Decoding anaphylatoxins: unveiling the molecular mechanisms of complement receptor activation and signaling.

Recent advances in cryo-electron microscopy (Cryo-EM) have revolutionized our understanding of the complement C5a/C3a receptors that are crucial in inflammation. A recent report by Yadav et al. has elucidated the activation, ligand binding, selectivity, and signaling bias of these receptors, thereby enhancing structure-guided drug discovery. This paves the way for more effective anti-inflammatory therapies that target these receptors with unprecedented precision.

Mechanism of nanoparticle-induced hypersensitivity in pigs: complement or not complement?

A recent study on nanoparticle-induced hypersensitivity reactions in pigs showed robust pulmonary intravascular macrophage clearance of Polybead® carboxylate microspheres in mediating the adverse cardiopulmonary distress, irrespective of the ability of these particles to activate the complement (C) system in vitro. Focusing on this observation, this article highlights the controversies in projecting in vitro C assay data to in vivo conditions and applying data on polystyrene particles to therapeutic nanopharmaceuticals. Based on overwhelming evidence of a role of anaphylatoxins in hypersensitivity reactions, the need to further explore the role of C activation in the reported and other reactions is highlighted. C-activation-related and C-independent pseudoallergies (CARPA and CIPA) can proceed simultaneously, as outlined by the 'double-hit' hypothesis.

Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction.

A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption.

Evolution of the complement system C3 gene in Antarctic teleosts.

Notothenioidei are typical Antarctic teleosts evolved to adapt to the very low temperatures of the Antarctic seas. Aim of the present paper is to investigate sequence and structure of C3, the third component of the complement system of the notothenioid Trematomus bernacchii and Chionodraco hamatus. We determined the complete nucleotide sequence of two C3 isoforms of T. bernacchii and a single C3 isoform of C. hamatus. These sequences were aligned against other homologous teleost sequences to check for the presence of diversifying selection. Evidence for positive selection was observed in the evolutionary lineage of Antarctic teleost C3 sequences, especially in that of C. hamatus, the most recently diverged species. Adaptive selection affected numerous amino acid positions including three residues located in the anaphylatoxin domain. In an attempt to evaluate the link between sequence variants and specific structural features, we constructed molecular models of Antarctic teleost C3s, of their proteolytic fragments C3b and C3a, and of the corresponding molecules of the phylogenetically related temperate species Epinephelus coioides, using human crystallographic structures as templates. Subsequently, we compared dynamic features of these models by molecular dynamics simulations and found that the Antarctic C3s models show higher flexibility, which likely allows for more pronounced movements of both the TED domain in C3b and the carboxyl-terminal region of C3a. As such dynamic features are associated to positively selected sites, it appears that Antarctic teleost C3 molecules positively evolved toward an increased flexibility, to cope with low kinetic energy levels of the Antarctic marine environment.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Structural and functional characterization of human and murine C5a anaphylatoxins.

Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8(Δ71-73), and of murine C5a and C5a-desArg are reported. Whereas A8(Δ71-73) adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

Human C3a and C3a desArg anaphylatoxins have conserved structures, in contrast to C5a and C5a desArg.

Complement is a part of innate immunity that has a critical role in the protection against microbial infections, bridges the innate with the adaptive immunity and initiates inflammation. Activation of the complement, by specific recognition of molecular patterns presented by an activator, for example, a pathogen cell, in the classical and lectin pathways or spontaneously in the alternative pathway, leads to the opsonization of the activator and the production of pro-inflammatory molecules such as the C3a anaphylatoxin. The biological function of this anaphylatoxin is regulated by carboxypeptidase B, a plasma protease that cleaves off the C-terminal arginine yielding C3a desArg, an inactive form. While functional assays demonstrate strikingly different physiological effects between C3a and C3a desArg, no structural information is available on the possible conformational differences between the two proteins. Here, we report a novel and simple expression and purification protocol for recombinant human C3a and C3a desArg anaphylatoxins, as well as their crystal structures at 2.3 and 2.6 Å, respectively. Structural analysis revealed no significant conformational differences between the two anaphylatoxins in contrast to what has been reported for C5a and C5a desArg. We compare the structures of different anaphylatoxins and discuss the relevance of their observed conformations to complement activation and binding of the anaphylatoxins to their cognate receptors.

Expression of receptors for anaphylatoxins C3a and C5a on rat islet preparations.

BACKGROUND: Complement activation has been implicated in the development of the instant blood-mediated inflammatory reaction (IBMIR). In particular, anaphylatoxins C3a and C5a elicit a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. We have previously shown that 2 types of receptors for C5a are expressed on isolated islets. In the present study, we investigated this component in detail. METHODS: C3aR, C5aR, and C5L2, together with CD11b and CD31, on freshly isolated islets (fresh group) and islets cultured with (cytokine group) or without (culture group) TNF-α, IL-1ß, and IFN-γ for ∼12 hours were analyzed by flow cytometry. In addition, these 3 kinds of receptors were analyzed on nonendocrine cells. RESULTS: C5aR and C5L2 were expressed on the isolated islets (C5aR: 7.91 ± 2.83%; C5L2: 2.45 ± 1.34%) and the expression of both C5a receptors was markedly attenuated by culture for 12 hours (C5aR: P < .005; C5L2: P < .05). Compared with the culture group, the expression was significantly up-regulated in the cytokine group (C5aR: P < .05; C5L2: P = .05). C5aR-positive cells expressed CD11b but not CD31. In contrast to islets, nonendocrine cells expressed C5L2 predominantly. C3aR was scarcely expressed on isolated islets or nonendocrine cells. CONCLUSIONS: These data suggest that C5aR and C5L2 are expressed on CD11b-positive leukocytes in islet preparations. Depletion of C5a receptors by culturing appropriately could be an attractive therapeutic strategy in clinical islet transplantation.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis.

Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.

Generation of anaphylatoxins by human beta-tryptase from C3, C4, and C5.

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Preservation of antimicrobial properties of complement peptide C3a, from invertebrates to humans.

The human anaphylatoxin peptide C3a, generated during complement activation, exerts antimicrobial effects. Phylogenetic analysis, sequence analyses, and structural modeling studies paired with antimicrobial assays of peptides from known C3a sequences showed that, in particular in vertebrate C3a, crucial structural determinants governing antimicrobial activity have been conserved during the evolution of C3a. Thus, regions of the ancient C3a from Carcinoscorpius rotundicauda as well as corresponding parts of human C3a exhibited helical structures upon binding to bacterial lipopolysaccharide permeabilized liposomes and were antimicrobial against gram-negative and gram-positive bacteria. Human C3a and C4a (but not C5a) were antimicrobial, in concert with the separate evolutionary development of the chemotactic C5a. Thus, the results demonstrate that, notwithstanding a significant sequence variation, functional and structural constraints imposed on C3a during evolution have preserved critical properties governing antimicrobial activity.

Ligand specificity of the anaphylatoxin C5L2 receptor and its regulation on myeloid and epithelial cell lines.

During complement activation the pro-inflammatory anaphylatoxins C3a and C5a are generated, which interact with the C3a receptor and C5a receptor (CD88), respectively. C5a and its degradation product C5a-des-Arg(74) also bind to the C5a receptor-like 2 (C5L2). C3a and C3a-des-Arg(77), also called acylation-stimulating protein, augment triglyceride synthesis and glucose uptake in adipocytes and skin fibroblasts. Based on data obtained using transfected HEK293 and RBL cells, C5L2 is additionally proposed as a functional receptor for C3a and C3a-des-Arg(77). Here we use (125)I-ligand binding assays and flow cytometry with fluorescently labeled ligands to demonstrate that neither C3a nor C3a-des-Arg(77) binds to C5L2. C5L2 expression and its regulation are investigated on various cell lines by a novel C5L2-restricted binding assay and quantitative real time PCR. Dibutyryl cAMP and interferon-gamma induce up-regulation of this receptor on myeloblastic cell lines (U937 and HL-60), whereas tumor necrosis factor-alpha (TNF-alpha) has no effect. In contrast, epithelial HeLa cells are found to constitutively express C5L2 but not the C5a receptor. In HeLa cells, interferon-gamma and TNF-alpha drastically reduce C5L2 expression. No C5a-dependent Ca(2+) signaling is observed even in these cells endogenously expressing C5L2. Taken together, C5L2 is not a receptor for C3a or C3a-des-Arg(77). Thus, this receptor is unlikely to be directly involved in lipid metabolism. Instead, the identification of stimuli modifying C5L2 expression indicates that C5L2 is a highly regulated scavenger receptor for C5a and C5a-des-Arg(74).

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2.

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5a(desArg) is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.

Evolution of anaphylatoxins, their diversity and novel roles in innate immunity: insights from the study of fish complement.

Anaphylatoxins are small molecules ( approximately 9 kDa) that are generated as a result of the activation of the complement system. These molecules play an important role in inflammation, and they are responsible for the activation of various innate and adaptive immune processes. The study of these important inflammatory molecules has been restricted to mammalian species so far. Recent studies have shown that teleost fish, unlike any other known animal species, contain multiple forms of the C3a anaphylatoxin, all of which are functionally active and play a prominent role in inducing superoxide production in fish leukocytes. The C5a anaphylatoxin has also been characterized in these animals, and like in mammals, it plays an important role in leukocyte chemotaxis and in triggering the respiratory burst of leukocytes. Interestingly, it has been shown that rainbow trout anaphylatoxins play an unexpected role in enhancing phagocytosis of particles. C5a and C3a receptors have recently been cloned and characterized in rainbow trout, suggesting that the duplication of these receptors from a common ancestor occurred before the emergence of teleosts. The studies derived from these molecules in teleost fish indicate that the basic structure and function of anaphylatoxins and their receptors, have been conserved for more than 300 million years.

Generation, purification and functional characterization of three C3a anaphylatoxins in rainbow trout: role in leukocyte chemotaxis and respiratory burst.

Activation of the complement system leads to cleavage of the C3 molecule into C3b and C3a fragments. The C3a fragment plays a major role in immunity by inducing chemotaxis of eosinophils and mast cells and stimulating the respiratory burst in leukocytes. Although this anaphylotoxin has been well studied in mammals, there is currently a lack of information about the structure and function of C3a anaphylotoxins in non-mammalian vertebrate species. Therefore, in the present study, we have isolated and characterized three different C3a anaphylatoxin molecules from rainbow trout, a teleost fish. C3a was generated from the trout C3-1, C3-3, and C3-4 isoforms by incubating each individual C3 molecule with purified trout factor B/C2 and factor D in the presence of Mg2+, then purifying the resulting C3a molecules by gel filtration. SDS-PAGE, N-terminal sequence and mass spectrometric analysis demonstrated the high degree of purity and expected molecular masses of the three C3a molecules. We showed that although activated trout serum was able to induce complement-dependent chemotaxis in trout head kidney leukocytes, none of the three isolated C3a molecules induced chemotaxis in the same cells. In contrast, all three C3a molecules strongly stimulated the respiratory burst of head kidney leukocytes in a dose-dependent manner. When the carboxy-terminal Arg was removed from all three C3a molecules, their ability to induce the respiratory burst was lost. These studies, therefore, provide strong evidence for the existence of three functional C3a molecules in a non-mammalian vertebrate species and suggest that some of the basic mechanisms of action of the C3a molecule have been conserved for more than 300 million years.

Biocompatibility of fluorinated polyimide.

Contact between blood and biomaterial triggers a complex series of events including protein adsorption, leukocyte adhesion and activation, and complement activation. In this article, a series of fluorinated polyimides cured at a different temperatures was prepared, and the biocompatibility of the membranes was evaluated using in vitro protein adsorption, neutrophil adhesion, and complement activation experiments under static conditions. We found that protein adsorption, neutrophil adhesion, and complement activation for the polyimides significantly depends on the curing temperature and decreases with an increase in the temperature and that the polyimide has a good biocompatibility compared with poly(styrene) and polydimethylsiloxane. We concluded that the rearrangement of molecules such as CF(3), sulfone, and ketone at the outermost surface occurs because of curing, which induces an increase in the hydrophobicity and that the cured polyimide suppresses protein adsorption, neutrophil adhesion, and complement activation because of its high hydrophobicity and low surface free energy.