Serpins in the Spotlight: Novel Bioinformatic Insights Into Hyalomma dromedarii Sialotranscriptome

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.

Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins)

BackgroundSerine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option.ResultsA sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL) and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K) residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG) and/or midguts (MG) of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively.ConclusionGiven the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of advances on the molecular physiology of I. scapularis. Although the paper is descriptive, this study provides the first step toward a comprehensive understanding of serpins in tick physiology.

Serpins function as a trap for serine proteases, presenting the reactive center loop (RCL) as a target for individual proteases. When the protease cleaves the RCL, the serpin and protease become covalently linked leading to a loss of function of both the protease and the serpin; this suicide inhibition is often referred to as a "mouse trap." When the RCL P1-P1' scissile bond is cut by the protease, the resulting bond between the protease and the RCL leads to insertion of the cleaved RCL into the β-sheet A and relocation of the protease to the opposite pole of the serpin, forming a suicide complex. Only a relatively small part of the serpin molecule can be removed in deletion mutations before the serpin RCL inhibitory function is lost. Serpin RCL peptides have been developed to block formation of serpin aggregates in serpinopathies, genetic serpin mutations wherein the abnormal serpins insert their RCL into adjacent serpins forming aggregates of inactive serpins.We have further posited that this natural cleavage site in the serpin RCL may form active serpin metabolites with potential to add to the serpin's inhibitory functions. We have developed RCL peptides based upon predicted serpin RCL cleavage (or metabolism) sites and tested these serpins for inhibitory function. In this chapter we describe the development of RCL-derived peptides, peptides derived based upon the RCL sequences of two myxomaviral serpins. Methods used to develop peptides are described for RCL-derived peptides from Serp-1, a thrombotic and thrombolytic serine protease inhibitor, and Serp-2, a cross class serine and cysteine protease inhibitor (Subheadings 2.1 and 3.1). Approaches to testing RCL peptide functions, in vitro by molecular assays and in vivo in models of cell migration, MHV-68 infection, and aortic allograft transplant are described (Subheadings 2.2 and 3.2).

Complementary Structural Mass Spectrometry Techniques Reveal Local Dynamics in Functionally Important Regions of a Metastable Serpin

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an invivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central β-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of α(1)-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8-P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama α(1)-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8-P6 region is only a small part of an extensive domain swap.

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage

Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.

Protozoan and bacterial pathogens in tick salivary glands in wild and domestic animal environments in South Africa

The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1), is an important biomarker for cardiovascular disease and many cancers. It is therefore a desirable target for pharmaceutical intervention. However, to date, no PAI-1 inhibitor has successfully reached clinical trial, indicating the necessity to learn more about the mechanics of the serpin. Although its kinetics of inhibition have been extensively studied, less is known about the latency transition of PAI-1, in which the solvent-exposed reactive center loop (RCL) inserts into its central β-sheet, rendering the inhibitor inactive. This spontaneous transition is concomitant with a large translocation of the RCL, but no change in covalent structure. Here, we conjugated the fluorescent probe, NBD, to single positions along the RCL (P13-P5') to detect changes in solvent exposure that occur during the latency transition. The results support a mousetrap-like RCL-insertion that occurs with a half-life of 1-2 h in accordance with previous reports. Importantly, this study exposes unique transitions during latency that occur with a half-life of ∼5 and 25 min at the P5' and P8 RCL positions, respectively. We hypothesize that the process detected at P5' represents s1C detachment, while that at P8 results from a steric barrier to RCL insertion. Together, these findings provide new insights by characterizing multiple steps in the latency transition.

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin (serine protease inhibitor) superfamily. Like most serpins, the inhibitory function of PAI-1 relies on a flexible reactive centre loop (RCL) undertaking a striking conformational transition. We have investigated the conformational dynamics of the RCL of PAI-1 by time-resolved fluorescence anisotropy. A heterogeneous population model with three rotational correlation times has been employed to account for the "dip and rise" observed in fluorescence anisotropy decay curves. The RCL becomes almost fully solvent exposed and exhibits faster rotation when PAI-1 interacts with a RCL-mimicking octapeptide which blocks the loop insertion pathway, indicating that the RCL is well displaced from the protein surface; while the binding of Somatomedin B (SMB) domain of vitronectin, only induces small changes in the RCL. Comparison of the fluorescence lifetime and anisotropy decay of the wild-type PAI-1 with that of the stabilised mutant suggests that there would be no major structural differences between them. Our results indicate that in a native serpin, the P14 residue of the hinge region can flip in and out of the central beta-sheet A more readily than previously thought, which is likely an inherent property for serpins' protease inhibitory function.