RESUMO
The release of growth factors from platelets, mediated by the coagulation and the complement system, plays an important role in the bone formation around implants. This study aimed at exploring the thromboinflammatory response of H2O2-alkali soaked commercially pure titanium grade 2 discs exposed to whole human blood, as a way to assess the bioactivity of the discs. Commercially pure titanium grade 2 discs were modified by soaking in H2O2, NaOH and Ca(OH)2. The platelet aggregation, coagulation activation and complement activation was assessed by exposing the discs to fresh whole blood from human donors. The platelet aggregation was examined by a cell counter and the coagulation and complement activation were assessed by ELISA-measurements of the concentration of thrombin-antithrombin complex, C3a and terminal complement complex. The modified surface showed a statistically significant increased platelet aggregation, coagulation activation and complement activation compared to unexposed blood. The surface also showed a statistically significant increase of coagulation activation compared to PVC. The results of this study showed that the H2O2-alkali soaked surfaces induced a thromboinflammatory response that indicates that the surfaces are bioactive.
Assuntos
Coagulação Sanguínea , Osso e Ossos/metabolismo , Inflamação , Trombina/química , Trombose , Titânio/química , Álcalis/química , Antibacterianos/farmacologia , Antitrombinas/química , Infecções Bacterianas/prevenção & controle , Biofilmes , Plaquetas , Ativação do Complemento , Complemento C3a/química , Proteínas do Sistema Complemento , Heparina/química , Humanos , Peróxido de Hidrogênio/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Teste de Materiais , Microscopia Eletrônica de Varredura , Ativação Plaquetária , Agregação Plaquetária , Propriedades de SuperfícieRESUMO
Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.
Assuntos
Amidas/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/química , Fumaratos/farmacologia , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/terapia , Renina/química , Amidas/uso terapêutico , Quimiotaxia/efeitos dos fármacos , Criança , Complemento C3/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C4/química , Complemento C5a/química , Complemento C5a/metabolismo , Complemento C5b/química , Complemento C5b/metabolismo , Fator B do Complemento/química , Fator D do Complemento/química , Feminino , Fumaratos/uso terapêutico , Membrana Basal Glomerular/patologia , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Mastócitos/fisiologia , Renina/antagonistas & inibidores , Renina/metabolismoRESUMO
Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (Kd) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pKi of 8.6 ± 0.2 and Ki of 2.5 nM) and C3aR ligands TR16 (pKi of 6.8 ± 0.1 and Ki of 138 nM), BR103 (pKi of 6.7 ± 0.1 and Ki of 185 nM), BR111 (pKi of 6.3 ± 0.2 and Ki of 544 nM) and SB290157 (pKi of 6.3 ± 0.1 and Ki of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.
Assuntos
Complemento C3a/química , Európio/química , Corantes Fluorescentes/química , Receptores de Complemento/análise , Sinalização do Cálcio , Linhagem Celular , Humanos , Ligantes , Fosforilação , Ligação Proteica , Receptores de Complemento/metabolismoRESUMO
In this study, a robust and straightforward method to covalently attach multi-functional hydrogel thin layers onto substrates was provided. In our strategy, double bonds were firstly introduced onto substrates to provide anchoring points for hydrogel layers, and then hydrogel thin layers were prepared via surface cross-linking copolymerization of the immobilized double bonds with functional monomers. Sulfobetaine methacrylate (SBMA), sodium allysulfonate (SAS), and methyl acryloyloxygen ethyl trimethyl ammonium chloride (METAC) were selected as functional monomers to form hydrogel layers onto polyether sulfone (PES) membrane surfaces, respectively. The thickness of the formed hydrogel layers could be controlled, and the layers showed excellent long-term stability. The PSBMA hydrogel layer exhibited superior antifouling property demonstrated by undetectable protein adsorption and excellent bacteria resistant property; after attaching PSAS hydrogel layer, the membrane showed incoagulable surface property when contacting with blood confirmed by the activated partial thromboplastin time (APTT) value exceeding 600s; while, the PMETAC hydrogel thin layer could effectively kill attached bacteria. The proposed method provides a new platform to directly modify material surfaces with desired properties, and thus has great potential to be widely used in designing materials for blood purification, drug delivery, wound dressing, and intelligent biosensors.
Assuntos
Antibacterianos/síntese química , Anticoagulantes/síntese química , Materiais Biocompatíveis/síntese química , Hidrogéis/síntese química , Adulto , Antibacterianos/farmacologia , Anticoagulantes/farmacologia , Materiais Biocompatíveis/farmacologia , Complemento C3a/química , Complemento C5a/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Hidrogéis/farmacologia , Masculino , Membranas Artificiais , Mesilatos/química , Metacrilatos/química , Tempo de Tromboplastina Parcial , Polímeros/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Sulfonas/químicaRESUMO
In an in-vitro assay, rabbit serum, but not human serum, killed Listeria monocytogenes, a foodborne pathogen. The aim of our study was to purify and partially characterize this killing factor. Listericidin was purified from rabbit serum by a single-step ion-exchange chromatography with DEAE-Sephadex A-50 and its antimicrobial activity was assessed by a microdilution method. Listericidin is a protein with a molecular weight of 9 kDa and an isoelectric point of 8.1. It kills L. monocytogenes at 4°C, 25°C, and 37°C, and its activity is resistant to heat (boiling) and acidic conditions (pH <2). Listericidin's activity is inhibited by sodium chloride and various growth media, is sensitive to proteolytic enzymes and is enhanced by calcium chloride, and is neutralized by monoclonal antibodies to human complement C3a. However, the listericidin reacts weakly with these antibodies in an ELISA. The first 33 N-terminal residues of listericidin (SVQLTEKRMDKVGQYTNKELRKXXEDGMRDNPM) have homology to various complement C3a components. Listericidin also kills other Listeria spp., Vibrio spp., Salmonella spp., Escherichia spp., Cronobacter spp., and Bacillus spp. The listericidin peptide purified in a single-step chromatography is pH and heat stable, and has a broad antimicrobial spectrum against major foodborne pathogens in addition to L. monocytogenes.
Assuntos
Antibacterianos/sangue , Proteínas Sanguíneas/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Coelhos/sangue , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Complemento C3a/química , Complemento C3a/imunologia , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Análise de Sequência de Proteína , Homologia de Sequência , Cloreto de Sódio/farmacologiaRESUMO
Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance of coagulation and fibrinolysis. TAFI is a metallocarboxypeptidase that circulates in plasma as zymogen. Activated TAFI (TAFIa) cleaves C-terminal lysine or arginine residues from peptide substrates. The removal of C-terminal lysine residues from partially degraded fibrin leads to reduced plasmin formation and thus attenuation of fibrinolysis. TAFI also plays a role in inflammatory processes via the removal of C-terminal arginine or lysine residues from bradykinin, thrombin-cleaved osteopontin, C3a, C5a and chemerin. TAFI has been studied extensively over the past three decades and recent publications provide a wealth of information, including crystal structures, mutants and structural data obtained with antibodies and peptides. In this review, we combined and compared available data on structure/function relationships of TAFI.
Assuntos
Carboxipeptidase B2/metabolismo , Animais , Anticorpos/química , Arginina/química , Coagulação Sanguínea , Bradicinina/química , Catálise , Bovinos , Quimiocinas/química , Complemento C3a/química , Complemento C5a/química , Cristalografia por Raios X , Precursores Enzimáticos/química , Fibrinólise , Humanos , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/química , Lisina/química , Camundongos , Mutação , Osteopontina/química , Peptídeos/química , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por Substrato , Trombina/químicaRESUMO
Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (Poly-[Hb-SOD-CAT-CA]) contains all three major functions of red blood cells (RBCs) at an enhanced level. It transports oxygen, removes oxygen radicals and transports carbon dioxide. Our previous studies in a 90-min 30 mm Hg Mean Arterial Pressure (MAP) sustained hemorrhagic shock rat model shows that it is more effective than blood in the lowering of elevated intracellular pCO2, recovery of ST-elevation and histology of the heart and intestine. This paper is to analyze the storage and temperature stability. Allowable storage time for RBC is about 1 d at room temperature and 42 d at 4 °C. Also, RBC cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for 1 year even at room temperature. However, Poly-[Hb-SOD-CAT-CA] contains both Hb and enzymes and enzymes are particularly sensitive to storage and heat. We thus carried out studies to analyze its storage stability at different temperatures and heat pasteurization stability. Results of storage stability show that lyophilization extends the storage time to 1 year at 4 °C and 40 d at room temperature (compared to respectively, 42 d and 1 d for RBC). After the freeze-dry process, the enzyme activities of Poly-[SFHb-SOD-CAT-CA] was 100 ± 2% for CA, 100 ± 2% for SOD and 93 ± 3.5% for CAT. After heat pasteurization at 70 °C for 2 h, lyophilized Poly-[Hb-SOD-CAT-CA] retained good enzyme activities of CA 97 ± 4%, SOD 100 ± 2.5% and CAT 63.8 ± 4%. More CAT can be added during the crosslinking process to maintain the same enzyme ratio after heat pasteurization. Heat pasteurization is possible only for the lyophilized form of Poly-[Hb-SOD-CAT-CA] and not for the solution. It can be easily reconstituted by dissolving in suitable solutions that continues to have good storage stability though less than that for the lyophilized form. According to the P50 value, Poly-[SFHb-SOD-CAT-CA] retains its oxygen carrying ability before and after long-term storage.
Assuntos
Substitutos Sanguíneos/química , Anidrases Carbônicas/química , Catalase/química , Hemoglobinas/química , Complexos Multienzimáticos/química , Oxigênio/sangue , Superóxido Dismutase/química , Animais , Transporte Biológico , Anidrases Carbônicas/sangue , Catalase/sangue , Complemento C3a/química , Complemento C3a/metabolismo , Estabilidade de Medicamentos , Ensaios Enzimáticos , Liofilização , Congelamento , Complexos Multienzimáticos/sangue , Pasteurização , Ratos , Ratos Sprague-Dawley , Refrigeração , Superóxido Dismutase/sangue , TemperaturaRESUMO
(RADA)4 peptides are promising biomaterials due to their high degree of hydration (<99.5% (w/v)), programmability at the molecular level, and their subsequent potential to respond to external stimuli. Interestingly, these peptides have also demonstrated the ability to cause rapid (â¼15s) hemostasis when applied directly to wounds. General hemocompatibility of (RADA)4 nanofibers was investigated systematically using clot formation kinetics, C3a generation, and platelet activation (morphology and CD62P) studies. (RADA)4 nanofibers caused a rapid clot formation, but yielded a low platelet activation and low C3a activation. The study suggests that the rapid hemostasis observed when these materials are employed results principally from humoral coagulation, despite these materials having a net neutral charge and high hydration at physiological conditions. The observed rapid hemostasis may be induced due to the available nanofiber surface area within the hydrogel construct. In conclusion, our experiments strongly support further development of (RADA)4 peptide based biomaterials. STATEMENT OF SIGNIFICANCE: Biomedicine based applications of (RADA)4 peptides are being extensively studied for the purpose of improving drug carriers, and 3D peptide nanofiber scaffolds. However, this peptide's biocompatibility has not been investigated till now. One particular study has reported a revolutionary and very desirable ability of (RADA)4 peptide to achieve complete and rapid hemostasis, nevertheless, the literature remains inconclusive on the underlying molecular mechanism. In this manuscript we bridge these two main knowledge gaps by providing the much needed systematic biocompatibility analysis (morphology analysis, platelet and C3a activation) of the (RADA)4 based hydrogels, and also investigate the underlying hemostatic mechanism of this peptide-induced hemostasis. Our work not only provides the much-needed biocompatibility of the peptide for applicative research, but also explores the molecular mechanism of hemostasis, which will help us design novel biomaterials to achieve hemostasis.
Assuntos
Hemostasia , Hidrogéis/química , Peptídeos/química , Engenharia Tecidual/métodos , Materiais Biocompatíveis/química , Coagulação Sanguínea , Complemento C3/química , Complemento C3a/química , Proteínas do Sistema Complemento , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Humanos , Inflamação , Cinética , Teste de Materiais , Microscopia Eletrônica de Transmissão , Nanofibras/química , Nefelometria e Turbidimetria , Selectina-P/química , Ativação PlaquetáriaRESUMO
OBJECTIVE: To investigate the clinical significance of anaphylatoxin C3a in induced sputum in patients with asthma. METHODS: The patients with acute exacerbation of asthma treated at our department between September, 2006 and February, 2007 were included in the study. The demographic data, medical history, levels of lung function and C3a levels in induced sputum were assessed. RESULTS: A total of 33 patients were included in the study. The level of C3a in induced sputum was significantly higher in patients with acute exacerbation of asthma (2.24 ng/ml, range 1.68-5.58 ng/ml) than that in patients with asthma remission (0.7 ng/ml, range 0.24-2.31 ng/ml, P<0.05). Sputum C3a levels in the remission patients were significantly higher than those in the healthy controls (0.12 ng/ml, range 0.07-0.39 ng/ml, P<0.05). The levels of C3a in patients with severe exacerbation (4.69 ng/ml, range 2.69-6.59 ng/ml) were significantly higher than those in patients with mild exacerbation (0.25 ng/ml, range 0.09-0.40 ng/ml) and moderate exacerbation (2.21 ng/ml, range 1.16-3.41 ng/ml) (P<0.01), and were significantly higher in patients with moderate exacerbation than in those in mild exacerbation (P<0.01). The level of C3a in induced sputum was positively correlated with the number of total cell count (r=0.718, P<0.05), eosinophils (r=0.495, P<0.05) and macrophages (r=0.600, P<0.05) in patients with acute exacerbation of asthma. CONCLUSION: Induced sputum C3a level can serve as an important clinical biomarker for clinical asthma management.
Assuntos
Asma/fisiopatologia , Complemento C3a/química , Escarro/química , Biomarcadores/química , Estudos de Casos e Controles , Eosinófilos , Humanos , Contagem de Leucócitos , MacrófagosRESUMO
A significant challenge in chemistry is to rationally reproduce the functional potency of a protein in a small molecule, which is cheaper to manufacture, non-immunogenic, and also both stable and bioavailable. Synthetic peptides corresponding to small bioactive protein surfaces do not form stable structures in water and do not exhibit the functional potencies of proteins. Here we describe a novel approach to growing small molecules with protein-like potencies from a functionally important amino acid of a protein. A 77-residue human inflammatory protein (complement C3a) important in innate immunity is rationally transformed to equipotent small molecules, using peptide surrogates that incorporate a turn-inducing heterocycle with correctly positioned hydrogen-bond-accepting atoms. Small molecule agonists (molecular weight <500 Da) examined for receptor affinity and cellular responses have the same high potencies, functional profile and specificity of action as C3a protein, but greater plasma stability and bioavailability.
Assuntos
Complemento C3a/química , Complemento C3a/fisiologia , Mediadores da Inflamação/química , Mediadores da Inflamação/fisiologia , Receptores de Complemento/agonistas , Complemento C3a/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Relação Estrutura-AtividadeRESUMO
Al(OH)3 is the most common adjuvant in human vaccines, but its mode of action remains poorly understood. Complement involvement in the adjuvant properties of Al(OH)3 has been suggested in several reports together with a depot effect. It is here confirmed that Al(OH)3 treatment of serum depletes complement components and activates the complement system. We show that complement activation by Al(OH)3 involves the three major pathways by monitoring complement components in Al(OH)3-treated serum and in Al(OH)3-containing precipitates. Al(OH)3 activation of complement results in deposition of C3 cleavage products and membrane attack complex (MAC) and in generation of the anaphylatoxins C3a and C5a. Complement activation was time dependent and inhibited by chelation with EDTA but not EGTA+Mg(2+). We thus confirm that Al(OH)3 activates the complement system and show that the alternative pathway is of major importance.
Assuntos
Hidróxido de Alumínio/química , Ativação do Complemento , Soro/química , Complemento C3/química , Complemento C3/metabolismo , Complemento C3a/biossíntese , Complemento C3a/química , Complemento C5a/biossíntese , Complemento C5a/química , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/química , Humanos , Soro/imunologia , Soro/metabolismoRESUMO
Our current knowledge of the structure and function of C3a comes from the study of vertebrate C3a anaphylatoxins, virtually nothing is known about the structure and function of C3a molecules in invertebrates. Here we demonstrated that C3a from the invertebrate chordate Branchiostoma japonicum, BjC3a, was similar to vertebrate C3a possessing potential antibacterial activity, as revealed by sequence analysis and computational modeling. The antibacterial activity of BjC3a was definitely confirmed by both antibacterial assay and TEM observation showing that recombinant BjC3a was directly bactericidal. Additionally, recombinant BjC3a, like vertebrate C3a, was capable of inducing sea bass macrophage migration and enhancing macrophage phagocytosis and respiratory burst response. Moreover, recombinant BjC3a-desArg (generated by removal of the C-terminal arginine), like mammalian C3a-desArg, retained the immunological activities of BjC3a such as antibacterial and respiratory burst-stimulating activities, indicating that the immunological functions of C3a-desArg were conserved throughout chordate evolution. Altogether, our findings show that invertebrate (amphioxus) BjC3a is able to interact with vertebrate (sea bass) macrophages and mediate immune activities, suggesting the emergence of the inflammatory pathway of the complement system similar to that of vertebrates in the basal chordate amphioxus.
Assuntos
Bass/imunologia , Complemento C3a/genética , Anfioxos/genética , Anfioxos/imunologia , Macrófagos/metabolismo , Sequência de Aminoácidos , Animais , Bass/metabolismo , Clonagem Molecular , Complemento C3a/química , Complemento C3a/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Anfioxos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
We report the total chemical synthesis of human C3a by one-pot native chemical ligation of three unprotected peptide segments, followed by efficient in vitro folding that yielded the anaphylatoxin C3a in high yield and excellent purity. Synthetic C3a was fully active and its crystal structure at 2.1 Å resolution showed 3 helices and a C-terminal turn motif.
Assuntos
Complemento C3a/síntese química , Cálcio/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Cristalografia por Raios X , Humanos , Peptídeos/síntese química , Peptídeos/química , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Inflammation is the primary problem associated with blood-contacting artificial organs. Leucocytes play an essential role in the generation of the inflammatory response. Inflammation can be defined in a variety of ways. The goal of this research is to develop a biosensor system that is less complicated and faster responding than conventional methods. In this study, highly sensitive QCM crystals were chemically modified to measure changes in adsorbed mass on the surface and were used to detect activated neutrophils. Leucocyte activation was quantified by measuring the change in frequency of the QCM. QCM crystals with immobilized anti-C3a were tested in vitro using different concentrations of neutrophils. The measured frequency shifts were proportional to neutrophil number, indicating that activated neutrophils attach to the surface of the QCM. These results were supported by AFM surface topography measurements and SEM images. This method presents a rapid, inexpensive, and easy bioassay that tests the inflammatory response to blood-contacting artificial organs.
Assuntos
Anticorpos/química , Materiais Biocompatíveis/efeitos adversos , Técnicas Biossensoriais/instrumentação , Complemento C3a/química , Proteínas Imobilizadas/química , Imunoglobulina G/sangue , Inflamação/imunologia , Teste de Materiais/instrumentação , Neutrófilos/citologia , Técnicas de Microbalança de Cristal de Quartzo/métodos , Células Cultivadas , Complemento C3a/imunologia , Cistamina/química , Glutaral/química , Humanos , Inflamação/patologia , Dispositivos Lab-On-A-Chip , Contagem de Leucócitos , Microscopia de Força Atômica , Microscopia Eletroquímica de Varredura , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Técnicas de Microbalança de Cristal de Quartzo/instrumentaçãoRESUMO
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.
Assuntos
Anafilatoxinas/química , Complemento C3a/química , Complemento C5a/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Many studies have used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to search for blood-based proteins that are related to the presence of breast cancer. We review the biomarkers discovered or targeted measured by these methods and discuss the strengths and weaknesses of these studies. We highlight two proteins that were most often related to breast cancer: C3a des-arginine anaphylatoxin (C3adesArg) (molecular weight: 8,938 Da) and fragments of inter-alpha trypsin inhibitor heavy chain H4 (ITIH4). In addition, we elaborate on three important methodological aspects related to these studies: protein identification, specificity of the markers, and disease heterogeneity. Finally, we propose some points to be addressed in future studies. These include the use of other analytical measurement techniques, need of protein identification, the importance of identical sample handling protocols for cases and controls, and the stratification of the results according to molecular subtypes and stages of breast cancer. Ultimately this may lead to the discovery of new and valid breast cancer specific biomarkers.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/química , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Complemento C3a/química , Complemento C3a/metabolismo , Detecção Precoce de Câncer , Feminino , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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 â¼12 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.
Assuntos
Anafilatoxinas/química , Antígeno CD11b/biossíntese , Ilhotas Pancreáticas/metabolismo , Receptor da Anafilatoxina C5a/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Animais , Complemento C3a/química , Complemento C5a/química , Proteínas do Sistema Complemento , Citometria de Fluxo/métodos , Inflamação , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Leucócitos/citologia , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Although human mast cells express G protein coupled receptors for the anaphylatoxin C3a, previous studies indicated that C3a causes mast cell degranulation, at least in part, via a C3a receptor-independent mechanism similar to that proposed for polycationic molecules such as compound 48/80. The purpose of the present study was to delineate the receptor specificity of C3a-induced degranulation in human mast cells. We found that C3a, a C3a receptor "superagonist" (E7) and compound 48/80 induced Ca(2+) mobilization and degranulation in a differentiated human mast cell line, LAD2. However, C3a and E7 caused Ca(2+) mobilization in an immature mast cell line, HMC-1 but compound 48/80 did not. We have previously shown that LAD2 cells express MrgX1 and MrgX2 but HMC-1 cells do not. To delineate the receptor specificity for C3a and compound 48/80 further, we generated stable transfectants expressing MrgX1 and MrgX2 in a rodent mast cell line, RBL-2H3 cells. We found that compound 48/80 caused degranulation in RBL-2H3 cells expressing MrgX1 and MrgX2 but C3a did not. By contrast, E7 activated RBL-2H3 cells expressing MrgX2 but not MrgX1. These findings demonstrate that in contrast to previous reports, C3a and compound 48/80 do not use a shared mechanism for mast cell degranulation. It shows that while compound 48/80 utilizes MrgX1 and MrgX2 for mast cell degranulation C3a does not. It further reveals the novel finding that the previously characterized synthetic peptide, C3a receptor "superagonist" E7 activates human mast cells via two mechanisms; one involving the C3a receptor and the other MrgX2.
Assuntos
Degranulação Celular/efeitos dos fármacos , Complemento C3a/metabolismo , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , p-Metoxi-N-metilfenetilamina/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Complemento C3a/agonistas , Complemento C3a/química , Humanos , Mastócitos/citologia , Mastócitos/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proto-Oncogene Mas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: C3a, a complement component stimulates hepatocyte proliferation. In this study, we investigated whether a functional peptide from its C terminal, YAAALGLAR (C3aP), enhanced liver function after steatotic liver transplantation in a rat model. METHODS: Livers of Sprague-Dawley (SD) rats fed a high-fat diet for 2 months were used as donors. Their liver parenchyma displayed over 60% macrovesicular steatosis upon histopathologic examination. Weight and age-matched isogeneic SD rats were used as recipients of orthotopic liver transplantations (OLTs). Before OLT, the donor livers were perfused with University of Wisconsin solution supplemented with 10 mg/L C3aP. Postoperatively, 0.1 mL of 100 microg/mL C3aP was administered daily intraperitoneally to the experimental group. Recipients without C3aP intraperitoneal administration were the control group. We examined the survival rates, histopathologic changes, and hepatic functions of recipients. RESULTS: Six of the 10 control recipients died within 3 days posttransplantation; however, among the experimental group, 8 of 10 recipients survived beyond 7 days. Histopathologic examination showed that at day 3 posttransplantation, hepatocyte steatosis was notably reversed in the experimental group with cells undergoing active mitosis. In contrast, massive parenchymal necrosis was observed among the control group without notable hepatocyte proliferation. Furthermore, serum enzyme levels among the experimental group were significantly lower than those of the control group at day 3 postoperatively. CONCLUSION: C3aP stimulated hepatocyte proliferation and reversed fatty degradation of hepatocytes, thus enhancing hepatic function and prolonging the survival of recipients of steatotic liver transplantations.
Assuntos
Complemento C3a/química , Complemento C3a/uso terapêutico , Fígado Gorduroso/patologia , Transplante de Fígado/fisiologia , Fragmentos de Peptídeos/uso terapêutico , Anafilatoxinas/uso terapêutico , Animais , Aspartato Aminotransferases/sangue , Gorduras na Dieta , Fígado Gorduroso/cirurgia , Regeneração Hepática , Transplante de Fígado/mortalidade , Transplante de Fígado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Análise de SobrevidaRESUMO
C5L2 is a recently identified receptor for C5a/C5adesArg, C3a and C3adesArg (ASP). C5a/C5adesArg bind with high affinity, with no identified activation. By contrast, some studies demonstrate C3a/ASP binding/activation to C5L2; others do not. Our aim is to critically evaluate ASP/C3adesArg-C5L2 binding and bioactivity. Cell-associated fluorescent-ASP (Fl-ASP) binding to C5L2 increased from transiently transfectedAssuntos
Adipócitos/metabolismo
, Complemento C3a/metabolismo
, Ácidos Graxos/metabolismo
, Receptores de Quimiocinas/metabolismo
, Células 3T3-L1
, Animais
, Células CHO
, Complemento C3a/química
, Complemento C3a/genética
, Complemento C3a/farmacologia
, Cricetinae
, Cricetulus
, Ácidos Graxos/química
, Humanos
, Camundongos
, Camundongos Knockout
, Ligação Proteica/fisiologia
, Receptor da Anafilatoxina C5a
, Receptores de Quimiocinas/química
, Receptores de Quimiocinas/genética
, Proteínas Recombinantes/química
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/metabolismo
, Proteínas Recombinantes/farmacologia
, Viperidae