RESUMO
Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.
RESUMO
The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.
Assuntos
Vírus da Influenza A/efeitos dos fármacos , Proteína D Associada a Surfactante Pulmonar/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Linhagem Celular , Células Epiteliais/virologia , Furões , Humanos , Neuraminidase/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/metabolismo , Sus scrofa , Traqueia/citologiaRESUMO
Porcine surfactant protein D (pSP-D) displays distinctively strong, broad-range inhibitory activity against influenza A virus (IAV). N-Linked glycosylation of the carbohydrate recognition domain (CRD) of pSP-D contributes to the high affinity of this collectin for IAV. To investigate the role of the N-linked glycan further, HEK293E protein expression was used to produce recombinant pSP-D (RpSP-D) that has similar structural and antiviral properties as NpSP-D. We introduced an additional N-linked glycan in the CRD of RpSP-D but this modification did not alter the antiviral activity. Human SP-D is unglycosylated in its CRD and less active against IAV compared with pSP-D. In an attempt to modify its antiviral properties, several recombinant human SP-D (RhSP-D) mutants were constructed with N-linked glycans introduced at various locations within its CRD. To retain lectin activity, necessary for the primary interactions between SP-D and IAV, N-linked glycosylation of RhSP-D was shown to be restricted to the corresponding position in the CRD of either pSP-D or surfactant protein A (SP-A). These N-glycosylated RhSP-D mutants, however, did not show increased neutralization activity against IAV. By developing RhSP-D mutants that also have the pSP-D-specific Ser-Gly-Ala loop inserted in the CRD, we could demonstrate that the N-linked glycan-mediated interactions between pSP-D and IAV involves additional structural prerequisites of the pSP-D CRD. Ultimately, these studies will help to develop highly effective SP-D-based therapeutic and prophylactic drugs against IAV.
Assuntos
Vírus da Influenza A/metabolismo , Lectinas , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Animais , Cães , Glicosilação , Células HEK293 , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/terapia , Mutação , Polissacarídeos/química , Polissacarídeos/genética , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
In the mammalian ovary, oocytes are arrested at prophase of meiosis I until a hormonal stimulus triggers resumption of meiosis. During the subsequent meiotic maturation process, which includes completion of the first meiotic division and formation of the second metaphase spindle, oocytes acquire competence for fertilization. Recently, it was shown that clathrin, a cytosolic protein complex originally defined for its role in intracellular membrane traffic, is also involved in the stabilization of kinetochore fibers in mitotic spindles of dividing somatic cells. However, whether clathrin has a similar function in meiotic spindles in oocytes has not been investigated previously. Our results show that endogenous clathrin associates with the meiotic spindles in oocytes. To study the function of clathrin during meiotic maturation, we microinjected green fluorescent protein-tagged C-terminal and N-terminal dominant-negative clathrin protein constructs into isolated porcine oocytes prior to in vitro maturation. Both protein constructs associated with meiotic spindles similar to endogenous clathrin, but induced misalignment and clumping of chromosomes, occurrence of cytoplasmic chromatin and failure of polar body extrusion. These data demonstrate that clathrin plays a crucial role in meiotic spindle function in maturing oocytes, possibly through spindle stabilization.
Assuntos
Clatrina/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Fuso Acromático/fisiologia , Animais , Feminino , Técnica Direta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/genética , Microinjeções/métodos , Microscopia Confocal , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Análise de Regressão , SuínosRESUMO
UNLABELLED: Parvalbumin from carp, a major allergen, was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity >95% to 98% based on SDS-PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide-mass fingerprinting, and IgE-binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV-CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing alpha-helices and beta-structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food, as well as for use as a standard calibrator for such assays. PRACTICAL APPLICATION: Parvalbumin is a major allergen from fish. Here, we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens.
Assuntos
Carpas/metabolismo , Proteínas de Peixes/isolamento & purificação , Parvalbuminas/química , Parvalbuminas/isolamento & purificação , Alimentos Marinhos , Temperatura , Alérgenos/química , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Filtração/métodos , Hipersensibilidade Alimentar/imunologia , Humanos , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Peso Molecular , Parvalbuminas/imunologia , Mapeamento de Peptídeos , Dobramento de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência , EspectrofotometriaRESUMO
Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococcal superantigen-like (SSL) proteins are a family of 14 exotoxins with homology to superantigens, but with generally unknown function. Recently, we showed that SSL5 binds to P-selectin glycoprotein ligand 1 dependently of sialyl Lewis X and inhibits P-selectin-dependent neutrophil rolling. Here, we show that SSL5 potently and specifically inhibits leukocyte activation by anaphylatoxins and all classes of chemokines. SSL5 inhibited calcium mobilization, actin polymerization, and chemotaxis induced by chemokines and anaphylatoxins but not by other chemoattractants. Antibody competition experiments showed that SSL5 targets several chemokine and anaphylatoxin receptors. In addition, transfection studies showed that SSL5 binds glycosylated N-termini of all G protein-coupled receptors (GPCRs) but only inhibits stimuli of protein nature that require the receptor N-terminus for activation. Furthermore, SSL5 increased binding of chemokines to cells independent of chemokine receptors through their common glycosaminoglycan-binding site. Importance of glycans was shown for both GPCR and chemokine binding. Thus, SSL5 is an important immunomodulatory protein of S aureus that targets several crucial, initial stages of leukocyte extravasation. It is therefore a potential new antiinflammatory compound for diseases associated with chemoattractants and their receptors and disorders characterized by excessive recruitment of leukocytes.
Assuntos
Anafilatoxinas/imunologia , Proteínas de Bactérias/imunologia , Exotoxinas/imunologia , Fatores Imunológicos/imunologia , Migração e Rolagem de Leucócitos/imunologia , Glicoproteínas de Membrana/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Staphylococcus aureus/imunologia , Fatores de Virulência/imunologia , Anafilatoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Sinalização do Cálcio/imunologia , Linhagem Celular , Quimiocinas/imunologia , Quimiocinas/metabolismo , Exotoxinas/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Antígenos CD15/análogos & derivados , Glicoproteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Ligação Proteica/imunologia , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Trissacarídeos/imunologia , Trissacarídeos/metabolismo , Fatores de Virulência/metabolismoRESUMO
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.
Assuntos
Carboxipeptidase B2/química , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Linhagem Celular , Cristalografia por Raios X , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Biológicos , Mutação/genética , Precursores de Proteínas , Estrutura Secundária de ProteínaRESUMO
Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Regeneração Hepática/efeitos dos fármacos , Animais , Linhagem Celular , Cães , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatectomia , Fator de Crescimento de Hepatócito/genética , Fígado/efeitos dos fármacos , Fígado/fisiologia , Tamanho do Órgão , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas RecombinantesRESUMO
Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms. We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to alpha-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity.
Assuntos
Galinhas/metabolismo , Hemaglutinação por Vírus/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Lectinas/metabolismo , Pulmão/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Regulação da Expressão Gênica , Lectinas/farmacologia , Testes de Neutralização , Estrutura Terciária de Proteína , Proteínas RecombinantesRESUMO
Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1-2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas do Tecido Nervoso/química , Receptores Imunológicos/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Heparina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Proteínas RoundaboutRESUMO
Activation of Roundabout 1 (Robo1) by Slit proteins results in axon repulsion from the midline. Robo1 is a large transmembrane receptor expressed on the axon growth cone and the minimal Robo1-binding region required for Slit activation has been mapped to the N-terminal Ig1-2 domains. The cDNA encoding the first two Ig domains of Robo1 has been cloned and the protein has been expressed in HEK293 EBNA-1 mammalian cells. Here, the purification and crystallization conditions of this Robo1 construct are reported. The crystals are orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 38.8, b = 69.4, c = 103.3 A and one molecule in the asymmetric unit. X-ray diffraction data have been collected to 2.8 A resolution on beamline ID29 at the ESRF.
Assuntos
Clonagem Molecular , Imunoglobulinas/química , Imunoglobulinas/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Linhagem Celular , Cristalização , Cristalografia por Raios X , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Imunoglobulinas/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Mapeamento de Peptídeos , Estrutura Terciária de Proteína , Receptores Imunológicos/biossíntese , Proteínas RoundaboutRESUMO
Slit2 and Roundabout 1 (Robo1) provide a key ligand-receptor interaction for the navigation of commissural neurons during the development of the central nervous system. Slit2 is a large multidomain protein containing an unusual domain organization of four tandem leucine-rich repeat (LRR) domains at its N-terminus. These domains are well known to mediate protein-protein interactions; indeed, the Robo1-binding region has been mapped to the concave face of the second LRR domain. It has also been shown that the fourth LRR domain may mediate Slit dimerization and that both the first and second domains can bind heparin. Thus, while roles have been ascribed for three of the LRR domains, there is still no known role for the third domain. Each of the four LRR domains from human Slit2 have now been successfully expressed in milligram quantities using expression in mammalian cells. Here, the crystallization of the second and third LRR domains and the structure of the third LRR domain are presented. This is the first structure of an LRR domain from human Slit2, which has an extra repeat compared with the Drosophila homologue. It is proposed that a highly conserved patch of surface residues on the concave face may mediate any protein-protein interactions involving this LRR domain, a result that will be useful in guiding further studies on Slit2.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Leucina/química , Proteínas do Tecido Nervoso/química , Receptores Imunológicos/química , Sequências Repetitivas de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans , Drosophila , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Xenopus , Peixe-Zebra , Proteínas RoundaboutRESUMO
Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-A resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices. Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complement-amplification step in the immune response.
Assuntos
Fator B do Complemento/química , Fator B do Complemento/metabolismo , Proteínas do Sistema Complemento/imunologia , Domínio Catalítico , Convertases de Complemento C3-C5/química , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequências Reguladoras de Ácido Nucleico/genética , Relação Estrutura-Atividade , Fator de von Willebrand/químicaRESUMO
Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of beta-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken beta-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 mug/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken beta-defensin gallinacin-6 plays an important role in chicken innate host defense.
Assuntos
Galinhas/fisiologia , Sistema Digestório/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Leveduras/efeitos dos fármacos , beta-Defensinas/biossíntese , beta-Defensinas/farmacologia , Animais , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/ultraestrutura , Microbiologia de Alimentos , Vetores Genéticos , Cinética , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Peptídeos/síntese química , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/farmacologia , Compostos de Sulfidrila/metabolismoRESUMO
C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix alpha7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix alpha7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.
Assuntos
Complemento C2a/química , Modelos Moleculares , Aminoácidos/química , Aminoácidos/genética , Domínio Catalítico , Ativação do Complemento , Complemento C2a/genética , Humanos , Ligantes , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por SubstratoAssuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombina/metabolismo , Cristalografia por Raios X , Humanos , Complexo Glicoproteico GPIb-IX de Plaquetas/agonistas , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Mutação Puntual , Conformação Proteica , Trombina/química , Trombina/genéticaRESUMO
Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.
Assuntos
Anticorpos Monoclonais/química , Colágeno/metabolismo , Epitopos/química , Oligopeptídeos/química , Fator de von Willebrand/química , Fator de von Willebrand/imunologia , Sequência de Aminoácidos , Mapeamento de Epitopos , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Conformação Proteica , Fator de von Willebrand/metabolismoRESUMO
The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His(1023), located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin alpha(2) I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.
Assuntos
Colágeno Tipo III/metabolismo , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , Dimerização , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Mutação Puntual , Ligação Proteica/genética , Estrutura Terciária de Proteína , Transfecção , Fator de von Willebrand/genéticaRESUMO
Transient interactions of platelet-receptor glycoprotein Ibalpha (GpIbalpha) and the plasma protein von Willebrand factor (VWF) reduce platelet velocity at sites of vascular damage and play a role in haemostasis and thrombosis. Here we present structures of the GpIbalpha amino-terminal domain and its complex with the VWF domain A1. In the complex, GpIbalpha wraps around one side of A1, providing two contact areas bridged by an area of solvated charge interaction. The structures explain the effects of gain-of-function mutations related to bleeding disorders and provide a model for shear-induced activation. These detailed insights into the initial interactions in platelet adhesion are relevant to the development of antithrombotic drugs.