Parasitoids introduce various virulence factors when parasitism occurs, and some taxa generate teratocytes to manipulate the host immune system and metabolic homeostasis for the survival and development of their progeny. Host-parasitoid interactions are extremely diverse and complex, yet the evolutionary dynamics are still poorly understood. A category of serpin genes, named CvT-serpins, was discovered to be specifically expressed and secreted by the teratocytes of Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella. Genomic and phylogenetic analysis indicated that the C. vestalis serpin genes are duplicated and most of them are clustered into 1 monophyletic clade. Intense positive selection was detected at the residues around the P1-P1' cleavage sites of the Cv-serpin reactive center loop domain. Functional analyses revealed that, in addition to the conserved function of melanization inhibition (CvT-serpins 1, 16, 18, and 21), CvT-serpins exhibited novel functions, i.e. bacteriostasis (CvT-serpins 3 and 5) and nutrient metabolism regulation (CvT-serpins 8 and 10). When the host-parasitoid system is challenged with foreign bacteria, CvT-serpins act as an immune regulator to reprogram the host immune system through sustained inhibition of host melanization while simultaneously functioning as immune effectors to compensate for this suppression. In addition, we provided evidence that CvT-serpin8 and 10 participate in the regulation of host trehalose and lipid levels by affecting genes involved in these metabolic pathways. These findings illustrate an exquisite tactic by which parasitoids win out in the parasite-host evolutionary arms race by manipulating host immune and nutrition homeostasis via adaptive gene evolution and neofunctionalization.

Twenty-seven serpins belonging to clade A, B, C, D, E, F, G, H and I serpins are currently referenced in chicken genome databases. Phylogenetic analysis of chicken serpins revealed that ovalbumin (Serpinb14) and its paralogs ovalbumin-related protein Y (Serpinb14b) and ovalbumin-related protein X (Serpinb14c) are found in bird species. These clade B serpins are specifically expressed in reproductive tissues and exported in the egg where they constitute major protein components. These data suggest that these three paralogs have probably appeared in birds to face new environments and ensure the extra-uterine development of an embryo in a shell egg. Twelve other serpins have been identified in the newly produced egg, some of them having a specific distribution in the respective egg structures (eggshell, egg white, vitelline membrane and egg yolk). The physiological role of these egg serpins remain largely unexplored, but there is increasing evidence in literature or by homologies with their mammalian counterparts, that some of them participate in cell proliferation, tissue remodeling and/or angiogenesis associated with folliculogenesis and development of extraembryonic structures, eggshell biomineralization, egg defense and nutrition of the embryo. A better knowledge of the phylogenetic evolution of these 15 serpins in other oviparous species, on their egg distribution, on their regulation during embryonic development (activation/degradation/transfer) and on their functional specificity, is needed to better appreciate their role and their bird-specificity. These review shed light on the multiple possibilities that offer the avian egg model to study the role of serpins in reproduction and developmental biology.

Most members of the serpin family of proteins are potent, irreversible inhibitors of specific serine or cysteine proteinases. Inhibitory serpins are distinguished from members of other families of proteinase inhibitors by their metastable structure and unique suicide-substrate mechanism. Animal serpins exert control over a remarkable diversity of physiological processes including blood coagulation, fibrinolysis, innate immunity and aspects of development. Relatively little is known about the complement of serpin genes in plant genomes and the biological functions of plant serpins.

Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Most serpins inhibit serine and/or cysteine proteases, and their inhibitory activities are usually defined in vitro. However, the physiological protease targets of most serpins are unknown despite many years of research. This may be due to the rapid degradation of the inactive serpin:protease complexes and/or the conditions under which the serpin inhibits the protease. The model organism Caenorhabditis elegans is an ideal system for identifying protease targets due to powerful forward and reverse genetics, as well as the ease of creating transgenic animals. Using combinatorial approaches of genetics and biochemistry in C. elegans, the true in vivo protease targets of the endogenous serpins can be elucidated.

Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (Ki = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (Ki = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.

Serpins are a family of serine protease inhibitors that regulate physiological functions and are found widely in animals, plants and microorganisms. However, there are few reports on the role of serpins in the Chinese mitten crab, Eriocheir sinensis. We cloned two serpin genes from E. sinensis, named Esserpin-2 and Esserpin-3. Quantitative and semi-quantitative real-time PCR results showed that Esserpin-3 mRNA transcripts were detected in all the examined E. sinensis tissues, but were most highly expressed in immune tissues, including the gills, hepatopancreas and intestines. Lower levels of Esserpin-3 expression were detected in the accessory gland, testis and stomach, while the muscle and heart showed minimal expression. Esserpin-3 was differentially expressed throughout testis developmental stages. Esserpin-3 protein was localized in the tube wall and lumen of the accessory gland. In the testes, Esserpin-3 was detected in spermatogonia, spermatocytes and spermatids. As Esserpin-3 was also detected in the sperm contained within the seminal vesicles, it is suggested to be an intrinsic sperm protein. During the acrosome reaction, Esserpin-3 expression reduced gradually and completely disappeared after the reaction. Together, our results indicate that Esserpin-3 is an intrinsic sperm protein involved in regulation of sperm maturation and the acrosome reaction in E. sinensis.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.

Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF.

The serine protease inhibitor (serpin) gene family is the largest family of protease inhibitors. Serine protease inhibitors have an active, but under-characterized, role in grain development and defense against pathogen attack in cereal crops. By exploiting publicly available genomic, transcriptomic and proteomic data for wheat (Triticum aestivum), we have identified and annotated the entire 'serpinome' of wheat and constructed a high-quality and robust phylogenetic tree of the gene family, identifying paralogous and homeologous clades from the hexaploid wheat genome, including the Serpin-Z group that have been well characterized in barley. Using publicly available RNAseq data (http://www.wheat-expression.com/), expression profiles of the wheat serpins were explored across a variety of tissues from the developing grain, spikelet and spike. We show that the SERPIN-Z clade, among others, are highly expressed during grain development, and that there is homeologous and paralogous functional redundancy in this gene family. Further to their role in grain development, serpins play an important but under-explored role in response to fungal pathogens. Using 13 RNAseq datasets of wheat tissues infected by fungal pathogens, we identified 37 serpins with a significant disease response. The majority of the disease-responsive serpins were upregulated by Fusarium graminearum, a destructive fungal pathogen that attacks the spike and developing grain of wheat. As serpins are ubiquitous in wheat grain, the genes encoding serpins may be linked to grain development, with their disease response a result of pleiotropy.

Activated protein C (APC) is a powerful anticoagulant enzyme that proteolytically inactivates the cofactors of the Xase and prothrombinase complexes, factors VIIIa and Va. A common mutation in factor V, fVLeiden, confers resistance to APC leading to an increased risk of thrombosis in the normal population. However, when coinherited with haemophilia, fVLeiden reduces bleeding severity, suggesting that inhibition of APC may be a useful strategy for treatment of haemophilia. We previously reported on serpins that were rationally designed for improved specificity for APC over other coagulation serine proteases. Based on structural differences in the substrate binding pockets to either side of the P1 Arg, we mutated the P2 and P1' residues to Lys. Although this approach achieved APC specificity, it resulted in a reduction in the rate of APC inhibition relative to the parent containing only the P1 Arg. Here we conduct site-specific random mutagenesis at the P2 and P1' positions to determine if improvements could be made in the rate of APC inhibition. In addition to our original Lys mutations, we found that Arg and Gln also confer specificity for APC. However, in all cases specificity for APC resulted in a reduction in inhibition rate.

Members of serpin superfamily are involved in wide array of cellular processes to control proteolytic activities of eukaryotic organisms. Vertebrate serpins are extensively studied and reported to be classified into six groups (V1-V6) based on gene structures. However, there is no study conducted for serpins in urochordates (the closest living invertebrates related to vertebrates) to date. To unravel further the phylogenetic history of serpin genes, we characterized serpin genes from two urochordates (Ciona intestinalis and Ciona savignyi). There are 11 and 5 serpins in the C. intestinalis and C. savignyi, respectively. The exon/intron structures and genomic locus comparisons together with sequence phylogenetic analysis, suggested that urochordate serpins are classified into six groups (U1-U6), different from six groups (V1-V6) of vertebrate serpins. Human α1-antitrypsin shared lower sequence identities and similarities with urochordates serpins ranged from 14-29% and 30-49%, respectively. Based on protein sequences, genes and genomic architectures, we conclude that these two urochordates do not contain a single copy of genuine ortholog of the vertebrate serpins.

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.

Serine protease inhibitors, or serpins, target serine proteases, and are important regulators of intra- and extracellular proteolysis. For parasite survival, parasite-derived protease inhibitors have been suggested to play essential roles in evading the host's immune system and protecting against exogenous host proteases. The aim of this work was to identify serpins via high throughput transcriptome sequencing and elucidate their potential functions during the lifecycle of the salmon louse Caligus rogercresseyi. Eleven putative, partial serpin sequences in the C. rogercresseyi transcriptome were identified and denoted as Cr-serpins 1 to 11. Comparative analysis of the deduced serpin-like amino acid sequences revealed a highly conserved reactive center loop region. Interestingly, P1 residues suggest putative functions involved with the trypsin/subtilisin, elastase, or subtilisin inhibitors, which evidenced increasing gene expression profiles from the copepodid to adult stage in C. rogercresseyi. Concerning this, Cr-serpin 10 was mainly expressed in the copepodid stage, while Cr-serpins 3, 4, 5, and 11 were mostly expressed in chalimus and adult stages. These results suggest that serpins could be involved in evading the immune response of the host fish. The identification of these serpins furthers the understanding of the immune system in this important ectoparasite species.

Psoroptes ovis var. cuniculi infestation rapidly causes skin lesion, cutaneous inflammatory and subsequent adaptive immune response in rabbits. To success feeding and survive on the host skin, this mite should product bioactive molecules to confront host tissue repair and immune defense, but these molecules of this mite remains mostly unknown. Serpins have been proved to involve in diverse biological functions including parasite reproduction, survival and modulating host defense. Limited information is currently available on serpins from Psoroptes mites. Herein, we identified four novel serpins (PsoSP3-PsoSP6) in P. ovis var. cuniculi using bioinformatics and molecular biology techniques. Sequence analysis revealed that PsoSP3-PsoSP6 comprised the common features of typical serpins superfamily including serpin domains, signature or the reactive centre loop (RCL) domain. The recombinant PsoSP4-PsoSP6 (rPsoSP4-rPsoSP6) revealed variable potency inhibition on trypsin, chymotrypsin and elastase except for rPsoSP3 in inhibitory activity assays. By quantitative RT-PCR, the expressions of PsoSP3 and PsoSP4 were higher in juvenile mites (larva and nymph) than in adult mites, however, PsoSP5 and PsoSP6 appeared near-exclusive expression in adult female mites. Immunolocalization showed that native PsoSP4 protein was localized in uterus, whilst native PsoSP3, PsoSP5 and PsoSP6 were specifically localized in the ovarian nutritive cell (ONC) in ovary. Our findings indicated that PsoSP3-PsoSP6 might play critical roles in development and reproduction physiologies. rPsoSP4-rPsoSP6 might participate in modulating host inflammation, immune response and tissue repair.

The serpin superfamily is characterized by proteins that fold into a conserved tertiary structure and exploits a sophisticated and irreversible suicide-mechanism of inhibition. Vertebrate serpins are classified into six groups (V1-V6), based on three independent biological features-genomic organization, diagnostic amino acid sites and rare indels. However, this classification system was based on the limited number of mammalian genomes available. In this study, several non-mammalian genomes are used to validate this classification system using the powerful Bayesian phylogenetic method. This method supports the intron and indel based vertebrate classification and proves that serpins have been maintained from lampreys to humans for about 500 MY. Lampreys have fewer than 10 serpins, which expand into 36 serpins in humans. The two expanding groups V1 and V2 have SERPINB1/SERPINB6 and SERPINA8/SERPIND1 as the ancestral serpins, respectively. Large clusters of serpins are formed by local duplications of these serpins in tetrapod genomes. Interestingly, the ancestral HCII/SERPIND1 locus (nested within PIK4CA) possesses group V4 serpin (A2APL1, homolog of α 2-AP/SERPINF2) of lampreys; hence, pointing to the fact that group V4 might have originated from group V2. Additionally in this study, details of the phylogenetic history and genomic characteristics of vertebrate serpins are revisited.

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.

We studied the genetic variability of serine protease inhibitors (serpins) of Myxozoa, microscopic endoparasites of fish. Myxozoans affect the health of both farmed and wild fish populations, causing diseases and mortalities. Despite their global impact, no effective protection exists against these parasites. Serpins were reported as important factors for host invasion and immune evasion, and as promising targets for the development of antiparasitic therapies. For the first time, we identified and aligned serpin sequences from high throughput sequencing datasets of ten myxozoan species, and analyzed 146 serpins from this parasite group together with those of other taxa phylogenetically, to explore their relationship and origins. High intra- and interspecific variability was detected among the examined serpins. The average sequence identity was 25-30% only. The conserved domains (i.e., motif and signature) showed taxon-level differences. Serpins clustered according to taxonomy rather than to serpin types, and myxozoan serpins seemed to be highly divergent from that of other taxa. None of them clustered with their closest relative free-living cnidarians. The genetic distinction of myxozoan serpins further strengthens the idea of an independent origin of Myxozoa, and may indicate novel protein functions potentially related to parasitism in this animal group.

In eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied.