RESUMO
Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H2O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.
Assuntos
Complemento C3/química , Dissulfetos/química , alfa-Macroglobulinas/química , Sequência de Aminoácidos , Complemento C3/genética , Cisteína/química , Cisteína/genética , Células HEK293 , Humanos , Mutação , Conformação Proteica , Proteólise , Tripsina/química , alfa-Macroglobulinas/genéticaRESUMO
Complement component C3 is well recognized as the central mediator of complement system, whose activation is responsible for the immune surveillance and elimination of non-self-antigens. In this study, C3 gene (HcC3) from a pearl making mussel, Hyriopsis cumingii, was successfully identified. The putative HcC3 possessed the canonical domains and highly conserved functional residues of C3 family members. In phylogenetic analysis, HcC3 was also clustered into C3 subfamily and separated from α2 macroglobulin clade. HcC3 gene was constitutively expressed in a wide range of tissues of pearl mussels, among which the immune-related tissues like hemocytes got highest expression. After allograft surgery of mantle tissues for aquaculture pearl production, the gene expression of HcC3 exhibited a rapid upregulation on day 1, dropped back on day 3, peaked the value on day 7, and restored to the level similar to control samples on day 14 after mantle allograft. The biphasic expression within the two weeks post the surgery suggests the important roles for HcC3 in alloimmune responses and an intricate complement activation mechanism in mollusks during tissue allograft.
Assuntos
Complemento C3/genética , Complemento C3/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Unionidae/genética , Unionidae/imunologia , Imunidade Adaptativa/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Complemento C3/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de SequênciaRESUMO
The complement system consists of a series of soluble and cell-surface proteins that serve numerous roles in innate immunity, development, and homeostasis. Despite its many functions, the central event in the complement system is the proteolytic activation of the 185â¯kDa complement component 3 (C3) into its opsonin and anaphylatoxin fragments known as C3b (175â¯kDa) and C3a (10â¯kDa), respectively. The C3 protein is comprised of thirteen separate structural domains, several of which undergo extensive structural rearrangement upon activation to C3b. In addition to this, the C-terminal C345c domain found in C3, C3b, and the terminal degradation product, C3c (135â¯kDa), appears to adopt multiple conformations relative to the remainder of the molecule. To facilitate various structure/function studies, we designed two C3 analogs that could be activated to a C345c-less, C3c-like state following treatment with Tobacco Etch Virus (TEV) protease. We generated stably transfected Chinese Hamster Ovary (CHO) cell lines that secrete approximately 1.5â¯mg of the highest-expressing C3 analog per liter of conditioned culture medium. We purified this C3 analog by sequential immobilized metal ion affinity and size exclusion chromatographies, activated the protein by digestion with TEV protease, and purified the resulting C3c analog by a final size exclusion chromatography. The conformations and activities of our C3 and C3c analogs were assessed by measuring their binding profiles to known C3/b/c ligands by surface plasmon resonance. Together, this work demonstrates the feasibility of producing a C3 analog that can be site-specifically activated by an exogenous proteolytic enzyme.
Assuntos
Complemento C3 , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Cromatografia em Gel , Complemento C3/química , Complemento C3/farmacologia , Cricetulus , Humanos , Domínios Proteicos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de SuperfícieRESUMO
Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level).
Assuntos
Deutério/química , Espectrometria de Massas/métodos , Peptídeos/química , Amidas/química , Complemento C3/química , Ligação de Hidrogênio , Espectrometria de Massas/normasRESUMO
BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is an orphan disease with a high rate of recurrence after kidney transplantation. However, reports of successful prevention of posttransplant aHUS recurrence with eculizumab emerged a few years ago. To further delineate its optimal use, we describe the largest series of kidney transplant recipients treated with prophylactic eculizumab. METHODS: Twelve renal transplant recipients with aHUS-related end-stage renal disease received eculizumab: 10 from day 0 and 2 at the time of recurrence (days 6 and 25). Clinical and histological features, complement assessment, and free eculizumab measurements were analyzed. The median follow-up was 24.6 months. RESULTS: Five patients had failed at least 1 previous renal transplant from aHUS. A genetic mutation was identified in 9 patients, anti-H antibodies were found in 2. No patient demonstrated biological recurrence of thrombotic microangiopathy under treatment. Three antibody-mediated rejections (AMRs) occurred without detectable C5 residual activity. AMR was associated with subclinical thrombotic microangiopathy in 2 patients. One patient lost his graft after several complications, including AMR. One patient experienced posttransplant C3 glomerulonephritis. The last median serum creatinine was 128.2 ± 40.8 µmol/L. CONCLUSIONS: These data confirm that eculizumab is highly effective in preventing posttransplantation aHUS recurrence, yet may not fully block AMR pathogenesis.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/prevenção & controle , Falência Renal Crônica/cirurgia , Transplante de Rim , Adolescente , Adulto , Síndrome Hemolítico-Urêmica Atípica/complicações , Biópsia , Complemento C3/química , Complemento C5/química , Feminino , Seguimentos , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Lupus nephritis is identified in up to 75% of patients with juvenile systemic lupus erythematosus and may present with abnormal urinary findings (overt lupus nephritis) or be apparent only upon renal biopsy (silent lupus nephritis). We investigated whether serum complement levels correlate with renal pathology in pediatric patients with silent lupus nephritis. We performed baseline renal biopsy in 45 children diagnosed with juvenile systemic lupus erythematosus who were admitted to Kagoshima University Hospital between January 2000 and June 2015. Patients were classified as having overt or silent lupus nephritis based on urinary findings at renal biopsy. Silent lupus nephritis was identified in 55.5% (25/45) of cases. Of these, 6 (13.3%) were classified as class III nephritis, according to the International Society of Nephrology/Renal Pathology Society criteria. Decreased serum C3 levels were associated with the renal pathology classification for patients with silent but not with overt lupus nephritis. No differences in serum C4 levels were identified between cases of silent and overt lupus nephritis. Baseline renal biopsy is a critical component of the work-up of juvenile systemic lupus erythematosus as treatable renal pathology may be present in the absence of urinary signs. Serum C3 may be an important marker of the progression of silent lupus nephritis.
Assuntos
Complemento C3/química , Nefropatias/sangue , Lúpus Eritematoso Sistêmico/sangue , Nefrite Lúpica/sangue , Adolescente , Biomarcadores/sangue , Biópsia , Criança , Proteínas do Sistema Complemento , Feminino , Humanos , Japão , Rim/patologia , Nefropatias/complicações , Lúpus Eritematoso Sistêmico/complicações , Nefrite Lúpica/complicações , Masculino , Pediatria , Insuficiência RenalRESUMO
Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs∗23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD.
Assuntos
Segmento Anterior do Olho/anormalidades , Complemento C3/genética , Anormalidades do Olho/genética , Genes Recessivos/genética , Mutação , Inibidor da Tripsina Pancreática de Kazal/genética , alfa-Macroglobulinas/genética , Adolescente , Adulto , Sequência de Aminoácidos , Segmento Anterior do Olho/metabolismo , Criança , Pré-Escolar , Complemento C3/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor da Tripsina Pancreática de Kazal/química , Adulto Jovem , alfa-Macroglobulinas/químicaRESUMO
The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.
Assuntos
Células Dendríticas/metabolismo , Antígeno HLA-DR1/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Complemento C3/química , Complemento C3/metabolismo , Células Dendríticas/química , Gelsolina/química , Gelsolina/metabolismo , Antígeno HLA-DR1/química , Humanos , Linfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Proteoma/química , Proteômica , Transdução de Sinais , Timosina/química , Timosina/metabolismoRESUMO
(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
Proliferative GN is classified as immune complex-mediated or complement-mediated (C3 glomerulopathy). Immune complex-mediated GN results from glomerular deposition of immune-complexes/Ig and C3; the C3 is derived from activation of the classical and/or lectin pathways of complement. C3 glomerulopathy results from deposition of C3 and other complement fragments with minimal or no deposition of immune complexes/Ig; the C3 is derived from activation of the alternative pathway of complement. C4d is a byproduct of activation of the classic and lectin pathways. Although widely used as a marker for antibody-mediated rejection, the significance of C4d in C3 glomerulopathy is undetermined. We studied glomerular C4d staining in 18 biopsy specimens of immune-complex GN, 30 biopsy specimens of C3 GN, and 13 biopsy specimens of postinfectious GN. All specimens of immune complex-mediated GN, except two specimens of IgA nephropathy and one specimen of sclerosing membranoproliferative GN, showed bright (2-3+) C4d staining. The staining pattern of C4d mirrored the staining patterns of Ig and C3. Conversely, C4d staining was completely negative in 24 (80%) of 30 specimens of C3 glomerulopathy, and only trace/1+ C4d staining was detected in six (20%) specimens. With regard to postinfectious GN, C4d staining was negative in six (46%) of 13 specimens, suggesting an abnormality in the alternative pathway, and it was positive in seven (54%) specimens. To summarize, C4d serves as a positive marker for immune complex-mediated GN but is absent or minimally detected in C3 glomerulopathy.
Assuntos
Complemento C4b/química , Glomerulonefrite por IGA/sangue , Glomerulonefrite Membranoproliferativa/sangue , Glomerulonefrite/sangue , Glomérulos Renais/imunologia , Fragmentos de Peptídeos/química , Complexo Antígeno-Anticorpo , Biópsia , Complemento C3/química , Glomerulonefrite/diagnóstico , Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite Membranoproliferativa/diagnóstico , Humanos , Imunoglobulinas/química , Rim/patologia , Glomérulos Renais/patologia , Microscopia de FluorescênciaRESUMO
C3 glomerulopathy describes a spectrum of disorders with glomerular pathology associated with C3 cleavage product deposition and with defective complement action and regulation (Fakhouri et al., 2010; Sethi et al., 2012b). Kidney biopsies from these patients show glomerular accumulation or deposition of C3 cleavage fragments, but no or minor deposition of immunoglobulins (Appel et al., 2005; D'Agati and Bomback, 2012; Servais et al., 2007; Sethi and Fervenza, 2011). At present the current situation asks for a better definition of the underlining disease mechanisms, for precise biomarkers, and for a treatment for this disease. The complement system is a self activating and propelling enzymatic cascade type system in which inactive, soluble plasma components are activated spontaneously and lead into an amplification loop (Zipfel and Skerka, 2009). Activation of the alternative pathway is spontaneous, occurs by default, and cascade progression leads to amplification by complement activators. The system however is self-controlled by multiple regulators and inhibitors, like Factor H that control cascade progression in fluid phase and on surfaces. The activated complement system generates a series of potent effector components and activation products, which damage foreign-, as well as modified self cells, recruit innate immune cells to the site of action, coordinate inflammation and the response of the adaptive immune system in form of B cells and T lymphocytes (Kohl, 2006; Medzhitov and Janeway, 2002; Ogden and Elkon, 2006; Carroll, 2004; Kemper and Atkinson, 2007; Morgan, 1999; Muller-Eberhard, 1986; Ricklin et al., 2010). Complement controls homeostasis and multiple reactions in the vertebrate organism including defense against microbial infections (Diaz-Guillen et al., 1999; Mastellos and Lambris, 2002; Nordahl et al., 2004; Ricklin et al., 2010). In consequence defective control of the spontaneous self amplifying cascade or regulation is associated with numerous human disorders (Ricklin and Lambris, 2007; Skerka and Zipfel, 2008; Zipfel et al., 2006). Understanding the exact action and regulation of this sophisticated homeotic cascade system is relevant to understand disease pathology of various complement associated human disorders. Furthermore this knowledge is relevant for a better diagnosis and appropriate therapy. At present diagnosis of C3 glomerulopathy is primarily based on the kidney biopsy, and histological, immmunohistological and electron microscopical evaluation (D'Agati and Bomback, 2012; Fakhouri et al., 2010; Medjeral-Thomas et al., 2014a,b; Sethi et al., 2012b). The challenge is to define the actual cause of the diverse glomerular changes or damages, to define how C3 deposition results in the reported glomerular changes, the location of the cell damage and the formation of deposits.
Assuntos
Fator Nefrítico do Complemento 3/química , Complemento C3/química , Proteínas Inativadoras do Complemento C3b/química , Glomerulonefrite Membranoproliferativa/patologia , Glomérulos Renais/patologia , Agregação Patológica de Proteínas/patologia , Imunidade Adaptativa , Linfócitos B/imunologia , Linfócitos B/patologia , Biomarcadores/sangue , Ativação do Complemento , Complemento C3/genética , Fator Nefrítico do Complemento 3/genética , Proteínas Inativadoras do Complemento C3b/genética , Expressão Gênica , Glomerulonefrite Membranoproliferativa/diagnóstico , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite Membranoproliferativa/imunologia , Humanos , Glomérulos Renais/química , Glomérulos Renais/imunologia , Agregação Patológica de Proteínas/diagnóstico , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. In vitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. In vivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the â¼6 min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8 min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7-21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis.
Assuntos
Neoplasias Mamárias Experimentais/diagnóstico por imagem , Microbolhas , Imagem Molecular/métodos , Neovascularização Patológica , Neuropilina-1/química , Animais , Linhagem Celular Tumoral , Complemento C3/química , Feminino , Humanos , Ligantes , Lipídeos/química , Masculino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Transplante de Neoplasias , Peptídeos/química , Neoplasias da Próstata/patologia , Ligação Proteica , Espalhamento de Radiação , UltrassonografiaRESUMO
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
Complement component C3 is a key protein in the complement system whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the miiuy croaker (Miichthys miiuy), which was high similarity to other complement C3. In this study, we report the primary sequence, the tissue expression profile, the polypeptide domain architecture and the phylogenetic analysis of miiuy croaker C3 gene. Rapid amplification of the cDNA ends (RACE) yielded the full open reading frame of this protein (4974 bp), and subsequent analysis indicated that the M. miiuy C3 gene encoded a protein of 1657 amino acids. The deduced amino acid sequence showed that M. miiuy C3 has conserved residues and domains known to be critical for C3 function. Phylogenetic analysis showed that miiuy croaker was most closely related to Epinephelus coioides. Expression analysis showed that C3 was expressed differentially in miiuy croaker tissues, while liver was the main source of C3 expression. Infection of miiuy croaker with Vibrio anguillarum resulted in significant changes expression of C3 gene in the immune-related tissues. These results showed that C3 gene might play an important role in immune mechanisms.
Assuntos
Complemento C3/genética , Proteínas de Peixes/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Complemento C3/química , Complemento C3/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Especificidade de Órgãos , Perciformes/metabolismo , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Alinhamento de Sequência/veterinária , Vibrio/fisiologiaRESUMO
Liver cancer remains the leading cause of cancer-related mortality worldwide. Early detection of liver cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The present study was designed to determine the differently expressed proteins at early stage in the serum of animals with liver cancer vis-à-vis controls and figure out the function of the proteins. One-dimensional electrophoresis (1D), two-dimensional electrophoresis (2DE) and liquid chromatography mass spectrometry (LC-MS/MS) were used to screen the serum proteins of liver cancer induced in animals by diethyl nitrosamine (DEN)+2-acetyl amino fluorine (2-AAF). From optimized 2DE image and computer assisted PD Quest analysis were found to be differentially expressed spots when the serum from normal and treated animals were compared. Among these, one spot was selected whose expression level was higher in DEN+2-AAF treated animal sera than in adjacent normal animal sera. The target spot was excised from the 2D gel of liver cancer sera and the peptide mass fingerprinting as obtained LC-MS/MS analysis after digesting the chosen protein spot. This was identified to be complement C3 protein. The changes in complement C3 expression level were validated by Western blot analysis. We reported that the changes in complement C3 concentration start at very early stage of tumorogenesis. The fully grown tumors were developed at 120 days and hepatotumorogenesis was confirmed by histopathological examination. This protein may therefore represent a powerful tool in search for candidate biomarkers for HCC.
Assuntos
Complemento C3/biossíntese , Neoplasias Hepáticas Experimentais/sangue , Neoplasias Hepáticas Experimentais/patologia , 2-Acetilaminofluoreno/toxicidade , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Western Blotting , Complemento C3/química , Dietilnitrosamina/toxicidade , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Dados de Sequência Molecular , Proteômica , Ratos , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
The C3 toxin produced by Clostridium botulinum (C3bot) catalyzes the mono-ADP-ribosylation of the small GTPases Rho A, B and C, resulting in their inactivation. Recently, a specific endocytotic uptake mechanism of C3bot was identified in macrophages and myeloid leukemia cells. Here, we present a novel delivery system based upon a mutant C3bot devoid of ADP-ribosylation activity (C3Mut) and wild-type streptavidin (Stv). The C3Mut moiety mediates endocytosis into macrophages, whereas Stv functions as an adaptor protein for attaching biotinylated molecules to facilitate their subsequent internalization. First, a bioconjugate consisting of recombinant C3Mut and Stv was generated via a thioether linkage that tightly interacted with biotinylated bovine serum albumin as demonstrated by dot blot analysis. We then showed the internalization of C3Mut-Stv into J774A.1 macrophages by confocal microscopy and observed translocation into the cytosol using cell fractionation. The C3Mut-Stv bioconjugate did not affect cell viability. Next, we prepared mono-biotinylated RNase A, which was attached to the C3Mut-Stv transporter, and demonstrated its C3Mut-Stv-mediated delivery into the cytosol of J774A.1 cells. Finally, C3Mut-Stv also promoted the efficient uptake of mono-biotinylated lysozyme into J774A.1 cells, highlighting its versatility. This study intriguingly demonstrates the use of the novel C3Mut-Stv delivery system for protein transduction and may provide a basis for future applications, in particular, of cytotoxic RNase A mutants.
Assuntos
Biotinilação , Complemento C3/química , Complemento C3/metabolismo , Técnicas de Transferência de Genes , Macrófagos/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Estreptavidina/química , Animais , Biotina/química , Biotina/metabolismo , Linhagem Celular , Complemento C3/genética , Citosol/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Muramidase/química , Muramidase/metabolismo , Ribonuclease Pancreático/genética , Estreptavidina/metabolismoRESUMO
BACKGROUND: Diabetic patients are at increased risk for bacterial infections; these studies provide new insight into the role of the host defense complement system in controlling bacterial pathogens in hyperglycemic environments. METHODS: The interactions of complement C3 with bacteria in elevated glucose were assayed for complement activation to opsonic forms, phagocytosis and bacterial killing. C3 was analyzed in euglycemic and hyperglycemic conditions by mass spectrometry to measure glycation and structural differences. RESULTS: Elevated glucose inhibited S. aureus activation of C3 and deposition of C3b and iC3b on the bacterial surface. S. aureus-generated C5a and serum-mediated phagocytosis by neutrophils were both decreased in elevated glucose conditions. Interestingly, elevated glucose increased the binding of unactivated C3 to S. aureus, which was reversible on return to normal glucose concentrations. In a model of polymicrobial infection, S. aureus in elevated glucose conditions depleted C3 from serum resulting in decreased complement-mediated killing of E. coli. To investigate the effect of differing glucose concentration on C3 structure and glycation, purified C3 incubated with varying glucose concentrations was analyzed by mass spectrometry. Glycation was limited to the same three lysine residues in both euglycemic and hyperglycemic conditions over one hour, thus glycation could not account for observed changes between glucose conditions. However, surface labeling of C3 with sulfo-NHS-biotin showed significant changes in the surface availability of seven lysine residues in response to increasing glucose concentrations. These results suggest that the tertiary structure of C3 changes in response to hyperglycemic conditions leading to an altered interaction of C3 with bacterial pathogens. CONCLUSIONS: These results demonstrate that hyperglycemic conditions inhibit C3-mediated complement effectors important in the immunological control of S. aureus. Mass spectrometric analysis reveals that the glycation state of C3 is the same regardless of glucose concentration over a one-hour time period. However, in conditions of elevated glucose C3 appears to undergo structural changes.
Assuntos
Complemento C3/imunologia , Hiperglicemia/imunologia , Hiperglicemia/microbiologia , Staphylococcus aureus/imunologia , Sequência de Aminoácidos , Complemento C3/química , Complemento C3/isolamento & purificação , Convertases de Complemento C3-C5/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Glucose/farmacologia , Glicosilação/efeitos dos fármacos , Humanos , Espectrometria de Massas , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Opsonizantes/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Ligação Proteica/efeitos dos fármacos , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Peripheral nerve injuries are frequently seen in trauma patients and due to delayed nerve repair, lifelong disabilities often follow this type of injury. Innovative therapies are needed to facilitate and expedite peripheral nerve regeneration. The purpose of this study was to determine the effects of a 1-time topical application of a 26-amino-acid fragment (C3(156-181)), derived from the Clostridium botulinum C3-exoenzyme, on peripheral nerve regeneration in 2 models of nerve injury and repair in adult rats. After sciatic nerve crush, different dosages of C3(156-181) dissolved in buffer or reference solutions (nerve growth factor or C3(bot)-wild-type protein) or vehicle-only were injected through an epineurial opening into the lesion sites. After 10-mm nerve autotransplantation, either 8.0 nmol/kg C3(156-181) or vehicle were injected into the proximal and distal suture sites. For a period of 3 to 10 postoperative weeks, C3(156-181)-treated animals showed a faster motor recovery than control animals. After crush injury, axonal outgrowth and elongation were activated and consequently resulted in faster motor recovery. The nerve autotransplantation model further elucidated that C3(156-181) treatment accounts for better axonal elongation into motor targets and reduced axonal sprouting, which are followed by enhanced axonal maturation and better axonal functionality. The effects of C3(156-181) are likely caused by a nonenzymatic down-regulation of active RhoA. Our results indicate the potential of C3(156-181) as a therapeutic agent for the topical treatment of peripheral nerve repair sites.
Assuntos
Axônios/efeitos dos fármacos , Complemento C3/uso terapêutico , Atividade Motora/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Potenciais de Ação/efeitos dos fármacos , Animais , Complemento C3/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Lateralidade Funcional , Imunoprecipitação , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Condução Nervosa/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Peptídeos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologia , Neuropatia Ciática/cirurgia , Estatísticas não Paramétricas , Fatores de Tempo , Transplante de Tecidos/métodos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The increased interest in chemical cross-linking for probing protein structure and interaction has led to a large increase in literature describing new cross-linkers and search programs. However, this has not led to a corresponding increase in the analysis of large and complex proteins. A major obstacle is that the new cross-linkers are either not readily available and/or have a low reactivity. In combination with aging search programs that are slow and have low sensitivity, or new search programs that are described but not released, these efforts do little to advance the field of cross-linking. Here we present a method pipeline for chemical cross-linking, using two standard cross-linkers, BS3 and BS2G, combined with our freely available CrossWork search program. By this approach we generate cross-link data sufficient to derive structural information for large and complex proteins. CrossWork searches batches of tandem mass-spectrometric data, and identifies cross-linked and non-cross-linked peptides using a standard PC. We tested CrossWork by searching mass-spectrometric datasets of cross-linked complement factor C3 against small (1 protein) and large (1000 proteins) search spaces, and show that the resulting distance constraints agree with the established structures. We further investigated the structure of the multi-domain ERp72, and combined the individual domains of ERp72 into a single structure.
Assuntos
Reagentes de Ligações Cruzadas/química , Peptídeos/química , Software , Espectrometria de Massas em Tandem , Complemento C3/química , Glutaratos/química , Humanos , Glicoproteínas de Membrana/química , Modelos Moleculares , Peptídeos/análise , Estrutura Terciária de Proteína , Succinimidas/químicaRESUMO
In suspected vasculitic neuropathy, vasculitis is demonstrated in only 30% of superficial peroneal nerve (SPN)/peroneus brevis muscle (PBM) specimens. Pathologic predictors of vasculitis are thus needed for non-diagnostic cases. Immune deposits in epineurial vessels have an established sensitivity but unknown specificity. In this study we assessed specificity using direct immunofluorescence (DIF) in SPN/PBM biopsies for suspected vasculitic neuropathy. Biopsies from 13 patients with vasculitis, 13 without vasculitis, and 6 with diabetic radiculoplexus neuropathy (DRPN) were stained for immunoglobulin G (IgG), IgM, and complement 3 (C3), and analyzed in a blinded manner. Vascular immunoglobulin or C3 deposits occurred in 12 of 13 nerve or muscle biopsies (11 of 13 nerves, 5 of 13 muscles) in vasculitis vs. 1 of 13 (1 of 13 nerves, 0 of 13 muscles) in controls (P = 0.00003). Specificity was 92%. For DRPN, vascular immune deposits occurred in 5 of 6 nerves or muscles (4 of 6 nerves, 1 of 5 muscles), similar to vasculitis but significantly different from controls. Epineurial/perimysial vascular deposits of immunoglobulin/C3 by DIF are a specific marker of vasculitic neuropathy.