RESUMO
Snake venoms consist of a complex mixture of many bioactive molecules. Among them are disintegrins, which are peptides without enzymatic activity, but with high binding affinity for integrins, transmembrane receptors that function to connect cells with components of the extracellular matrix. Integrin-mediated cell attachment is critical for cell migration and dissemination, as well as for signal transduction pathways involved in cell growth. During tumor development, integrins play key roles by supporting cancer cell proliferation, angiogenesis, and metastasis. The recognition that snake venom disintegrins can block integrin functions has spawned a number of studies to explore their cancer therapeutic potential. While dozens of different disintegrins have been isolated, none of them as yet has undergone clinical evaluation in cancer patients. Among the best-characterized and preclinically most advanced disintegrins is vicrostatin (VCN), a recombinant disintegrin that was rationally designed by fusing 62 N-terminal amino acids derived from the disintegrin contortrostatin with 6 C-terminal amino acids from echistatin, the disintegrins from another snake species. Bacterially produced VCN was shown to target multiple tumor-associated integrins, achieving potent anti-tumor and anti-angiogenic effects in in vitro and in vivo models in the absence of noticeable toxicity. This review will introduce the field of snake venom disintegrins as potential anticancer agents and illustrate the translational development and cancer-therapeutic potential of VCN as an example.
Assuntos
Antineoplásicos/farmacologia , Desintegrinas/farmacologia , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Desintegrinas/química , Desintegrinas/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Venenos de Serpentes/química , Venenos de Serpentes/metabolismoRESUMO
A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.
Assuntos
Radioisótopos de Cobre/farmacocinética , Desintegrinas/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/diagnóstico por imagem , Animais , Radioisótopos de Cobre/química , Desintegrinas/química , Avaliação Pré-Clínica de Medicamentos , Compostos Heterocíclicos/química , Humanos , Masculino , Camundongos , Camundongos Nus , Especificidade de Órgãos , Células PC-3 , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Polietilenoglicóis/química , Neoplasias da Próstata/metabolismo , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Sarcosina/análogos & derivados , Sarcosina/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Herpes simplex virus (HSV) causes significant health problems from periodical skin and corneal lesions to encephalitis. HSV entry provides a unique opportunity for therapeutic intervention. In this study, we evaluated contortrostatin (CN), an Arg-Gly-Asp motif containing disintegrin isolated from snake venom, as a novel therapeutic agent with ability to block HSV entry and related membrane fusion. METHODS: In vitro efficacy of CN against HSV was determined using an HSV type-1 (HSV-1) entry assay based on the measurement of ß-galactosidase reporter activity originating from the genome of a recombinant strain of HSV-1(KOS) gL86. HSV-1 glycoprotein-mediated cell-to-cell fusion was used to study the effect of CN on polykaryocyte formation. Primary as well as transformed cell lines were used for this study. RESULTS: Pre-treatment of Chinese hamster ovary (CHO-K1) cells expressing HSV-1 glycoprotein D receptors and primary cultures of human corneal fibroblasts (CF) with CN resulted in the inhibition of entry, cell-to-cell fusion, and polykaryocyte formation. Interestingly, a more pronounced anti-HSV-1 effect was observed in naturally susceptible CF than CHO-K1 cells. CONCLUSIONS: CN, a novel venom-derived peptide, exhibits the ability to block two key steps, entry and cell-to-cell fusion, in HSV infection. Showing strong promise for development as an anti-HSV agent, it also demonstrates better prophylactic efficacy in primary cells.
Assuntos
Desintegrinas/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Venenos de Serpentes/química , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacologia , Células CHO , Fusão Celular , Córnea/citologia , Cricetinae , Desintegrinas/química , Desintegrinas/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Células Gigantes/química , Células Gigantes/efeitos dos fármacos , Células HeLa , Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Cultura Primária de Células , Receptores de Superfície Celular/química , Transfecção , Proteínas do Envelope Viral/química , Ensaio de Placa ViralRESUMO
Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However, although bites can be deadly, snake venom is a natural biological resource that contains several components of potential therapeutic value. Venom has been used in the treatment of a variety of pathophysiological conditions in Ayurveda, homeopathy and folk medicine. With the advent of biotechnology, the efficacy of such treatments has been substantiated by purifying components of venom and delineating their therapeutic properties. This review will focus on certain snake venom components and their applications in health and disease.
Assuntos
Hemostasia , Neurotoxinas/química , Venenos de Serpentes/química , Animais , Sistema Cardiovascular/efeitos dos fármacos , Desintegrinas/química , Desintegrinas/farmacologia , Fibrinogênio/química , Fibrinogênio/farmacologia , Lectinas Tipo C/química , Músculos/efeitos dos fármacos , Fosfolipases A/metabolismo , Fosfolipases A/farmacologia , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Estrutura Terciária de Proteína , Protrombina/química , Protrombina/farmacologia , Transdução de Sinais , Trombina/química , Trombina/farmacologiaRESUMO
Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional (1)H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of alpha(1)-K562 cells to collagen IV with IC(50) values of 180 and 703 nm, respectively. The IC(50) values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 microm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including alpha(IIb)beta(3), alpha(v)beta(3), alpha(2)beta(1), alpha(5)beta(1), alpha(4)beta(1), alpha(6)beta(1), and alpha(9)beta(1) up to a concentration of 24 mum. Although the RTS motif appears to be more potent than KTS inhibiting the alpha(1)beta(1) integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity.
Assuntos
DNA Complementar/genética , Desintegrinas/genética , Integrina alfa1beta1/antagonistas & inibidores , Trimeresurus/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Venenos de Crotalídeos , Cisteína/análise , Desintegrinas/química , Desintegrinas/farmacologia , Dissulfetos/análise , Glândulas Exócrinas/química , Expressão Gênica , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Mapeamento de Peptídeos , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes , Tripsina/metabolismoRESUMO
We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64/Gly and Arg233/Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of alpha2-macroglobulin, although physiological substrates are presently unknown.
Assuntos
Proteínas ADAM/química , Proteínas ADAM/metabolismo , Metaloproteases/química , Proteínas ADAMTS , Sequência de Aminoácidos , Animais , Biotinilação , Northern Blotting , Western Blotting , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , Cisteína/química , DNA Complementar/metabolismo , Desintegrinas/química , Embrião de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Furina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Trombospondinas/química , Fatores de Tempo , Distribuição Tecidual , Transfecção , alfa-Macroglobulinas/metabolismoRESUMO
The adamalysins are involved in proteolysis, adhesion, fusion, and intracellular signaling. Human ADAM19/adamalysin-19 (A disintegrin and metalloproteinase 19) was identified from primary dendritic cell cDNA libraries. It has a signal sequence, a pro-domain with a "cysteine-switch" residue, a metalloproteinase domain with a zinc-binding site, a disintegrin, a cysteine-rich domain, an epidermal-growth-factor-like domain, a transmembrane domain, and a cytoplasmic domain with putative SH3 ligand binding sites. Its mRNA was expressed in the placenta, heart, bladder, lymph nodes, and leukocytes, colorectal adenocarcinoma SW 480, and other organs/cells. The hADAM19 recombinant protein was expressed in human cells. It formed a complex with and cleaved alpha-2 macroglobulin (alpha2-M). Its proteolytic activity was blocked by 1,10-phenanthroline, EDTA, EGTA, and a synthetic matrix metalloproteinase (MMP) inhibitor and not by the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. It did not cleave the MMP substrates tested, e.g., type I collagen and gelatin, casein, and four peptide substrates. Thus, hADAM19 is an active metalloproteinase and may have a specific substrate profile.
Assuntos
Desintegrinas/genética , Desintegrinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas ADAM , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Desintegrinas/química , Feminino , Expressão Gênica , Humanos , Técnicas In Vitro , Masculino , Proteínas de Membrana/química , Metaloendopeptidases/química , Dados de Sequência Molecular , Proteínas Musculares/química , Gravidez , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Distribuição Tecidual , alfa-Macroglobulinas/metabolismoRESUMO
ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 modules) proteins constitute a family of zinc metalloproteinases which target and process extracellular matrix proteins. We cloned and characterized a novel human ADAMTS gene, ADAMTS14, which is located on human chromosome 10q2. ADAMTS14 exhibits the characteristic multidomain structure of ADAMTS proteins including four thrombospondin modules and shows highest similarity to ADAMTS3 and ADAMTS2. By RT-PCR analysis we demonstrated that ADAMTS14 is expressed in human retina and also at low levels in adult brain, lung and placenta.
Assuntos
Metaloendopeptidases/genética , Proteínas ADAM , Proteínas ADAMTS , Proteína ADAMTS4 , Sequência de Aminoácidos , Cromossomos Humanos Par 10 , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Desintegrinas/química , Endopeptidases/química , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Pró-Colágeno N-Endopeptidase/química , Retina/metabolismo , Trombospondinas/químicaRESUMO
Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.
Assuntos
Venenos de Crotalídeos/química , DNA Complementar/metabolismo , Metaloendopeptidases/genética , Sequência de Aminoácidos , Apoptose , Proteínas Reguladoras de Apoptose , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Clonagem Molecular , Venenos de Crotalídeos/metabolismo , Fragmentação do DNA , Desintegrinas/química , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Fibrinogênio/metabolismo , Biblioteca Gênica , Humanos , Hidrólise , Metaloendopeptidases/química , Dados de Sequência Molecular , Oligopeptídeos/química , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Veias Umbilicais/metabolismo , Zinco/metabolismoRESUMO
ADAMs (a disintegrin and metalloproteases) mediate several important processes (e.g. tumor necrosis factor-alpha release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin alpha(9)beta(1) specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and alpha(9)beta(1) supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for alpha(9)beta(1)/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/alpha(6)beta(1) interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that alpha(9)beta(1) may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and alpha(9)beta(1), this interaction may be of potential biological and pathological significance.
Assuntos
Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Oligopeptídeos/metabolismo , Proteínas ADAM , Proteína ADAM12 , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cálcio/metabolismo , Cátions , Adesão Celular , Comunicação Celular , Cricetinae , DNA Complementar/metabolismo , Desintegrinas/química , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Glutationa Transferase/metabolismo , Humanos , Células K562 , Cinética , Magnésio/metabolismo , Manganês/metabolismo , Proteínas de Membrana/química , Metaloendopeptidases/química , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
By means of DEAE-Sepharose CL-6B column chromatography, gel filtration on Sephadex G-75 and Superose 12 FPLC, halysetin, an antiplatelet protein, was purified from the venom of Agkistrodon halys Pallas with molecular mass of 29 kDa on SDS-PAGE and 23,168 Da by mass spectrometry. The p1 was about 5.0. Halysetin was devoid of phospholipase A2, fibrino-(geno)lytic, esterase, hemorrhagenic activities. Halysetin dose-dependently inhibited the aggregation of human platelet, which was stimulated by collagen with IC50 of 420 nM, but not that stimulated by ADP. The N-and C-terminal sequences of halysetin were characterized. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. It encoded a protein of 212-amino-acid residues with disintegrin-like/cysteine-rich domains and was highly homologous with SYMPs (snake venom metalloprotease).
Assuntos
Cisteína/química , Desintegrinas/química , Venenos de Víboras/química , Venenos de Víboras/isolamento & purificação , Difosfato de Adenosina/metabolismo , Agkistrodon , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Agarose , Cromatografia Líquida , Colágeno/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Esterases/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Espectrometria de Massas , Dados de Sequência Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Agregação Plaquetária , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/química , Venenos de Víboras/genéticaRESUMO
Metalloproteinase-disintegrins (ADAMs) are type 1 transmembrane proteins that contain a unique domain structure including a zinc-binding metalloproteinase domain. We have isolated cDNAs encoding two novel members of this family, ADAM29 and ADAM30 which show testis-specific expression. Three forms of ADAM29 were found that encode proteins of 820, 786 and 767 amino acids. All of the amino acid differences are located in the cytoplasmic domain. Two forms of ADAM30 were isolated that encode proteins of 790 and 781 amino acids, with the difference in the coding region occurring in the cytoplasmic domain. ADAM29 and ADAM30 map to human chromosome 4q34 and 1p11-13, respectively. An ancestral analysis of all known mammalian ADAMs indicates that the zinc-binding motif in the catalytic domain arose once in a common ancestor and was subsequently lost by those members lacking this motif.
Assuntos
Mapeamento Cromossômico , DNA Complementar , Desintegrinas/metabolismo , Metaloendopeptidases/genética , Testículo/metabolismo , Proteínas ADAM , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cromossomos Humanos , Clonagem Molecular , Citoplasma/metabolismo , Desintegrinas/química , Evolução Molecular , Feminino , Biblioteca Gênica , Humanos , Masculino , Mamíferos , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
In this paper, we present a cDNA sequence encoding a full-length precursor form of a new member (ACLD) of the metalloproteinase-disintegrin-like protein family from the venom glands of Agkistrodon contortrix laticinctus (broad-banded copperhead) snake. Comparison of the deduced amino acid sequence of ACLD with those of other members of the metalloproteinase-disintegrin protein family from both mammalian and snake venom origin suggests that some conserved residues may be involved in processing of the disintegrin domain.
Assuntos
Venenos de Crotalídeos/química , Cisteína/química , DNA Complementar/análise , Desintegrinas/química , Metaloendopeptidases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Venenos de Crotalídeos/genética , Cisteína/genética , DNA Complementar/isolamento & purificação , Desintegrinas/genética , Humanos , Metaloendopeptidases/genética , Dados de Sequência Molecular , Família Multigênica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.
Assuntos
Antígenos CD40/imunologia , Células Dendríticas/enzimologia , Desintegrinas/química , Desintegrinas/genética , Centro Germinativo/enzimologia , Metaloendopeptidases/genética , Proteínas ADAM , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Antígenos CD11/imunologia , Clonagem Molecular , DNA Antissenso , DNA Complementar/química , Células Dendríticas/imunologia , Desintegrinas/biossíntese , Regulação da Expressão Gênica/genética , Centro Germinativo/imunologia , Humanos , Hibridização In Situ , Metaloendopeptidases/biossíntese , Metaloendopeptidases/química , Dados de Sequência Molecular , Tonsila Palatina , Reação em Cadeia da Polimerase , Análise de Sequência , Homologia de Sequência de Aminoácidos , Células-Tronco/químicaRESUMO
Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.