Microvascular effects of anaphylatoxins C3a and C5a

The direct microvascular effects of human C3a and C5a were investigated by using hamster cheek pouches prepared for intravital microscopy. Topical application of 10 nM C3a resulted in pronounced microcirculatory alterations, characterized by vasoconstriction, platelet aggregation, and an increase in macromolecular leakage at postcapillary venules, as assessed by extravasation of intravascular fluorescein-labeled dextran (m.w. 150,000). Exposure of the preparation to 500 nM COOH-terminal octapeptide analogs of C3a resulted in a microvascular response almost identical to that of C3a, supporting the view that the active site of this anaphylatoxin resided within the COOH-terminal portion. Changes similar to those caused by C3a were also induced by 20 or 100 nM C5a and, in addition, the higher concentration of C5a caused accumulation of polymorphonuclear leukocytes (PMNL) in small venules and somewhat prolonged vascular leakage from venules exhibiting PMNL accumulation. Histamine was found to partially mediate the vascular leakage induced by C3a and the initial (first 5 min) permeability response to the high concentration of C5a, whereas the subsequent leakage induced by the latter anaphylatoxin was unaffected by mepyramine pretreatment. In neutropenic and mepyramine-treated animals exposed to the high concentration of C5a, a partial reduction of both the early and the subsequent vascular leakage was seen, indicating that accumulated PMNL play a role in the prolonged phase of leakage. The pronounced microvascular alterations induced by low concentrations of C3a and C5a strengthen the view that these anaphylatoxins act as mediators of inflammatory reactions in which the complement system is activated.     

Comparative structural anatomy of the complement anaphylatoxin proteins C3a, C4a and C5a

The anaphylatoxins are a family of proteins produced during the course of complement activation as the result of cleavage by specific serine proteases. These proteins are involved in a variety of biological functions, including inflammation. Comparative modeling techniques have been used to produce structures for C4a and C5a from the crystal structure of C3a. All three structures have conserved interior residues but very different external side chains and surface shapes and properties. Comparison of the anaphylatoxin structures and of the sequence conservation among different species suggests possible locations for their receptor binding sites and for their specificity residues which permit regulated proteolytic cleavage from precursor.     

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.     

The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and β-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the β-arrestin pathway. Association of C5L2 with β-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the β-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.     

Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.     

Inactivation of C5a anaphylatoxin by a peptide that is complementary to a region of C5a

PL37 (RAARISLGPRCIKAFTE) is an antisense homology box peptide composed of aa 37-53 of C5a-anaphylatoxin and is considered to be the region essential for C5a function. Using a computer program, we designed the complementary peptides ASGAPAPGPAGPLRPMF (Pep-A) and ASTAPARAGLPRLPKFF (Pep-B). Pep-A bound to PL37 and to C5a with very slow dissociation as determined by analysis using surface plasmon resonance, whereas Pep-B failed to bind at all. C5a was inactivated by concentrations of 7 nM or more of Pep-A, and this concentration of Pep-A inhibited induction of intracellular Ca(2+) influx in neutrophils. Patch clamp electrophysiology experiments also showed the effectiveness of Pep-A in C5aR-expressing neuroblastoma cells. Furthermore, Pep-A administration prevented rats from C5a-mediated rapid lethal shock induced by an Ab to a membrane inhibitor of complement after LPS sensitization.     

Structure-function relationships of human C5a and C5aR

Using peptides that represent linear regions of the powerful complement activation product, C5a, or loops that connect the four alpha helices of C5a, we have defined the ability of these peptides to reduce binding of (125)I-C5a to human neutrophils, inhibit chemotactic responses of neutrophils to C5a, and reduce H(2)O(2) production in neutrophils stimulated with PMA. The data have defined likely sites of interaction of C5a with C5aR. The peptides had no functional activity per se on neutrophils and did not interfere with neutrophil responses to the unrelated chemotactic peptide, N-formyl-Met-Leu-Phe. Although previous data have suggested that there are two separate sites on C5a reactive with C5aR, the current data suggest that C5a interacts with C5aR in a manner that engages three discontinuous regions of C5a.     

Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or β-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a–C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.     

Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor.

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C-terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N-terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site-directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (beta-glucuronidase release). Mutants that contained the single residue substitutions Ile-6-->Gly or Tyr-13-->Gly were reduced in potency to 4-30% compared with wild-type rC5a. Other single-site glycine substitutions at positions Leu-2, Ala-10, Lys-4, Lys-5, Glu-7, Glu-8, and Lys-14 showed little effect on C5a potency. The double mutant, Ile-6-->Gly/Tyr-13-->Gly, was reduced in potency to < 0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in alpha-helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N-terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.     

Growth of Pseudomonas C on C1 compounds: a correction.

On reexamination Pseudomonas C was found to be incapable of growth on formaldehyde or formate as a sole carbon source and to contain a hexose phosphate synthase activity when grown on methanol.     

Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13

Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical andphysical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N^H102F, W^H103Y); M2 (W^H103Y) and M3 (R^L90G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3?nmol/L) than C4C13 (130?nmol/L) and M3 had weaker affinity (234.5?nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.     

Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N

Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.     

Leaf structure and translocation of dry matter in a C3 and a C4 grass

Summary Dry weight analyses and ¹⁴CO₂ were used to study translocation in leaves of the C₃ grass Lolium temulentum L. and the C₄ grass Panicum maximum Jacq. and the results related to the distribution and amount of phloem in the lamina. The rate of specific mass transfer rose from the tips to the bases of leaf blades, in both species high rates were recorded. Major veins were responsible for the bulk of longitudinal translocation and minor veins were important in collecting and loading photosynthate. Transverse veins stored ¹⁴C-assimilate and may have coordinated the functioning of the longitudinal veins. The bearing of the results on the mechanism of translocation is discussed.     

Cloning and characterization of a novel gene (C8orf2), a human representative of a novel gene family with homology to C. elegans C42.C1.9.

Large-scale sequencing of selected genomic regions, coupled with in silico gene trapping, is a robust approach to identifying previously unknown genes. In this way we have found a gene (C8orf2) that is highly homologous to C. elegans C42C1.9. C8orf2 was situated on 8p11. 2 between STS markers NIB1979 (proximal) and AFMA295ZD5 (distal), oriented toward the centromere. C8orf2 consisted of 16 exons spanning more than 16.5 kb of genomic DNA, and was expressed ubiquitously in human tissues. The gene encoded 339-and 152-amino acid polypeptides by alternative splicing; the larger variant contained a region extremely rich in charged amino acids, in particular lysine and glutamic acid. C8orf2 also bore sequence homology to the human KE04p gene. Its conservation among highly divergent species suggests that C8orf2 belongs to a novel gene family.