Designing synthetic superagonists of C3a anaphylatoxin

An extensive structure-activity study of synthetic analogues of the C3a anaphylatoxin was conducted. Our goal was to map C3a-C3a receptor interactions by designing synthetic analogue molecules having maximal biologic potency. Nonspecific binding of the polycationic C3a to polyanionic molecules on cellular surfaces often obscures specific binding to the receptor. Less cationic synthetic C3a analogues would be useful tools in identifying and characterizing the various cell types having C3a receptors. These factors should also be useful as pharmacologic probes for mechanism studies, as high-affinity ligands for target cell identification, and for receptor isolation. Attachment of amino-terminal hydrophobic groups such as Fmoc to C3a analogues [as orginally introduced by Gerardy-Schahn et al. (1988) Biochem. J. 255, 209] markedly enhanced the potency of synthetic C3a peptides. The enhancement effect on potency from introducing hydrophobic groups to C3a analogues was interpreted as possibly being nonspecific. Our systematic search for an optimal peptide length, composition, and N-terminal hydrophobic unit resulted in several superpotent C3a analogues having 200-1500% the potency of natural C3a. One particularly potent C3a peptide was designed by incorporating two tryptophanyl residues at the N-terminal end of a 15-residue C3a analogue. The superpotent peptide W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R has several residues differing (underlined) from the sequence corresponding to positions 63-77 in human C3a, a region that contains the essential functional site of the molecule. This 15-residue model peptide exhibited the greatest biological potency of all peptides tested, being 12-15 times more active than natural C3a. Since an optimal distance was found to exist between the N-terminal hydrophobic unit (W-W) and the C-terminal primary binding site (LGLAR), we concluded that the hydrophobic unit interacts specifically with a secondary binding site on the C3a receptor. The presence of both a primary (effector) and secondary (hydrophobic) binding site on these linear synthetic ligands, which can interact cooperatively with the C3a receptor, presumably accounts for the high relative potency of the analogues. Our design of superpotent analogues of C3a demonstrates the feasibility for constructing small synthetic peptides to mimic natural biologic factors that depend on secondary or tertiary structure for their activity. These synthetic peptide studies demonstrate that a linear array of amino acids (e.g., W-W) can successfully substitute for a conformation-dependent binding site on a bioactive factor.     

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.     

Complement-derived anaphylatoxin C5a protects against glutamate-mediated neurotoxicity.

Previous work from this laboratory indicates a role for the complement component C5 in neuroprotection against excitotoxicity. In the present study, we tested the hypothesis that the C5-derived anaphylatoxin C5a protects against kainic acid (KA)-induced neurodegeneration and investigated the mechanism of C5a neuronal activity in vitro. Brain intraventricular infusion of KA into adult mice caused neuronal morphological features of apoptosis in the pyramidal layer of the hippocampal formation as indicated by counts of neurons with pyknotic/condensed nuclei associated with cytoplasmic eosinophilia. Co-intraventricular infusion of human recombinant C5a with KA resulted in a marked reduction of morphological features of apoptotic neuronal death. In vitro studies confirmed C5a neuroprotection: treatment of primary murine corticohippocampal neurons with human or mouse recombinant C5a reduced glutamate neurotoxicity, as measured by trypan blue exclusion assay. This protection concurred with inhibition of glutamate-mediated induction of the caspase-3-related cysteine protease and coincided with marked reduction of neurons with morphological features of apoptosis, as found in vivo. Our studies indicate that C5a may inhibit glutamate-mediated neuronal death through partial inhibition of caspase-3 activity. These findings suggest a novel noninflammatory role for C5a in modulating neuronal responses to excitotoxins.     

Distribution of rat C5a anaphylatoxin receptor

The anaphylatoxin, complement 5a (C5a), plays a key role in mediating various inflammatory reactions following complement activation. Several investigators have reported that C5a receptor (C5aR) is expressed in non-myeloid cells under certain conditions or in different cell lines. In our study, the abundance of C5aR-positive myeloid cells in rats depended on the organs examined. C5aR was usually expressed at the site of exposure to pathogens, such as in salivary gland or lung, and was up-regulated in liver in the inflammatory state induced by lipopolysaccharide (LPS) administration. Furthermore, the increased expression of C5aR antigen was not accompanied by an increase in C5aR mRNA in Kupffer cells following LPS challenge.     

Circular dichroism of C5a anaphylatoxin of porcine complement

The far-ultraviolet circular dichroism spectrum of C5a anaphylatoxin of porcine complement implies that it has a substantial content of helical structure. The circular dichroism spectra of C5a in the 200–250 nm region at pH 7.2 and 3.7 are nearly identical and resemble those of C3a anaphylatoxin. Treatment of C5a with 2-mercaptoethanol progressively diminishes the ellipticity at 222 nm and its anaphylatoxic activity to limiting values. Removal of the reducing agent by dialysis completely restored both the ellipticity at 222 nm and the activity. This finding indicates that the integrity of the secondary conformation of the C5a molecule is largely dependent on disulfide bonds and is essential for its full activity.     

A functional C5a anaphylatoxin receptor in a teleost species

The anaphylatoxins are potent, complement-derived low m.w. proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions. C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes. Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate. To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes. Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression. The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5a(desArg), demonstrating that sites other than the C-terminal of tC5a interact with the C5aR. Both tC5a and tC5a(desArg) were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active. Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a. Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive G(i) proteins in tC5a-mediated chemotaxis.     

Modeling molecular mechanisms of binding of the anaphylatoxin C5a to the C5a receptor

This study presents the 3D model of the complex between the anaphylatoxin C5a and its specific receptor, C5aR. This is the first 3D model of a G-protein-coupled receptor (GPCR) complex with a peptide ligand deduced by a molecular modeling procedure analyzing various conformational possibilities of the extracellular loops and the N-terminal segment of the GPCR. The modeling results indicated two very different ways of interacting between C5a and C5aR at the two interaction sites suggested earlier based on the data of site-directed mutagenesis. Specifically, C5a and C5aR can be involved in "mutual-induced fit", where the interface between the molecules is determined by both the receptor and the ligand. The rigid core of the C5a ligand selects the proper conformations of the highly flexible N-terminal segment of C5aR (the first interaction site). At the same time, the binding conformation of the flexible C-terminal fragment of C5a is selected by well-defined interactions with the TM region of the C5aR receptor (the second interaction site). The proposed 3D model of C5a/C5aR complex was built without direct use of structural constraints derived from site-directed mutagenesis reserving those data for validation of the model. The available data of site-directed mutagenesis of C5a and C5aR were successfully rationalized with the help of the model. Also, the modeling results predicted that the full-length C5a and C5a-des74 metabolite would have different binding modes with C5aR. Modeling approaches employed in this study are readily applicable for studies of molecular mechanisms of binding of other polypeptide ligands to their specific GPCRs.     

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.     

Characterization of a receptor for C5a anaphylatoxin on human eosinophils

The complement anaphylatoxin peptide C5a is well known to activate human polymorphonuclear leukocytes through receptor-mediated processes. C5a has also been reported to activate eosinophils for both chemotaxis and hexose uptake. We characterized the receptor molecule for human C5a on human eosinophils and compared it with the receptor on human neutrophils. At 4 degrees C, uptake of 1 nM 125I-C5a reaches equilibrium within 10 min on both cell types. Binding of 125I-C5a occurs over a concentration range comparable to that which stimulates lysosomal enzyme release and hexose uptake in both cell types. Scatchard analyses of the data indicate the presence of two receptor populations on eosinophils; a high affinity receptor with 15,000-20,000 sites/cell and a Kd of 3.1 +/- 0.6 x 10(-11) M, and a low affinity receptor with approximately 375,000 sites/cell and a Kd of 1 x 10(-7) M. Parallel experiments with neutrophils indicate the presence of a single receptor population with approximately 90,000 sites/cell and a Kd of 4.8 +/- 0.1 x 10(-10)M. The eosinophil receptor molecule was further characterized by covalently cross-linking 125I-C5a to cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material. Autoradiography indicates the presence of a dominant C5a-eosinophil receptor complex with an apparent mass of 60-65 kDa. The corresponding neutrophil-C5a receptor complex has an apparent mass of 50-52 kDa as observed by others. When the cross-linked 125I-C5a-receptor complex was treated with cyanogen bromide, different patterns were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for neutrophils and eosinophils. Thus, human eosinophils have a receptor for C5a anaphylatoxin which appears to be distinct from the C5a receptor present on human neutrophils.     

Structure and function of human C5a anaphylatoxin. Selective modification of tyrosine 23 alters biological activity but not antigenicity

Reaction of either human C5a or its des-Arg74 derivative (des-Arg74-C5a) with tetranitromethane under nondenaturing conditions results in selective nitration of only 1 of the 2 tyrosine residues found in these glycopolypeptides. This reactive tyrosyl residue was identified as that found in position 23 of the sequence. Nitrotyrosyl23-C5a and -des-Arg74-C5a were separated from their respective unmodified precursors by cation-exchange fast protein liquid chromatography. These purified derivatives served as reagents for the subsequent preparation of both aminotyrosyl23-C5a and -des-Arg74-C5a as well as photoreactive analogs of C5a. Radioimmunoassays performed with C5a derivatives serving as competing ligands and a murine antihuman C5a monoclonal antibody employed as first antibody demonstrated that selective modification of tyrosine23 did not produce a detectible alteration in the antigenic properties of C5a. By contrast, either nitro- or aminotyrosyl23-C5a was approximately 3-fold less active than native C5a in both bioassays and competitive ligand-receptor binding assays. Additionally, photoreactive derivatives prepared by coupling either N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate or p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate to aminotyrosyl23-C5a at pH 5.0 failed to interact with the neutrophil C5a receptor. These observations suggest that the tyrosyl23 residue of C5a may participate importantly in the binding interactions of this chemotactic factor and its granulocyte receptor.     

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.     

Design and biological activity of a new generation of synthetic C3a analogues by combination of peptidic and non-peptidic elements.

Based on published X-ray crystallographic data of the anaphylatoxic complement peptide C3a, we have synthesized a series of peptides with appropriate amino acid exchanges and a maximal length of 13 amino acids. N-terminal acylation of these optimized structures with epsilon-aminohexanoic acid and complex aromatic structures like fluorenylmethoxycarbonyl, 2-nitro-4-azidophenyl, fluoresceinyl and rhodaminyl leads to a dramatic increase in biological activity. The culmination of our synthetic efforts is a C3a analogue with 13 amino acid residues and a biological activity six times that of native C3a.     

Expression and induction of anaphylatoxin C5a receptors in the rat liver

The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and in-vivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-beta1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.     

Characterization of human C4a anaphylatoxin

Human C4a anaphylatoxin was isolated from a Cls digest of the fourth component of complement. Isolation required a two-step procedure involving ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. Characterization of C4a indicated it is a highly cationic polypeptide (pI = 9.0-9.5) containing 77 residues with Mr = 8,759. C4a is devoid of tryptophan, histidine, and carbohydrate. Judged by the shape and magnitude of its circular dichroism spectrum, 54% of the polypeptide backbone of C4a assumes an alpha-helical conformation. Partial NH2-terminal sequence determination of C4a revealed a sequence identical with that published by Bolotin et al. (Bolotin, C., Morris, S., Tack, B., and Prahl, J. (1977) Biochemistry 16, 2008-2015) for the NH2 terminus of the alpha-subunit of human C4. Comparison of the NH2-terminal sequence of C4a with the sequences of complement activation fragments C3a (Hugli, T.E. (1975) J. Biol. Chem. 250, 8293-8301) and C5a (Fernandez, H.N., and Hugli, T.E. (1978) J. Biol. Chem, 253-6955-6962) showed that of the first 24 NH2-terminal residues of C4a, 6 were identical with those of C3a (25% homology) and 8 were identical with those of C5a (33% homology). These data represent the first chemical evidence for the existence of an evolutionary relationship among anaphylatoxins C3a, C4a, and C5a, and imply that a similar relationship exists among their precursor proteins.     

Anticancer activity of synthetic analogues of the phorboxazoles

The phorboxazoles are recently described natural products that are extremely cytostatic towards the National Cancer Institute's panel of 60 tumor cell lines. We report here the results of preliminary structure-activity studies of synthetic analogues of the phorboxazoles against BT-20 breast cancer, NALM-6 B-lineage ALL, U373 brain tumor, and U373 glioblastoma cell lines. These data indicate the importance of several of the natural products' structural moieties for potent anticancer activity.     

Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells

A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.     

Synthetic peptides as antagonists of the anaphylatoxin C3a.

Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.     

Complement anaphylatoxin C5a neuroprotects through mitogen-activated protein kinase-dependent inhibition of caspase 3

We previously reported that pretreatment of murine cortico-hippocampal neuronal cultures with the complement-derived anaphylatoxin C5a, protects against glutamate neurotoxicity. In this study we explored the potential mechanisms involved in C5a-mediated neuroprotection. We found that C5a neuroprotects in vitro through inhibition of apoptotic death because pretreatment with human recombinant (hr)C5a prevented nuclear DNA fragmentation coincidental to inhibition of the pro-apoptotic caspase 3 activity mediated by glutamate treatment. Also, hrC5a-mediated responses appeared to be receptor-mediated because pretreatment of cultures with the specific C5a receptor antagonist C177, prevented hrC5a-mediated neuroprotection. Based on this evidence, we further explored possible signaling pathways involved in hrC5a inhibition of caspase 3 activation and apoptotic neuronal death. We found that treatment of cultures with the mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059 prevented hrC5a-mediated inhibition of caspase 3 and apoptotic neuron death. MAPK pathways, whose activation by hrC5a is inhibited by PD98059 and C177, include the extracellular signal-regulated kinase (ERK)2 and, to a lesser extent, ERK1. The study suggests that C5a may protect against glutamate-induced apoptosis in neurons through MAPK-mediated regulation of caspase cascades.     

Inactivation of the complement anaphylatoxin C5a by secreted products of parasitic nematodes

Given the importance of the complement anaphylatoxins in cellular recruitment during infection, the ability of secreted products from larval stages of Brugia malayi and Trichinella spiralis to influence C5a-mediated chemotaxis of human peripheral blood granulocytes in vitro was examined. Secreted products from B. malayi microfilariae almost completely abolished chemotaxis. This inhibition was blocked by phenylmethylsulphonyl fluoride, indicating the presence of a serine protease, which was subsequently shown to cleave C5a. In contrast, secreted products from T. spiralis infective larvae showed modest inhibition of C5a-mediated granulocyte chemotaxis, and this was blocked by potato carboxypeptidase inhibitor, an inhibitor of several metallocarboxypeptidases. Adult and larval stages of both parasites were demonstrated to secrete carboxypeptidases which cleaved hippuryl-l-lysine and hippuryl-l-arginine, and the T. spiralis enzyme was partially characterised. The data are discussed with reference to inflammation in parasitic nematode infection.     

Potentiation of the primary humoral immune response in vitro by C5a anaphylatoxin

The effect of the anaphylatoxin C5a on the primary humoral immune response to SRBC was studied in culture of spleen cells from C3H mice. The addition of human C5a to antigen-stimulated cultures resulted in a significant, dose-dependent augmentation of the primary PFC response to antigen. The specificity of this effect was affirmed by the ability of C5ades Arg, but not of the structurally analogous C3a anaphylatoxin, to act in a parallel fashion. Enhancement could be observed over a range of doses of antigen. Brief preincubation of macrophages, but not of lymphoid cells, with C5a was sufficient to cause subsequent enhancement of the primary humoral immune responses. The duration of preincubation required for this effect closely paralleled that for binding of C5a to peritoneal cells. Immunopotentiation by C5a appears to involve the function of C5a receptor-bearing, Ia- accessory cells as well as Ia+ antigen-presenting cells. Immunopotentiation could be observed when the addition of C5a was delayed for up to 24 hr after initiation of culture. Additionally, augmentation of tritiated thymidine uptake in mixed lymphocyte reactions was enhanced by the addition of C5a in a fashion parallel to that for the primary response to SRBC. These observations support a role for C5a as a modulator of cellular immunity in addition to its role in acute inflammation. Possible cellular mechanisms and implications for immunomodulation of immune responses are discussed.