RESUMO
Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.
Assuntos
Aminopeptidases/metabolismo , Exossomos/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Aminopeptidases/genética , Animais , Citocinas/genética , Citocinas/metabolismo , Exossomos/genética , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Fagocitose , Células RAW 264.7RESUMO
Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Dipeptidil Peptidase 4/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Exossomos/metabolismo , Saliva/citologia , Adulto , Membrana Celular/efeitos dos fármacos , Temperatura Baixa , Detergentes/farmacologia , Exossomos/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Octoxinol/farmacologia , Polietilenoglicóis/farmacologia , Adulto JovemRESUMO
Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.
Assuntos
Exossomos/genética , RNA/genética , Saliva/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteínas/genética , Análise de Sequência de RNA , TranscriptomaRESUMO
Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.
RESUMO
Small non-coding RNAs, such as microRNAs (miRNAs), are involved in diverse processes, including organ development and tissue differentiation. Exosomes are small membrane vesicles (30-100 nm in diameter) produced by numerous cells. Recently, exosomes have been shown to contain miRNAs. However, the small RNAs contained in exosomes are not fully characterized. In a previous study, we found at least two types of salivary exosome that are different in size and have different proteomes. Studies of salivary exosomal small RNAs are limited to miRNAs. In this study, we examined small RNA transcriptomes using next generation sequencing technology to elucidate a full transcriptome set of small RNAs expressed in the two types of salivary exosomes and in whole saliva (WS). Many types of small RNA, such as miRNA, piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA) and other small RNAs are contained in salivary exosomes and WS. Among these small RNAs we identified novel miRNA candidates.
Assuntos
Exossomos/metabolismo , Pequeno RNA não Traduzido/genética , Saliva/metabolismo , Adulto , Feminino , Humanos , Análise de Sequência de RNA , TranscriptomaRESUMO
Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.
Assuntos
Exossomos/metabolismo , Proteômica , Saliva/química , Proteínas e Peptídeos Salivares/metabolismo , Biomarcadores , Exossomos/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas e Peptídeos Salivares/análise , Proteínas e Peptídeos Salivares/genéticaRESUMO
BACKGROUND: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. METHODS: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. RESULTS: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. CONCLUSION: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract.
RESUMO
SSJ-127, a novel antimalarial rhodacyanine derivative, has shown potent antimalarial activity against chloroquine-resistant Plasmodium strains in vitro and subcutaneous administration of SSJ-127 results in a complete cure of a mouse malaria model. SSJ-127 was detected by fluorescence microscopy in the mouse malaria parasites Plasmodium berghei after exposure of infected red blood cells to the compound in vitro and in vivo. Selective accumulation of SSJ-127 in an organelle is observed in all blood stages of live malaria parasites. The organelle is clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways.
Assuntos
Antimaláricos/química , Benzotiazóis/química , Oxazóis/química , Plasmodium berghei/efeitos dos fármacos , Compostos de Piridínio/química , Tiazóis/química , Animais , Antimaláricos/síntese química , Antimaláricos/farmacologia , Benzotiazóis/síntese química , Benzotiazóis/farmacologia , Eritrócitos/parasitologia , Camundongos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Oxazóis/síntese química , Oxazóis/farmacologia , Compostos de Piridínio/síntese química , Compostos de Piridínio/farmacologia , Rodamina 123/metabolismo , Tiazóis/síntese química , Tiazóis/farmacologiaRESUMO
Six allergic conjunctivitis patients (12 eyes) and 4 healthy volunteers (8 eyes) were investigated in terms of the effect of cooling sheets on eye itching and tear histamine concentration, before and 5 min after cooling the eyelids with cooling sheets. The severity of itching was evaluated with a five-level itching score. The combination treatment of levocabastine with cooling sheets significantly reduced eye itching, while no significant change in tear histamine concentration was observed before and after cooling sheet use. The cooling sheets are useful for reducing eye itching in the therapy of allergic conjunctivitis. The tear histamine concentration did not correlate with the antiitching effect of cooling sheets in this study.
Assuntos
Conjuntivite Alérgica/terapia , Crioterapia/métodos , Antagonistas não Sedativos dos Receptores H1 da Histamina/administração & dosagem , Histamina/análise , Piperidinas/administração & dosagem , Prurido/terapia , Lágrimas/química , Ensaio de Imunoadsorção Enzimática , Humanos , Soluções Oftálmicas , Índice de Gravidade de DoençaRESUMO
Exosomes are small membrane vesicles (30-100 nm) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We provide here the first evidence for the presence of exosome-like vesicles in snake venom. We isolated vesicles from fresh venom from Gloydius blomhoffii blomhoffii by gel-filtration. We found that the vesicles showed a typical exosome-like size and morphology as analyzed by electron microscopy. We observed that the vesicles contained dipeptidyl peptidase IV, aminopeptidase A, ecto-5'-nucleotidase and actin. Vesicle preparations truncated bioactive peptides such as angiotensin II, substance P, cholecystokinin-octapeptide, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. The role of these vesicles is still unknown, but they may affect blood pressure and glucose homeostasis following envenomation.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Homeostase/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Vesículas Secretórias/metabolismo , Viperidae , 5'-Nucleotidase/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Pressão Sanguínea/fisiologia , Colecistocinina/metabolismo , Cromatografia em Gel , Venenos de Crotalídeos/metabolismo , Dipeptidil Peptidase 4/metabolismo , Eletroforese em Gel de Poliacrilamida , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glutamil Aminopeptidase/metabolismo , Homeostase/fisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Vesículas Secretórias/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância P/metabolismoRESUMO
Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), is classified into two subgroups, Stx1 and Stx2. Clinical data clearly indicate that Stx2 is associated with more severe toxicity than Stx1, but the molecular mechanism underlying this difference is not fully understood. Here, we found that after being incorporated into target cells, Stx2, can be transported by recycling endosomes, as well as via the regular retrograde transport pathway. However, transport via recycling endosome did not occur with Stx1. We also found that Stx2 is actively released from cells in a receptor-recognizing B-subunit dependent manner. Part of the released Stx2 is associated with microvesicles, including exosome markers (referred to as exo-Stx2), whose origin is in the multivesicular bodies that formed from late/recycling endosomes. Finally, intravenous administration of exo-Stx2 to mice causes more lethality and tissue damage, especially severe renal dysfunction and tubular epithelial cell damage, compared to a free form of Stx2. Thus, the formation of exo-Stx2 might contribute to the severity of Stx2 in vivo, suggesting new therapeutic strategies against EHEC infections.
Assuntos
Exossomos/metabolismo , Toxina Shiga II/toxicidade , Fatores de Virulência/toxicidade , Animais , Transporte Biológico , Endossomos/metabolismo , Rim/efeitos dos fármacos , Camundongos , Toxina Shiga II/metabolismo , Fatores de Virulência/metabolismoRESUMO
The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.
Assuntos
Venenos de Crotalídeos/enzimologia , Glutamil Aminopeptidase/genética , Proteínas de Membrana/genética , Filogenia , Viperidae , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Gel , Clonagem Molecular , Análise por Conglomerados , Primers do DNA/genética , DNA Complementar/genética , Glutamil Aminopeptidase/análise , Proteínas de Membrana/análise , Dados de Sequência Molecular , Conformação Proteica , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.
Assuntos
Bungarotoxinas/toxicidade , Clonagem Molecular/métodos , DNA Complementar/isolamento & purificação , Sequência de Aminoácidos , Bungarotoxinas/química , Bungarotoxinas/isolamento & purificação , Filogenia , Reação em Cadeia da PolimeraseRESUMO
Dipeptidyl peptidase activity was investigated in snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis and Crotalus atrox. The strongest dipeptidyl peptidase IV (DPP IV) activity was found in venom from G. blomhoffi brevicaudus. The substrate specificity, susceptibility to inhibitors, and pH optimum of the partially purified enzyme were similar to those of known DPP IVs from bacteria and eukaryotes. The G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on amino acid sequences highly conserved in known DPP IVs. Two cDNA species encoding DPP IV were obtained, and designated as DPP IVa and DPP IVb. This is the first study to report the primary structure of DPP IV from a reptile. The deduced amino acid sequences for DPP IVa and DPP IVb both consist of 751amino acid residues and are highly homologous to each other. A putative catalytic triad for serine proteases, Ser-616, Asp-694, and His-726, is present. It is of particular interest that the deduced NH(2)-terminal sequence associated with the characteristic signal peptide is identical to that determined from the purified DPP IV. This indicates that the signal peptide of snake venom DPP IV is not cleaved off during biosynthesis, unlike those of other snake venom proteins.
Assuntos
Venenos de Crotalídeos/enzimologia , Dipeptidil Peptidase 4/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Venenos de Crotalídeos/química , DNA Complementar/química , DNA Complementar/metabolismo , Dipeptidil Peptidase 4/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.
Assuntos
Bungarus , Venenos Elapídicos/isolamento & purificação , Venenos Elapídicos/toxicidade , Neurotoxinas/isolamento & purificação , Neurotoxinas/toxicidade , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/toxicidade , Sequência de Aminoácidos , Animais , Bungarus/genética , Venenos Elapídicos/enzimologia , Feminino , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Neurotoxinas/genética , Fosfolipases A/metabolismo , Fosfolipases A2 , TailândiaRESUMO
The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.
Assuntos
Proteínas Neurotóxicas de Elapídeos/genética , Elapidae/genética , Filogenia , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Proteínas Neurotóxicas de Elapídeos/química , Dose Letal Mediana , Dados de Sequência Molecular , Compostos Organofosforados , Alinhamento de Sequência , Análise de Sequência de ProteínaAssuntos
Venenos de Cnidários/química , Peptídeos/química , Venenos de Serpentes/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Venenos de Crotalídeos/química , Citotoxinas/química , Citotoxinas/fisiologia , Venenos Elapídicos/química , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/fisiologia , Peptídeos/fisiologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Venenos de Víboras/químicaRESUMO
A Gloydius blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences from known ecto-5'-nucleotidases (ecto-5'-NTs). Molecular cloning of ecto-5'-NT from G. blomhoffi brevicaudus venom predicted that it was a glycosyl phosphatidylinositol-anchored membrane protein containing 588 amino acid residues with 7 potential N-linked glycosylation sites. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian ecto-5'-NT sequences. This is the first report of the primary structure of ecto-5'-NT from a reptile. Gel-filtration chromatography of fresh venom from Gloydius blomhoffi blomhoffi, a subspecies of G. blomhoffi, revealed that at least a part of ecto-5'-NT is bound to exosome-like vesicles.
Assuntos
5'-Nucleotidase/genética , Venenos de Crotalídeos/enzimologia , Viperidae/metabolismo , 5'-Nucleotidase/química , 5'-Nucleotidase/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Clonagem Molecular , Venenos de Crotalídeos/química , Exossomos/metabolismo , Biblioteca Gênica , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Saliva contains a large number of proteins that participate in the protection of oral tissue. We found, for the first time, small vesicles (30-130 nm in diameter) in human whole saliva. Vesicles from saliva were identified by electron microscopy after isolation by gel-filtration on Sepharose CL-4B. They resemble exosomes, which are vesicles with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We performed a biochemical characterization of these vesicles by amino acid sequence analysis and Western blot analysis. We found that they contain dipeptidyl peptidase IV (DPP IV), galectin-3 and immunoglobulin A, which have potential to influence immune response. The DPP IV in the vesicles was metabolically active in cleaving substance P and glucose-dependent insulinotropic polypeptide to release N-terminal dipeptides. Our results demonstrate that human whole saliva contains exosome-like vesicles; they might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Saliva/enzimologia , Vesículas Secretórias/enzimologia , Sequência de Aminoácidos , Western Blotting , Cromatografia em Gel , Galectina 3/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Humanos , Imunoglobulina A/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Vesículas Secretórias/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância P/metabolismoRESUMO
We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.