RESUMO
Essentials Nitric oxide synthesis controls protein disulfide isomerase (PDI) function. Nitric oxide (NO) modulation of PDI controls endothelial thrombogenicity. S-nitrosylated PDI inhibits platelet function and thrombosis. Nitric oxide maintains vascular quiescence in part through inhibition of PDI. SUMMARY: Background Protein disulfide isomerase (PDI) plays an essential role in thrombus formation, and PDI inhibition is being evaluated clinically as a novel anticoagulant strategy. However, little is known about the regulation of PDI in the vasculature. Thiols within the catalytic motif of PDI are essential for its role in thrombosis. These same thiols bind nitric oxide (NO), which is a potent regulator of vessel function. To determine whether regulation of PDI represents a mechanism by which NO controls vascular quiescence, we evaluated the effect of NO on PDI function in endothelial cells and platelets, and thrombus formation in vivo. Aim To assess the effect of S-nitrosylation on the regulation of PDI and other thiol isomerases in the vasculature. Methods and results The role of endogenous NO in PDI activity was evaluated by incubating endothelium with an NO scavenger, which resulted in exposure of free thiols, increased thiol isomerase activity, and enhanced thrombin generation on the cell membrane. Conversely, exposure of endothelium to NO+ carriers or elevation of endogenous NO levels by induction of NO synthesis resulted in S-nitrosylation of PDI and decreased surface thiol reductase activity. S-nitrosylation of platelet PDI inhibited its reductase activity, and S-nitrosylated PDI interfered with platelet aggregation, α-granule release, and thrombin generation on platelets. S-nitrosylated PDI also blocked laser-induced thrombus formation when infused into mice. S-nitrosylated ERp5 and ERp57 were found to have similar inhibitory activity. Conclusions These studies identify NO as a critical regulator of vascular PDI, and show that regulation of PDI function is an important mechanism by which NO maintains vascular quiescence.
Assuntos
Células Endoteliais/metabolismo , Óxido Nítrico/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Trombose/metabolismo , Músculos Abdominais/metabolismo , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Membrana Celular/metabolismo , Fator Xa/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Proteínas Recombinantes/metabolismo , Compostos de Sulfidrila/metabolismo , Trombina/metabolismoRESUMO
Over the past six decades research employing in vitro assays has identified enzymes, cofactors, cell receptors and associated ligands important to the haemostatic process and its regulation. These studies have greatly advanced our understanding of the molecular and cellular bases of haemostasis and thrombosis. However, in vitro assays cannot simultaneously reproduce the interactions of all of the components of the haemostatic process that occur in vivo nor do they reflect the importance of haemodynamic factors resulting from blood flow. To overcome these limitations investigators have increasingly turned to animal models of haemostasis and thrombosis. In this article we describe some advances in the visualisation of platelet and endothelial cell activation and blood coagulation in vivo and review what we have learned from our intravital microscopy experiments using primarily the laser-induced injury model for thrombosis.
Assuntos
Vasos Sanguíneos/patologia , Células Endoteliais/metabolismo , Microscopia , Trombose/diagnóstico , Animais , Vasos Sanguíneos/lesões , Diagnóstico por Imagem/métodos , Diagnóstico por Imagem/tendências , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/patologia , Fibrina/metabolismo , Hemostasia Cirúrgica/tendências , Humanos , Lasers/efeitos adversos , Ativação Plaquetária , Trombose/sangue , Trombose/etiologia , Trombose/patologiaRESUMO
Thrombus formation, including platelet adhesion, activation, secretion and aggregation as well as tissue factor-initiated thrombin generation and fibrin formation, has been studied in the past using in vitro systems, often with isolated components. Given the complexity of hemostasis and thrombosis, many of the concepts that have been developed to explain these processes are being revisited by studying thrombus formation in live animals using intravital microscopy and genetically altered mice. Although much of the dogma that has evolved has been confirmed by in vivo studies of thrombus formation, there have also been conflicts between old concepts and new direct observations. In vivo studies of the initiation of thrombus formation, platelet accumulation and thrombin generation have provided evidence for the participation of novel proteins and identified new pathways and mechanisms.
Assuntos
Trombose/fisiopatologia , Coagulação Sanguínea , Humanos , Agregação Plaquetária , Trombose/etiologiaRESUMO
We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo.
Assuntos
Processamento de Imagem Assistida por Computador/instrumentação , Microscopia Confocal/instrumentação , Trombose/metabolismo , Animais , Arteríolas/lesões , Arteríolas/patologia , Fibrina/metabolismo , Fibrina/ultraestrutura , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador/métodos , Lasers , Leucócitos/metabolismo , Leucócitos/ultraestrutura , Camundongos , Microcirculação , Microscopia Confocal/métodos , Selectina-P/metabolismo , Selectina-P/ultraestrutura , Ativação Plaquetária/fisiologia , Fatores de TempoRESUMO
gamma-Carboxyglutamic acid (Gla) is an unusual amino acid that is synthesized post-translationally from glutamate in a vitamin K-dependent reaction. The dicarboxylic side chain of Gla chelates Ca(2+), a property important for the biological activity of vitamin K-dependent proteins. To date, Gla-containing polypeptides have been identified in venom from two groups of organisms: elapid snakes, and snails of the genus Conus. In certain elapid snakes, a gamma-carboxylated coagulation factor Xa-like protein is a component of the venom whereas cone snails utilize Gla in a range of peptide neurotoxins. Using a monoclonal antibody that specifically recognizes Gla residues, venom samples from various organisms were screened by western blotting and immunofluorescence assays. Amino acid analyses were also performed on most samples. A survey of 21 snake species from 12 genera detected gamma-carboxylated polypeptides only in venom of snakes from the elapid subfamily Acanthophiinae. Gla-containing polypeptides were also observed in cone snail venom but not in venom or toxic salivary secretions from several other organisms. The Gla-specific antibody used here provides a simple immunochemical means to detect gamma-carboxylated polypeptides in venom and may allow new species to be identified that utilize Gla in the biosynthesis of toxic polypeptides.
Assuntos
Ácido 1-Carboxiglutâmico/análise , Venenos de Anfíbios/química , Venenos Elapídicos/química , Fator Xa/metabolismo , Venenos de Moluscos/química , Vitamina K/farmacologia , Ácido 1-Carboxiglutâmico/imunologia , Ácido 1-Carboxiglutâmico/farmacologia , Animais , Anticorpos Monoclonais , Anuros , Bioensaio/métodos , Proteínas Sanguíneas/metabolismo , Western Blotting , Fator Xa/análise , Imunofluorescência , Peptídeos/análise , Peptídeos/química , Glândulas Salivares/química , Caramujos , SerpentesRESUMO
P-selectin is a transmembrane protein that resides within the alpha granule membrane of unstimulated platelets. The "extracellular" domains face into the lumen of the granule and the cytoplasmic tail extends into the platelet cytoplasm. Upon platelet stimulation, P-selectin is phosphorylated and translocated to the plasma membrane via a secretory pathway. P-selectin in the plasma membrane surface is exposed and serves as a cell adhesion receptor to interact with other cell receptors, including PSGL-1 and GPIb. P-selectin upregulates tissue factor in monocytes and leads to leukocyte accumulation in areas of vascular injury associated with thrombosis and inflammation.
Assuntos
Plaquetas/química , Selectina-P/sangue , Proteínas de Transporte Vesicular , Adesão Celular/fisiologia , Humanos , Proteínas de Membrana/fisiologia , Selectina-P/metabolismo , Selectina-P/farmacologia , Transporte Proteico , Proteínas SNARE , Vesículas Secretórias/metabolismo , Transdução de SinaisRESUMO
The hydrophobic omega-loop within the prothrombin gamma-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1-46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-(1-46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1-46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.
Assuntos
Protrombina/química , Protrombina/metabolismo , Ácido 1-Carboxiglutâmico/química , Corantes Fluorescentes/metabolismo , Humanos , Modelos Biológicos , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Iodeto de Potássio/química , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de FluorescênciaRESUMO
P-selectin glycoprotein ligand 1 (PSGL-1) is a sialomucin expressed on leukocytes that mediates neutrophil rolling on the vascular endothelium. Here, the role of PSGL-1 in mediating lymphocyte migration was studied using mice lacking PSGL-1. In a contact hypersensitivity model, the infiltration of CD4(+) T lymphocytes into the inflamed skin was reduced in PSGL-1-deficient mice. In vitro-generated T helper (Th)1 cells from PSGL-1-deficient mice did not bind to P-selectin and migrated less efficiently into the inflamed skin than wild-type Th1 cells. To assess the role of PSGL-1 in P- or E-selectin-mediated migration of Th1 cells, the cells were injected into E- or P-selectin-deficient mice. PSGL-1-deficient Th1 cells did not migrate into the inflamed skin of E-selectin-deficient mice, indicating that PSGL-1 on Th1 cells is the sole ligand for P-selectin in vivo. In contrast, PSGL-1-deficient Th1 cells migrated into the inflamed skin of P-selectin-deficient mice, although less efficiently than wild-type Th1 cells. This E-selectin-mediated migration of PSGL-1-deficient or wild-type Th1 cells was not altered by injecting a blocking antibody to L-selectin. These data provide evidence that PSGL-1 on Th1 cells functions as one of the E-selectin ligands in vivo.
Assuntos
Quimiotaxia de Leucócito , Dermatite Alérgica de Contato/imunologia , Selectina E/imunologia , Glicoproteínas de Membrana/imunologia , Selectina-P/imunologia , Células Th1/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Células CHO , Cricetinae , Dermatite Alérgica de Contato/patologia , Modelos Animais de Doenças , Selectina E/genética , Selectina E/metabolismo , Ligantes , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxazolona/efeitos adversos , Selectina-P/genética , Selectina-P/metabolismo , Pele/imunologia , Pele/patologia , Células Th1/metabolismo , Células Th1/fisiologiaRESUMO
Phospholipid membranes play a significant role during the proteolytic activation of blood coagulation proteins. This investigation identifies a role for phosphatidylethanolamine (PE) during the activation of factor X by the tenase complex, an enzymatic complex composed of the serine protease, factor IXa, a protein cofactor, factor VIIIa, a phospholipid membrane, and Ca(2+). Phospholipid vesicles composed of PE, phosphatidylserine (PS), and phosphatidylcholine support factor Xa generation. The K(m) and k(cat) for factor X activation by the tenase complex are independent of PE in the presence of 20% PS. At lower PS concentrations, the presence of 20 or 35% PE lowers the K(m) and increases the k(cat) as compared to those in vesicles without PE. The effect of PE on the k(cat) of the tenase complex is mediated through factor VIIIa. PE also enhances factor Xa generation by facilitating tenase complex formation; PE lowers the K(d(app)) of factor IXa for both phospholipid/Ca(2+) and phospholipid/Ca(2+)/factor VIIIa complexes in the presence of suboptimal PS. In addition, the K(d)s of factor IXa and factor X were lower for phospholipid vesicles containing PE. N-Methyl-PE increased the k(cat) and decreased the K(d(app)), whereas N,N-dimethyl-PE had no effect on either parameter, indicating the importance of headgroup size. Lyso-PE had no effect on kinetic parameters, indicating the sn-2 acyl chain dependence of the PE effect. Together, these results demonstrate a role for PE in the assembly and activity of the tenase complex and further extend the understanding of the importance of PE-containing membranes in hemostasis.
Assuntos
Fator IXa/metabolismo , Fator VIIIa/metabolismo , Lipossomos/química , Fosfatidiletanolaminas/química , Fosfolipídeos/química , Sítios de Ligação , Cálcio/metabolismo , Transferência de Energia , Fator IXa/química , Fator VIIIa/química , Humanos , Cinética , Espectrometria de FluorescênciaRESUMO
The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.
Assuntos
Fator Va/metabolismo , Protrombina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Protrombina/químicaRESUMO
Blood coagulation factor IXa gains proteolytic efficiency upon binding to a phospholipid membrane. We have found that an amphipathic, membrane-binding peptide from the C2 domain of factor VIII, fVIII(2303)(-23), enhances proteolytic efficiency of factor IXa in the absence of phospholipid membranes. This enhancement is the result of a reduction in the K(M) for the substrate, factor X, with little effect on the k(cat). Enhanced function requires interaction of the gamma-carboxyglutamic acid (Gla) domains of factor IXa and factor X since (i) a synthetic peptide comprising the Gla domain of factor IXa and antibodies directed to the Gla domain of factor IXa inhibit this acceleration, (ii) the acceleration is Ca(II) dependent, and (iii) conversion of Gla-domainless factor X is not affected by the presence of fVIII(2303)(-23). The effect of fVIII(2303)(-23) on factor IXa parallels the enhanced function produced by phosphatidylserine-containing bilayers, and fVIII(2303)(-23) does not further enhance function of factor IXa when phospholipid vesicles are present. The critical feature of fVIII(2303)(-23) is apparently its amphipathic helix-forming structure [Gilbert, G. E., and Baleja, J. D. (1995) Biochemistry 34, 3022-3031] because other alpha-helical peptides such as a homologous peptide from the C2 domain of factor V and melittin have similar effects. Diastereomeric analogues of fVIII(2303)(-23) and melittin, which have reduced helical content, do not support factor IXa activity. A truncated peptide of fVIII(2303)(-23) with three C-terminal residues deleted retains alpha-helical content but loses capacity to enhance factor X cleavage, suggesting that a minimum length of alpha-helix is required. Although these results probably do not illuminate the physiologic function of the factor VIII peptide corresponding to fVIII(2303)(-23), they demonstrate a novel, membrane-mimetic role of amphipathic helical peptides in supporting function of factor IXa.
Assuntos
Venenos de Crotalídeos , Fator IXa/química , Fragmentos de Peptídeos/química , Proteínas de Répteis , Ácido 1-Carboxiglutâmico/química , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Catálise , Fator IX/química , Fator IX/metabolismo , Fator IXa/metabolismo , Fator VIII/química , Fator VIII/metabolismo , Fator X/química , Fator X/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid, an amino acid critical to the function of the vitamin K-dependent blood coagulation proteins. Given the functional similarity of mammalian vitamin K-dependent carboxylases and the vitamin K-dependent carboxylase from Conus textile, a marine invertebrate, we hypothesized that structurally conserved regions would identify sequences critical to this common functionality. Furthermore, we examined the diversity of animal species that maintain vitamin K-dependent carboxylation to generate gamma-carboxyglutamic acid. We have cloned carboxylase homologs in full-length or partial form from the beluga whale (Delphinapterus leucas), toadfish (Opsanus tau), chicken (Gallus gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus polyphemus), and cone snail (Conus textile) to compare these structures to the known bovine, human, rat, and mouse cDNA sequences. Comparison of the predicted amino acid sequences identified a nearly perfectly conserved 38-amino acid residue region in all of these putative carboxylases. In addition, this amino acid motif is also present in the Drosophila genome and identified a Drosophila homolog of the gamma-carboxylase. Assay of hagfish liver demonstrated vitamin K-dependent carboxylase activity in this hemichordate. These results demonstrate the broad distribution of the vitamin K-dependent carboxylase gene, including a highly conserved motif that is likely critical for enzyme function. The vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid appears to be a highly conserved function in the animal kingdom.
Assuntos
Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/genética , Sequência Conservada , Peixes , Vitamina K/metabolismo , Baleias , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Galinhas , Clonagem Molecular , Peixes/genética , Feiticeiras (Peixe)/genética , Humanos , Invertebrados/química , Invertebrados/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Baleias/genéticaRESUMO
Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.
Assuntos
Conotoxinas/química , Venenos de Moluscos/química , Alquilação , Sequência de Aminoácidos , Aminoácidos/análise , Conotoxinas/isolamento & purificação , Dissulfetos/química , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredução , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin-like selectin counterreceptor that binds to P-selectin, E-selectin, and L-selectin. To determine its physiological role in cell adhesion as a mediator of leukocyte rolling and migration during inflammation, we prepared mice genetically deficient in PSGL-1 by targeted disruption of the PSGL-1 gene. The homozygous PSGL-1-deficient mouse was viable and fertile. The blood neutrophil count was modestly elevated. There was no evidence of spontaneous development of skin ulcerations or infections. Leukocyte infiltration in the chemical peritonitis model was significantly delayed. Leukocyte rolling in vivo, studied by intravital microscopy in postcapillary venules of the cremaster muscle, was markedly decreased 30 min after trauma in the PSGL-1-deficient mouse. In contrast, leukocyte rolling 2 h after tumor necrosis factor alpha stimulation was only modestly reduced, but blocking antibodies to E-selectin infused into the PSGL-1-deficient mouse almost completely eliminated leukocyte rolling. These results indicate that PSGL-1 is required for the early inflammatory responses but not for E-selectin-mediated responses. These kinetics are consistent with a model in which PSGL-1 is the predominant neutrophil P-selectin ligand but is not a required counterreceptor for E-selectin under in vivo physiological conditions.
Assuntos
Movimento Celular/fisiologia , Selectina E/fisiologia , Glicoproteínas de Membrana/fisiologia , Neutrófilos/citologia , Neutrófilos/fisiologia , Selectina-P/fisiologia , Animais , Deleção de Genes , Marcação de Genes , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.
Assuntos
Plaquetas/citologia , Plaquetas/fisiologia , Calpaína/fisiologia , Adesividade Plaquetária/fisiologia , Agregação Plaquetária , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio , Humanos , Dados de Sequência Molecular , Mutação , Fragmentos de PeptídeosRESUMO
To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.
Assuntos
Conotoxinas/química , Venenos de Moluscos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Bovinos , Dicroísmo Circular , Clonagem Molecular , Conotoxinas/genética , Conotoxinas/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Dados de Sequência Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Caramujos/genéticaRESUMO
Tandem mass spectrometry (MS/MS) of 28 residue peptides harboring gamma-carboxylated glutamic acid residues, a posttranslational modification of several proenzymes of the blood coagulation cascade, using either collisions or infrared photons results in complete ejection of the gamma-CO2 moieties (-44 Da) before cleavage of peptide-backbone bonds. However, MS/MS using electron capture dissociation (ECD) in a Fourier transform mass spectrometer cleaves backbone bonds without ejecting CO2, allowing direct localization of this labile modification. Sulfated side chains are also retained in ECD backbone fragmentations of a 21-mer peptide, although CAD causes extensive SO3 loss. ECD thus is a unique complement to conventional methods for MS/MS, causing less undesirable loss of side-chain functionalities as well as more desirable backbone cleavages.
Assuntos
Ácido 1-Carboxiglutâmico/análise , Ácido 1-Carboxiglutâmico/química , Espectrometria de Massas/métodos , Peptídeos/análise , Peptídeos/metabolismo , Ácido 1-Carboxiglutâmico/metabolismo , Sequência de Aminoácidos , Íons , Dados de Sequência Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , SulfatosRESUMO
The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.
Assuntos
Carbono-Carbono Ligases/metabolismo , Cisteína/metabolismo , Compostos de Epóxi/metabolismo , Ácido Glutâmico/metabolismo , Vitamina K/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carbono-Carbono Ligases/antagonistas & inibidores , Ativação Enzimática , Compostos de Epóxi/antagonistas & inibidores , Etilmaleimida/química , Etilmaleimida/metabolismo , Ácido Glutâmico/química , Humanos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Precursores de Proteínas/química , Especificidade por Substrato , Reagentes de Sulfidrila/química , Vitamina K/antagonistas & inibidoresRESUMO
Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Conotoxinas , Venenos de Moluscos/química , Peptídeos/química , Processamento de Proteína Pós-Traducional/genética , Ácido 1-Carboxiglutâmico/química , Animais , Aplysia/metabolismo , Cálcio/metabolismo , Dissulfetos/química , Espectroscopia de Ressonância Magnética , Peptídeos/farmacologia , Caramujos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cell-cell interactions mediating leukocyte trafficking, thrombogenesis and inflammation are crucial for the host defense mechanism. The selectin family of integral membrane proteins includes E-selectin, L-selectin and P-selectin. Selectins mediate tethering and rolling of leukocytes to the vessel wall at the site of inflammation. The counter-receptor for P-selectin and possibly the other selectins is P-selectin glycoprotein ligand-1 (PSGL-1). This review focuses on the properties and biology of PSGL- 1.