelling study by Martzen et al. (Reference Martzen, Geise, Hogan and Peanasky1985) showed that host chymotrypsin co-localized with A. suum chymotrypsin/elastase isoinhibitors in the muscle sarcolemma, in developing eggs and larvae, as well as at the epithelial surface of the gut of the adult parasite; inactive complexes were formed, indicating a possible role in protecting the parasite from host digestive attack. The serpin-host protease complex formation may also mask the surface of the developing migrating larvae and promote effective evasion from the host immune system (Martzen et al. Reference Martzen, Geise, Hogan and Peanasky1985, Reference Martzen, Geise and Peanasky1986).
Five isoinhibitors (1–5) of chymotrypsin/elastase have been isolated and purified from A. lumbricoides by CM-Sephadex C-25 column affinity chromatography (Peanasky et al. Reference Peanasky, Bentz, Paulson, Graham and Babin1984). They comprise 63–66 amino acids with 10 cysteine residues (Babin et al. Reference Babin, Peanasky and Goos1984), the characteristic feature of smapins. Protease inhibition assays carried out with these 5 isolated isoinhibitors showed that each reacted more strongly with chymotrypsin than any other serine protease tested. The assays showed also that these isoinhibitors reacted very strongly with porcine elastase-1 suggesting that chymotrypsin and elastase may be the possible targets of these inhibitors. Nevertheless, the precise roles that these isoinhibitors might play in the survival of A. lumbricoides in the host intestine remain unknown.
Smapins from Anisakis simplex
Anisakis simplex is a marine nematode worm parasite of fish that frequently causes gastrointestinal symptoms in humans, which may be associated with mild to severe immunological, usually allergic-type, reactions (Audicana and Kennedy, Reference Audicana and Kennedy2008). To date, 8 A. simplex allergens have been described at the molecular level (Ani s 1 to Ani s 8) (Audicana and Kennedy, Reference Audicana and Kennedy2008). Of these, the ES-derived Ani s 6 is a smapin and the first identified nematode serpin causing allergy in humans; it was shown to inhibit α-chymotrypsin but not trypsin in a dose-dependent manner, and may act as a blood anticoagulant inhibiting the serine proteases, factors Xa and VIIa (Audicana and Kennedy, Reference Audicana and Kennedy2008; Kobayashi et al. Reference Kobayashi, Ishizaki, Shimakura, Nagashima and Shiomi2007a,Reference Kobayashi, Shimakura, Ishizaki, Nagashima and Shiomib). Earlier, Lu et al. (Reference Lu, Nguyen, Morris, Hill and Sakanari1998) isolated 3 elastase isoinhibitors from A. simplex and reported the presence of a hypervariable region within the reactive site centres; sharing 95–98% amino acid sequence identity, these serpins may be involved in reproduction although the serine proteases they inhibit have not been determined.
Smapins from Ancylostoma caninum
Hookworms cause anaemia in their mammalian hosts as they feed on blood from capillaries of the small intestine (Cappello et al. Reference Cappello, Vlasuk, Bergum, Huang and Hotez1995). Like other haematophagous invertebrates, hookworms have evolved potent anti-clotting strategies to facilitate blood feeding. Three different smapins with anticoagulant properties (NAP5, NAP6 and NAPc2) were identified and characterized from the dog hookworm, Ancylostoma caninum by Duggan et al. (Reference Duggan, Dyson and Wright1999). These NAPs are 75–84 residues long and contain the 10 cysteine residues, paired into 5 disulfides, typical of smapins. Being highly potent and specific inhibitors of the serine proteases, factors VIIa and Xa, the key physiological initiators of blood coagulation, they have been targeted as novel anticoagulants for treatment of thrombotic disorders.
Earlier, Cappello et al. (Reference Cappello, Vlasuk, Bergum, Huang and Hotez1995) purified and biochemically characterized another hookworm-derived blood-clotting inhibitor of human coagulation factor Xa, termed A. caninum anticoagulant peptide (AcAP). Amino acid analysis of the purified protein showed that this inhibitor was made up of 71 amino acids with a molecular weight of 16·5 kDa. Protease inhibition assays carried out with several serine proteases indicated that AcAP specifically inhibited factor Xa and not trypsin, chymotrypsin or thrombin. Pro-thrombin time (PT) and activated partial thromboplastin time (PTT) are standard blood-clotting time assays used to measure the time it takes for blood to clot and AcAP was shown to prolong both, suggesting that interfering with the ability of the adult worm to feed on host blood may lessen the morbidity of chronic hookworm infection. Determination of the first 30 amino acids of the recombinant AcAP revealed a unique partial sequence with heterogeneity at 2 distinct positions suggesting the presence of more than one protein responsible for the anticoagulant activity observed.
This hypothesis was subsequently confirmed by Stassens et al. (Reference Stassens, Bergum, Gansemans, Jespers, Laroche, Huang, Maki, Messens, Lauwereys, Cappello, Hotez, Lasters and Vlasuk1996) who identified and characterized 3 homologous small protein anticoagulants from A. caninum, termed AcAPc2, AcAPc5 and AcAPc6; these authors showed that AcAPc5 and AcAPc6 directly inhibited factor Xa while AcAPc2 predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor fVIIa/TF. Homologues of AcAPc2 (AcAPc3 and AcAPc4) with the same substrate specificity have also been characterised (Mieszczanek et al. Reference Mieszczanek, Harrison, Vlasuk and Cappello2004). Very recently, another novel small serine protease inhibitor anticoagulant peptide, designated Ac-AP-12, was identified and shown to be expressed exclusively in the adult stage of the parasite (Jiang et al. Reference Jiang, Zhan, Mayor, Gillespie, Keegan, Bottazzi and Hotez2011). RT-PCR, Western blotting and immunolocalization studies with an anti-Ac-AP-12 rabbit anti-serum showed that the protein was expressed only in the adult stage of the parasite. Multiple sequence analysis of the predicted amino acid sequence of the protein showed 43–60% identity to the other anticoagulant peptides previously described in A. caninum. Phylogenetic analysis showed that Ac-Ap-12 belongs to the group of factor Xa inhibitors (Jiang et al. Reference Jiang, Zhan, Mayor, Gillespie, Keegan, Bottazzi and Hotez2011) and, like the other A. caninum serpins, it may be suitable for development as a blood-clotting agent.
Serpins from Trichuris suis
The swine whipworm, Trichuris suis, inhabits the caecum and colon of infected pigs and can cause severe mucohaemorrhagic enteritis. Rhoads et al. (Reference Rhoads, Fetterer and Hill2000a) identified a trypsin inhibitor, termed TsTCI, in extracts of adult T. suis and culture fluid from a 24-h in vitro cultivation of adult parasites. Elastase, thrombin, and factor Xa were not inhibited. The cDNA-derived amino acid sequence of the mature TsTCI consisted of 61 residues including 8 cysteine residues with a molecular weight of 6·687 kDa.
The same group (Rhoads et al. Reference Rhoads, Fetterer, Hill and Urban2000b) purified another serpin, termed TsCEI, with an estimated molecular weight of 6·437 kDa from adult T. suis. TsCEI potently inhibited both chymotrypsin and pancreatic elastase. Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1) and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6·196 kDa. TsCEI displayed 48% sequence identity to TsTCI. These two smapins from T. suis may function as components of a parasite defence mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses (Rhoads et al. Reference Rhoads, Fetterer and Hill2000a,Reference Rhoads, Fetterer, Hill and Urbanb).
TREMATODE SERPINS
Serpins from Schistosoma spp
Schistosomes have evolved highly efficient mechanisms, including the expression of serpins to counteract potentially damaging host proteases, which allow them to persist long term in their hosts (Blanton et al. Reference Blanton, Licate and Aman1994).
In an attempt to identify protein(s) in schistosomes that may be involved in inhibiting host clotting mechanisms, Blanton et al. (Reference Blanton, Licate and Aman1994) screened a cDNA library constructed from Schistosoma haematobium with specific human antisera and identified a clone termed SHW 4-2, with a predicted amino acid sequence belonging to the serpin gene superfamily. Analysis of the cDNA clone showed that the sequence had 1 open reading frame predicting a 409 amino acid protein. Multiple sequence alignment revealed that the SHW 4-2 cDNA exhibited greatest sequence similarity to the glial-derived nexins and anti-thrombin whose specific targets are thrombin (Monard et al. Reference Monard, Reinhard, Meier, ▪, ▪, Farmer, Rovelli and Ortmann1990), indicating a possible role in inhibition of blood coagulation. Immunolocalization studies showed that the S. haematobiun serpin was present on the surface of the parasite and, therefore, able to interact with host cells and proteases. The serpin was species-specific being recognized only by sera from S. haematobium-infected individuals (Blanton et al. Reference Blanton, Licate and Aman1994). The species specificity of this serpin was subsequently confirmed by Li et al. (1995) who, additionally, characterized the human IgG4 and IgE antibody isotype responses to the molecule. The crystal structure of this S. haematobium serpin was obtained by Huang et al. (Reference Huang, Haas, Biesterfeldt, Mankawsky, Blanton and Lee1999) who demonstrated that the protein formed a tight covalent complex with human trypsin in vitro, suggesting that the parasite might be using this serpin-trypsin complex to evade the host immune response by reducing the immunogenicity of the exposed serpin. Another possibility might be that the parasite uses this serpin-host trypsin complex to reduce the proteolytic activity of the host proteases.
Ghendler et al. (Reference Ghendler, Arnon and Fishelson1994) isolated and characterized another novel serpin from S. mansoni. The serpin was partially purified from an adult worm extract by gel filtration on an HPLC superose-12 column as a complex with a 28 kDa protease, and the protease-inhibitor complex immunoprecipitated with rabbit anti-28 kDa protease antibodies. Analysis of the immunoprecipitated proteins by SDS-PAGE and autoradiography demonstrated a major band at 74 kDa which represented a protease-inhibitor complex. Incubation of [35S] methionine-labelled adult worm extracts with biotinylated elastase and subsequent precipitation with streptavidin-agarose isolated the 74 kDa band and 2 other smaller bands of 64 kDa and 56 kDa. Antibodies raised in rabbits against the inhibitor-biotinylated elastase-streptavidin-agarose complex immunoprecipitated a protein of 56 kDa from metabolically labelled and extracted AW proteins; hence this novel AW protease inhibitor was named S. mansoni protease inhibitor56 (Smpi56) (Ghendler et al. Reference Ghendler, Arnon and Fishelson1994). Protease inhibition assays showed that Smpi56 strongly bound and inhibited human neutrophil elastase suggesting that Smpi56 might protect the parasite from elastase released from neutrophils.
A schistosome homologue of mouse contrapsin – a serpin present in serum that reacts specifically with trypsin-like proteases (Nathoo et al. Reference Nathoo, Rasums, Katz, Ferguson and Finlay1982; Takahara and Sinohara, Reference Takahara and Sinohara1983a,Reference Takahara and Sinoharab) – has been identified in S. mansoni adult worm homogenates (Modha and Doenhoff, Reference Modha and Doenhoff1994). Modha and Doenhoff (Reference Modha and Doenhoff1994) demonstrated that contrapsin from mouse serum and from S. mansoni homogenates were immunologically identical, despite the significant difference in their molecular weights. These authors showed that contrapsin is a tegumental protein which bound to and inhibited host trypsin with high specificity and the binding caused the serpin to lose its immunogenicity so that an antibody response was not mounted (Modha and Doenhoff, Reference Modha and Doenhoff1994). Additional studies are required to determine the precise biological function(s) of this S. mansoni serpin and to investigate further its possible role in host immune evasion.
Microtus fortis is an Asian vole that is naturally resistant to S. japonicum infection (He et al. Reference He, Luo, Zhang, Yu, Lin, Li, Li and Liu1999). With the aim of identifying S. japonicum molecules associated with this resistance, Yan et al. (Reference Yan, Liu, Song, Xu and Dissous2005) screened an adult worm cDNA expression library with sera from M. fortis and identified a cDNA clone that encoded a sequence homologous to the serpin superfamily. Full-length sequence analysis of the Sj serpin clone revealed a 1200-bp open reading frame encoding a protein of 400 amino acids. Multiple sequence alignment of the S. japonicum reactive centre loop (RCL) showed high sequence similarity with serpins from S. mansoni (Smserpin Accession number AAA29938) and S. haematobium (SH serpin) (Huang et al. Reference Huang, Haas, Biesterfeldt, Mankawsky, Blanton and Lee1999). Sj serpin is a tegumental protein that is only expressed in the adult and cercarial stages of the parasite. C57BL/6 mice immunized with the Sj serpin induced the production of high levels of specific IgE and IgG1 antibodies as well as a marked IL-4 response. Lymphocyte surface marker analysis revealed proliferation of CD19-expressing B cells, indicating a predominant Th2-type response to the serpin. Immunized mice developed some protection against S. japonicum suggesting a potential role for Sj serpin as a vaccine candidate or as a novel target for anti-schistosome drugs although additional study is required to characterize the precise biological function(s) of this protein as well as its possible role in host immune modulation.
Using phylogenetic analysis, published sequences and information from the completed and annotated genomes of S. mansoni (Berriman et al. Reference Berriman, Haas, LoVerde, Wilson, Dillon, Cerqueira, Mashiyama, Al-Lazikani, Andrade, Ashton, Aslett, Bartholomeu, Blandin, Caffrey, Coghlan, Coulson, Day, Delcher, DeMarco, Djikeng, Eyre, Gamble, Ghedin, Gu, Hertz-Fowler, Hirai, Hirai, Houston, Ivens, Johnston, Lacerda, Macedo, McVeigh, Ning, Oliveira, Overington, Parkhill, Pertea, Pierce, Protasio, Quail, Rajandream, Rogers, Sajid, Salzberg, Stanke, Tivey, White, Williams, Wortman, Wu, Zamanian, Zerlotini, Fraser-Liggett, Barrell and El-Sayed2009) and S. japonicum (Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium, 2009), Quezada and McKerrow (Reference Quezada and McKerrow2011) identified 2 major serpin clades with homology to the gene encoding human α1-antitrypsin; there were 8 serpin gene sequences in the S. mansoni database compared with 4 serpin genes in the S. japonicum gene database. Most of the variation in serpin genes occurred in the reactive centre loop (RCL) and these authors suggested the greater multiplicity of serpin genes in S. mansoni perhaps reflects adaptation to infection of the human host.
Serpins from Clonorchis sinensis
Clonorchis sinensis is endemic to Southeast Asia, resides in the liver of humans and many other mammals, and causes clonorchiasis (Lun et al. Reference Lun, Gasser, Lai, Li, Zhu, Yu and Fang2005). A cDNA of 1149 bp encoding a novel serpin of 42·2 kDa (CsproSERPIN) has been isolated and characterized from C. sinensis (Yang et al. Reference Yang, Hu, Wang, Liang, Hu, Wang, Chen, Xu and Yu2009). Semi-quantitative RT-PCR analysis of the infective metacercaria and adults showed a higher level of CsproSERPIN expression in the former suggesting an important biological role, possibly in metacercarial excystment (Yang et al. Reference Yang, Hu, Wang, Liang, Hu, Wang, Chen, Xu and Yu2009). Kang et al. (Reference Kang, Sohn, Ju, Kim and Na2010) biochemically characterized another serpin (CsSERPIN) with a molecular weight of 44 kDa from C. sinensis. While transcriptional analysis of CsSERPIN showed expression in all developmental stages of the parasite, the highest levels were seen in adults and eggs. Amino acid sequence analysis demonstrated that CsSERPIN lacked a N-terminal signal peptide, a C-terminal extension and a transmembrane domain, suggesting a cytosolic location, a feature supported by phylogenetic and immunoblotting analyses (Kang et al. Reference Kang, Sohn, Ju, Kim and Na2010). Immunofluorescence studies showed that CsSERPIN was localized in the eggs within the uterus and in the vitelline glands of adult worms (Kang et al. Reference Kang, Sohn, Ju, Kim and Na2010). Protease inhibition assays carried out with a panel of mammalian serine proteases revealed that CsSERPIN inhibited the enzymatic activity of chymotrypsin in a dose-dependent manner but showed little or no inhibitory activity against trypsin, thrombin, elastases or cathepsin G. Due to its localization in the uterine eggs, CsSERPIN may be involved in the development and/or maturation of the miracidia within the egg by modulating the activities of the parasite's endogenous serine proteases (Kang et al. Reference Kang, Sohn, Ju, Kim and Na2010).
A serpin from Paragonimus westermani
Paragonimus westermani is of both public health and veterinary importance, causing pulmonary and/or extrapulmonary granulomatous disease in humans and other mammalian hosts. Hwang et al. (Reference Hwang, Lee, Na, Lee, Cho and Kim2009) obtained a complete cDNA sequence encoding a novel serine protease inhibitor (PwSERPIN) from P. westermani during analysis of EST sequences randomly selected from an adult worm cDNA library. Subsequent analysis of the PwSERPIN sequence indicated that it was probably a cytosolic protein (Hwang et al. Reference Hwang, Lee, Na, Lee, Cho and Kim2009). Although PwSERPIN was shown to be expressed in all stages of the life cycle there was a clear gradual increase in transcription levels as the parasite developed from metacercaria to adult. It effectively inhibited porcine trypsin, bovine chymotrypsin and human thrombin but had little inhibitory activity against human neutrophil cathepsin G or human and porcine elastases, suggesting a role in the regulation of endogenous cytosolic serine proteases (Hwang et al. Reference Hwang, Lee, Na, Lee, Cho and Kim2009).
CESTODE SERPINS
The cestodes are a highly diversified group and cause a range of diseases including echinococcocosis, and taeniasis/cysticercosis (Spakulová et al. Reference Spakulová, Orosová and Mackiewicz2011).
Serpins from Echinococcus spp
Antigen B (EgAgB) is a highly immunogenic protein produced in great abundance by the larval hydatid cyst of Echinococcus granulosus (Li et al. Reference Li, Zhang, Wilson, Ito and McManus2003; Zhang et al. Reference Zhang, Li and McManus2003, Reference Zhang, Li, Jones, Zhang, Zhao, Blair and McManus2010). The protein, encoded by 5 subclasses of at least 10 genes (Zhang et al. Reference Zhang, Li, Jones, Zhang, Zhao, Blair and McManus2010), is synthesized and secreted by the cyst germinal layer and protoscoleces (Sanchez et al. Reference Sanchez, March, Mercader, Coll, Munoz and Prats1991) but its precise function remains unclear.
With the aim of identifying E. granulosus antigens that might interfere with the host immune response, Shepherd et al. (Reference Shepherd, Aitken and McManus1991) isolated and characterized the smallest 12 kDa subunit of EgAgB. Multiple sequence alignment of the deduced amino acid sequence of the 12 kDa-subunit with baboon and human α−1 antitrypsin amino acid sequences showed some shared sequence homology but not with the reactive site. Subsequent protease inhibition assays demonstrated that the electrophoretically purified 12-kDa antigen inhibited the activity of porcine elastase at similar concentrations as commercially produced α−1 antitrypsin; furthermore, the 12 kDa antigen inhibited human neutrophil chemotaxis indicating that the native protein might play an important role in the survival of the parasite in an immunocompetent host (Shepherd et al. Reference Shepherd, Aitken and McManus1991). Although these data suggested that E. granulosus antigen B was a serpin due to its sequence similarity with other well characterized serpins as well as its capacity to inhibit serine proteases, further studies are required to determine the precise role of this protein family in the biology of E. granulosus.
Merckelbach and Ruppel (Reference Merckelbach and Ruppel2007) cloned and bacterially expressed a serpin gene (serpinEmu) from E. multilocularis and tested the inhibitory potential of the purified recombinant protein against a number of mammalian proteases involved in cellular immune defense, blood clotting and digestion. Multiple sequence alignment of its deduced amino acid sequence with mammalian serpins suggested serpinEmu is an intracellular protein due to the lack of a signal sequence and no N- or C-terminal extensions. Protease inhibition assays showed that serpinEmu inhibited mammalian trypsin and pancreatic elastase (PE) with high specificity but no inhibition was evident with cathepsin G or chymotrypsin. SerpinEmu was highly expressed in E. multilocularis oncospheres, likely playing a similar role to that of the human intracellular serpin B9 in cytotoxic lymphocytes, which is thought to protect immune effector cells against endogenous proteases (Hirst et al. Reference Hirst, Buzza, Bird, Warren, Cameron, Zhang, Ashton-Rickardt and Bird2003; Zhang et al. Reference Zhang, Park, Wang, Shah, Liu, Murmann, Wang, Peter and Ashton-Rickardt2006).
Trypsin and PE, which were most readily inhibited by serpinEmu are mammalian digestive enzymes, suggesting a probable extracellular role for serpinEmu, a hypothesis supported by the fact that plasminogen-activator inhibitor 2, an intracellular serpin, has been shown to be secreted by monocytes through a pathway independent of the endoplasmic reticulum and Golgi apparatus (Ritchie and Booth, Reference Ritchie and Booth1998). A serpin lacking a signal sequence has also been shown to be excreted into the saliva of the ectoparasitic tick, Ixodes ricinus (Prevot et al., Reference Prevot, Adam, Boudjeltia, Brossard, Lins, Cauchie, Brasseur, Vanhaeverbeek, Vanhamme and Godfroid2006). Merckelbach and Ruppel (Reference Merckelbach and Ruppel2007), therefore, suggested that, if serpinEmu were to be excreted by E. multilocularis oncospheres, it might be able to block attack by host digestive enzymes thereby making this serpin an important target of the intestinal immune system and a possible candidate for vaccine development.
A PHYLOGENY OF HELMINTH PARASITE SERPINS
Comprehensive phylogenetic analysis of a number of the helminth serpins discussed in this review was undertaken in order to shed some light on their evolutionary relationships. The phylogenetic analysis assigned the serpins to 4 major branches (Fig. 3). Branch 1 consists of Hc-Serpin and Tv-SERP clustering closely together with Bmserp and BmSERPIN more distantly related but still falling within this grouping. The second major branch comprises Ts11-1 and A. suum serpin. The third major branch includes sm_serpin, (gi256082483), sm_serpin (gi256082483), Sj serpin and SJCHGC00560 which cluster closely together suggesting a possible common ancestry. Branch 4 consists of CsproSERPIN, CsSERPIN and PwSERPIN. Suprisingly, the phylogenetic tree reveals that CsSERPIN is more closely related to PwSERPIN than CsproSERPIN (Fig. 3). The clustering pattern of serpins from branches 1 and 2 as well as those of branches 3 and 4 is not surprising given the representatives belong to the same nematode or trematode classes, respectively. The E. multilocularis serpin (serpin_Emu) is distantly related to those of the other helminths, suggesting early evolutionary divergence.
Fig. 3. Multifurcating phylogenetic tree showing relationships between a number of the helminth parasite serpins described in this review. The 14 serpins were aligned using MUSCLE and a bootstrapped maximum likelihood tree was generated using PhyML 3.0. The branch bootstrap support values are shown on branch splits. The analysis was carried out as described by Dereeper et al. (Reference Dereeper, Guignon, Blanc, Audic, Buffet, Chevenet, Dufayard, Guindon, Lefort, Lescot, Claverie and Gascuel2008).
FINAL COMMENTS
Although recognized for their involvement in many important endogenous regulatory processes, it has been suggested that serpins from pathogens, including those of helminth parasite origin, may have evolved specifically to limit or hinder the activation of the host immune response by inhibiting enzymes involved in generating immuno-stimulatory signals (Chopin et al. Reference Chopin, Bilfinger, Stefano, Matias and Salzet1997; Chopin, Reference Chopin, Matias, Stefano and Salzet1998a,Reference Chopin, Stefano and Salzetb). Many of the studies presented here strongly support the idea that serpins not only perform endogenous physiological and regulatory functions in parasitic helminths but may also be actively involved in host-parasite interplay as well as possible host immune modulation and/or evasion processes. These findings highlight the potential of serpins and smapins as possible drug targets as well as potential anti-helminthic vaccine candidates. Additional studies, building on the findings presented in this review are, however, needed to functionally characterize the biological importance of the native molecules from each of the parasitic helminth species. With the recent publication of the draft genomes of B. malayi (Ghedin et al. Reference Ghedin, Wang, Spiro, Caler, Zhao, Crabtree, Allen, Delcher, Guiliano, Miranda-Saavedra, Angiuoli, Creasy, Amedeo, Haas, El-Sayed, Wortman, Feldblyum, Tallon, Schatz, Shumway, Koo, Salzberg, Schobel, Pertea, Pop, White, Barton, Carlow, Crawford, Daub, Dimmic, Estes, Foster, Ganatra, Gregory, Johnson, Jin, Komuniecki, Korf, Kumar, Laney, Li, Li, Lindblom, Lustigman, Ma, Maina, Martin, McCarter, McReynolds, Mitreva, Nutman, Parkinson, Peregrin-Alvarez, Poole, Ren, Saunders, Sluder, Smith, Stanke, Unnasch, Ware, Wei, Weil, Williams, Zhang, Williams, Fraser-Liggett, Slatko, Blaxter and Scott2007) T. spiralis (Mitreva et al. Reference Mitreva, Jasmer, Zarlenga, Wang, Abubucker, Martin, Taylor, Yin, Fulton, Minx, Yang, Warren, Fulton, Bhonagiri, Zhang, Hallsworth-Pepin, Clifton, McCarter, Appleton, Mardis and Wilson2011) and A. suum (Jex et al. Reference Jex, Liu, Li, Young, Hall, Li, Yang, Zeng, Xu, Xiong, Chen, Wu, Zhang, Fang, Kang, Anderson, Harris, Campbell, Vlaminck, Wang, Cantacessi, Schwarz, Ranganathan, Geldhof, Nejsum, Sternberg, Yang, Wang and Gasser2011), more serpin genes are likely to be identified by data mining and, with their subsequent biochemical characterization, more light will be shed on their roles in the biology of the parasitic helminths. In turn, this may lead to the identification of further intervention targets against this important group of pathogens.
FINANCIAL SUPPORT
The authors’ research has received financial support from various sources including: the UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases; the National Health and Medical Research Council (NHMRC) of Australia; the Wellcome Trust (UK); the Sandler Foundation (USA); the Dana Foundation (USA); and the National Institute of Allergy and Infectious Diseases. Donald P. McManus is a NHMRC senior principal research fellow. A. J. Molehin is supported by a University of Queensland International Research Tuition Award (UQIRTA) and University of Queensland Research Scholarship (UQRS).
