Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade

Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.     

昆虫hemocytin蛋白研究进展

本课题组前期研究发现,绿僵菌素A与家蚕Bombyx mori 的hemocytin蛋白互作,强烈抑制血淋巴免疫,预示hemocytin可能成为一种杀虫剂的新型作用靶标。因此,进一步了解hemocytin十分必要。Hemocytin是昆虫血淋巴免疫的重要因子,作为一种凝集素,介导血淋巴中的凝血、结节和囊胞化过程,防止表皮破损造成的血淋巴外溢和微生物入侵,并参与对已入侵病原的固定与清除。昆虫的hemocytin一般由3 000~4 000个氨基酸组成,是一个巨大的多结构域蛋白,含有多个重复排列的结构域,包括FA58C (coagulation factor 5 or 8 C-terminal), VWD (von Willebrand factor type D), TIL (trypsin inhibitor like cysteine rich), VWC (von Willebrand factor type C), CT (C-terminal cystine knot like), C8 (8 conserved cysteine residues), ChtBD2 (chitin-binding domain type 2)和MUC (mucin-2 protein WxxW repeating region);不同昆虫间hemocytin的氨基酸序列相似性较小,但其结构域序列具有较高的保守性。Hemocytin由血细胞生物合成,以成熟形式分泌至血淋巴中。Hemocytin是凝血块的主要成分,通过其纤维结构凝聚血细胞和凝血因子形成软凝块封闭伤口,再通过交联作用形成硬凝块和结痂。Hemocytin在结节和囊胞化过程中发挥重要作用,将血细胞、免疫因子和病原体凝聚,最后联合黑化作用隔绝和杀死病原体。总体上,昆虫hemocytin的研究还不够深入。解析hemocytin调控昆虫免疫的分子机理,对于丰富昆虫免疫学基础研究,促进基于hemocytin为靶点的新型杀虫剂研发具有重要意义。     

Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects

Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.     

Pathogen Entrapment by Transglutaminase—A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.     

Coagulation and survival in Drosophila melanogaster fondue mutants

Drosophila larval coagulation factors have been identified in vitro. Better understanding of insect hemolymph coagulation calls for experiments in vivo. We have characterized a fondue (fon) mutation and null alleles isolated by imprecise excision of a transposable element. Loss of fon was pupal lethal, but adults could be recovered by expressing the UAS::fonGFP construct of Lindgren et al. (2008). Despite their lethality, fon mutations did not affect larval survival after wounding either when tested alone or in combination with a mutation in the hemolectin clotting factor gene. This reinforces the idea of redundant hemostatic mechanisms in Drosophila larvae, and independent pleiotropic functions of the fondue protein in coagulation and a vital process in metamorphosis.     

Differential protein expression in the honey bee head after a bacterial challenge

Insect immune proteins and peptides induced during bacterial infection are predominantly synthesized by the fat body or by haemocytes and released into the hemolymph. However, tissues other than the "immune-related" ones are thought to play a role in bacteria-induced responses. Here we report a proteomic study of honey bee heads designed to identify the proteins that are differentially expressed after bacterial challenge in a major body segment not directly involved in insect immunity. The list of identified proteins includes structural proteins, an olfactory protein, proteins involved in signal transduction, energy housekeeping, and stress responses, and also two major royal jelly proteins. This study revealed a number of bacteria-induced responses in insect head tissue directly related to typical functions of the head, such as exocrine secretion, memory, and senses in general.     

Leureptin: A soluble,extracellular leucine-rich repeat protein from Manduca sexta that binds lipopolysaccharide

Leucine-rich repeat containing proteins are involved in immune response in many capacities. In insects, these include Toll-like receptors and the Anopheles gambiae proteins APL1 and LRIM1. Here we describe the identification and characterization of leureptin, a novel extracellular protein with 13 leucine-rich repeats from hemolymph of the insect Manduca sexta. After injection of bacteria, leureptin mRNA level increased in fat body, but protein levels in plasma decreased, an indication that leureptin is consumed during the immune response. Leureptin bound to bacterial lipopolysaccharide (LPS). Microscopy using leureptin antiserum showed that leureptin associates with hemocytes after injection of bacteria, an indication that leureptin is involved in hemocyte responses to bacterial infection. Sequence database searches suggest similar proteins are present in other Lepidopteran species.     

Systematic investigation of the hemolymph proteome of Manduca sexta at the fifth instar larvae stage using one- and two-dimensional proteomics platforms

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.     

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.     

Proteomic analysis of the systemic immune response of Drosophila

Improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize proteins involved in a physiological process, such as the immune response of the insect model Drosophila melanogaster. Profiling of the proteins present in the hemolymph (insect blood) of noninfected flies versus flies infected with bacteria or fungi was performed by two-dimensional gel electrophoresis, silver or Coomassie staining, and image analysis. Through this differential analysis, more than 70 out of 160 spots were up- or down-regulated by at least 5-fold after microbial infection. Coomassie staining, in-gel digestion, and database searches yielded the identity of a series of proteins that are directly involved in the Drosophila immune system. This included proteases, protease inhibitors, and recognition molecules such as prophenoloxydase-activating enzymes, serpins, and Gram-negative binding protein-like. Proteins with a potential function in the immune response were also identified, such as an odorant binding protein, peptidylglycine alpha-hydroxylating monooxygenase, and transferrin, affording new candidates for further investigation of innate immune mechanisms. Moreover, several molecules resulting from the cleavage of proteins were detected after the fungal infection. Altogether, this first differential proteomic analysis of the immune response of Drosophila paves the way for the study of proteins affected during innate immunity.     

Staphylococcal Superantigen-like Protein 10 (SSL10) Inhibits Blood Coagulation by Binding to Prothrombin and Factor Xa via Their γ-Carboxyglutamic Acid (Gla) Domain

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10⁻⁷ m in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca²⁺ and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

Helix pomatia lectin and annexin V,two molecular probes for insect microparticles: possible involvement in hemolymph coagulation

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail (Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.     

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.     

Purification of Lysozyme from Hemolymph of Tobacco Cutworm, Spodoptera litura

ABSTRACT Lysozyme is one of the components of the innate immunity of the insects. The lysozyme has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae of Spodoptera litura. The hemolymph immunized with the insect nonpathogen, Escherichia coli was collected 2 days after the abdominal injection. The molecular weight of Spodoptera lysozyme was estimated to be about 15 kDa by SDS-PAGE and it is great similarity with Agrius lysozyme, which recently purified from larval hemolymph of Agrius convolvuli.     

昆虫差异蛋白质组: 进展和展望

黑腹果蝇Drosophila melanogaster、家蚕Bombyx mori、意大利蜜蜂Apis mellifera和冈比亚按蚊Anopheles gambiae等昆虫的基因组测序已经基本完成,蛋白质组学技术将是阐明这些基因组功能的重要工具。本文综述了应用差异蛋白质组学技术在昆虫诱导性免疫、抗性机制和分子病理研究方面取得的一些成果：（1） 细菌、真菌、脂多糖或机械损伤诱导的昆虫血淋巴或血细胞来源细胞系的蛋白质表达的改变; （2） Bt抗性昆虫中肠刷状缘膜和抗锥虫采采蝇Glossina morsitans唾液腺多种蛋白质表达的改变; （3）化学农药处理和姬蜂Chelonus inanitus携带的多分DNA病毒引起的昆虫蛋白质组的变化。最后讨论了昆虫差异蛋白质组学研究的瓶颈与对策。     

Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.