RESUMO
Structures similar to blood vessels in location, morphology, flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degradation of original organic components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel extracts is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.
Assuntos
Vasos Sanguíneos/anatomia & histologia , Vasos Sanguíneos/metabolismo , Dinossauros/anatomia & histologia , Dinossauros/metabolismo , Fósseis/anatomia & histologia , Actinas/genética , Actinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Vasos Sanguíneos/microbiologia , Osso e Ossos/irrigação sanguínea , Galinhas , Dinossauros/genética , Imunofluorescência/métodos , Espectrometria de Massas , Modelos Biológicos , Dados de Sequência Molecular , Miosinas/genética , Miosinas/isolamento & purificação , Filogenia , Proteômica/métodos , Alinhamento de Sequência , Especificidade da Espécie , Struthioniformes , Tropomiosina/genética , Tropomiosina/isolamento & purificação , Tubulina (Proteína)/genética , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
Whereas the roles of proangiogenic factors in carcinogenesis are well established, those of endogenous angiogenesis inhibitors (EAIs) remain to be fully elaborated. We investigated the roles of three EAIs during de novo tumorigenesis to further test the angiogenic balance hypothesis, which suggests that blood vessel development in the tumor microenvironment can be governed by a net loss of negative regulators of angiogenesis in addition to the well-established principle of up-regulated angiogenesis inducers. In a mouse model of pancreatic neuroendocrine cancer, administration of endostatin, thrombospondin-1, and tumstatin peptides, as well as deletion of their genes, reveal neoplastic stage-specific effects on angiogenesis, tumor progression, and survival, correlating with endothelial expression of their receptors. Deletion of tumstatin and thrombospondin-1 in mice lacking the p53 tumor suppressor gene leads to increased incidence and reduced latency of angiogenic lymphomas associated with diminished overall survival. The results demonstrate that EAIs are part of a balance mechanism regulating tumor angiogenesis, serving as intrinsic microenvironmental barriers to tumorigenesis.
Assuntos
Autoantígenos/metabolismo , Colágeno Tipo IV/metabolismo , Endostatinas/metabolismo , Neoplasias/metabolismo , Trombospondina 1/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/química , Autoantígenos/genética , Linhagem Celular , Colágeno Tipo IV/química , Colágeno Tipo IV/genética , Progressão da Doença , Endostatinas/química , Endostatinas/genética , Feminino , Humanos , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Estadiamento de Neoplasias , Neoplasias/genética , Neoplasias/prevenção & controle , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevenção & controle , Peptídeos/farmacologia , Propionatos/farmacologia , Análise de Sobrevida , Trombospondina 1/química , Trombospondina 1/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited. OBJECTIVE: To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint. DESIGN: Single blinded, randomized, placebo controlled pilot study. SETTING: Veterans Affairs Medical Center. PARTICIPANTS: Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint. METHODS: Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period. MAIN OUTCOME MEASURES: The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months. CONCLUSIONS: We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores.
Assuntos
Osteoartrite do Joelho , Plasma Rico em Plaquetas , Humanos , Ácido Hialurônico , Injeções Intra-Articulares , Articulação do Joelho , Osteoartrite do Joelho/tratamento farmacológico , Resultado do TratamentoRESUMO
The role of heparan sulfate (HS) in regulating blood coagulation has a wide range of clinical implications. In this study, we investigated the role of 3-O-sulfotransferase isoform 5 (3-OST-5) in generating anticoagulant HS in vivo. A Chinese hamster ovary cell line (3OST5/CHO) stably expressing 3-OST-5 was generated. The expression of 3-OST-5 in 3OST5/CHO cells was confirmed by Northern blot analysis, RT-PCR, and the disaccharide analyses of the HS from the cells. We also determined the effects of the HS from 3OST5/CHO on antithrombin-mediated inhibition of factor Xa. Fluorescently labeled antithrombin bound to the surface of 3OST5/CHO cells, suggesting that the antithrombin-binding HS is indeed present on the cell surface. Our results demonstrate that the 3-OST-5 gene is capable of synthesizing anticoagulant HS in CHO cells and has the potential to contribute to the biosynthesis of anticoagulant HS in humans.
Assuntos
Anticoagulantes/metabolismo , Heparitina Sulfato/biossíntese , Isoenzimas/metabolismo , Sulfotransferases/metabolismo , Animais , Anticoagulantes/química , Antitrombinas/metabolismo , Células CHO , Cricetinae , Dissacarídeos/análise , Eletroforese , Fator Xa/metabolismo , Heparitina Sulfato/química , Humanos , Isoenzimas/genética , Ligação Proteica , Sulfotransferases/genéticaRESUMO
Viral hepatitis resulting in chronic liver disease is an important clinical challenge and insight into the cellular processes that drive pathogenesis will be critical in order to develop new diagnostic and therapeutic options. Nuclear inclusions in viral and non-viral hepatitis are well documented and have diagnostic significance in some disease contexts. However, the origins and functional consequences of these nuclear inclusions remain elusive. To date the clinical observation of nuclear inclusions in viral and non-viral hepatitis has not been explored at depth in murine models of liver disease. Herein, we report that in a transgenic model of hepatitis B surface antigen mediated hepatitis, murine hepatocytes exhibit nuclear inclusions. Cells bearing nuclear inclusions were more likely to express markers of cell proliferation. We also established a correlation between these inclusions and oxidative stress. N-acetyl cysteine treatment effectively reduced oxidative stress levels, relieved endoplasmic reticulum (ER) stress, and the number of nuclear inclusions we observed in the transgenic mice. Our results suggest that the presence of nuclear inclusions in hepatocytes correlates with oxidative stress and cellular proliferation in a model of antigen mediated hepatitis.
Assuntos
Hepatite/patologia , Hepatite/virologia , Hepatócitos/patologia , Hepatócitos/virologia , Corpos de Inclusão Intranuclear/patologia , Aldeídos/metabolismo , Animais , Biomarcadores/metabolismo , Morte Celular , Núcleo Celular/metabolismo , Tamanho do Núcleo Celular , Senescência Celular , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Glicogênio/metabolismo , Hepatite/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Hepatócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Vacúolos/metabolismoRESUMO
The extracellular matrix undergoes extensive remodeling during hepatocellular carcinoma and functions as a critical component of the tumor microenvironment by providing a substratum for cell adhesion and serving as a reservoir for a variety of cytokines and growth factors. Despite the clinical correlation between ECM deposition and hepatocellular carcinoma progression, it remains unclear how global extracellular matrix gene expression is altered in hepatocellular carcinoma and the molecular pathways that govern this change. Herein, a comprehensive analysis of the extracellular matrix transcriptome using an RNA-sequencing dataset provided by The Cancer Genome Atlas consortium was conducted and indicates substantial differential gene expression of key extracellular matrix collagens, glycoproteins, and proteoglycans in hepatocellular carcinoma. This analysis also reveals alternative expression of extracellular matrix gene transcript variants that could impact biological activity and serves as a framework for exploring the dynamic nature of the extracellular matrix transcriptome in cancer and identifying candidate genes for future exploration.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Hepáticas/metabolismo , Transcriptoma/fisiologia , Sequência de Bases , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência de RNARESUMO
The regenerative response to drug- and toxin-induced liver injury induces changes to the hepatic stroma, including the extracellular matrix. Although the extracellular matrix is known to undergo changes during the injury response, its impact on maintaining hepatocyte function and viability in this process remains largely unknown. We demonstrate that recovery from toxin-mediated injury is impaired in mice deficient in a key liver extracellular matrix molecule, type XVIII collagen, and results in rapid death. The type-XVIII-collagen-dependent response to liver injury is mediated by survival signals induced by α1ß1 integrin, integrin linked kinase and the Akt pathway, and mice deficient in either α1ß1 integrin or hepatocyte integrin linked kinase also succumb to toxic liver injury. These findings demonstrate that type XVIII collagen is an important functional component of the liver matrix microenvironment and is crucial for hepatocyte survival during injury and stress.
Assuntos
Colágeno Tipo XVIII/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Fígado/metabolismo , Fígado/patologia , Animais , Tetracloreto de Carbono , Morte Celular , Colágeno Tipo XVIII/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Integrina alfa1beta1/metabolismo , Camundongos , Ligação Proteica , Transdução de Sinais , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismoRESUMO
The functional role of pericytes in cancer progression remains unknown. Clinical studies suggest that low numbers of vessel-associated pericytes correlated with a drop in overall survival of patients with invasive breast cancer. Using genetic mouse models or pharmacological inhibitors, pericyte depletion suppressed tumor growth but enhanced metastasis. Pericyte depletion was further associated with increased hypoxia, epithelial-to-mesenchymal transition (EMT), and Met receptor activation. Silencing of Twist or use of a Met inhibitor suppressed hypoxia and EMT/Met-driven metastasis. In addition, poor pericyte coverage coupled with high Met expression in cancer cells speculates the worst prognosis for patients with invasive breast cancer. Collectively, our study suggests that pericytes within the primary tumor microenvironment likely serve as important gatekeepers against cancer progression and metastasis.
Assuntos
Transição Epitelial-Mesenquimal , Metástase Neoplásica , Pericitos/fisiologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Animais , Antineoplásicos/farmacologia , Benzamidas , Benzenossulfonatos/farmacologia , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Crizotinibe , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Mesilato de Imatinib , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Niacinamida/análogos & derivados , Pericitos/patologia , Compostos de Fenilureia , Piperazinas/farmacologia , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirazóis , Piridinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Transdução de Sinais , Sorafenibe , Sunitinibe , Células Tumorais CultivadasRESUMO
Angiogenesis, the process of forming new blood vessels, is a well established and clinically relevant feature of a variety of disease states. Whether blood vessels sprout in a given tissue environment depends on the balance between factors that stimulate angiogenesis and those that impede it. Potent pro-angiogenic factors such as vascular endothelial growth factor (VEGF) have been identified, validated, and successfully used in the clinic. Likewise, anti-angiogenic factors are also emerging as biologically relevant and therapeutically useful entities. PAR1 is a G protein-coupled receptor (GPCR) that participates in hemostasis and vascular development and that mediates the angiogenic activity of thrombin. PAR1 is activated through proteolytic cleavage of its first forty-one extracellular residues by a variety of proteases, most notably thrombin. However, little effort has focused on the forty-one-residue peptide fragment liberated during PAR1 activation. Tsopanoglou and colleagues have now demonstrated that this peptide, parstatin, has intriguing antiangiogenic activity, and, in a follow-up study, they demonstrate its potential pharmacological utility using a rat model of ischemic heart disease.
Assuntos
Inibidores da Angiogênese/farmacologia , Tratamento Farmacológico , Fragmentos de Peptídeos/farmacologia , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Expressão Gênica , Humanos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptor PAR-1/genética , Receptor PAR-1/metabolismoRESUMO
Molecular preservation in non-avian dinosaurs is controversial. We present multiple lines of evidence that endogenous proteinaceous material is preserved in bone fragments and soft tissues from an 80-million-year-old Campanian hadrosaur, Brachylophosaurus canadensis [Museum of the Rockies (MOR) 2598]. Microstructural and immunological data are consistent with preservation of multiple bone matrix and vessel proteins, and phylogenetic analyses of Brachylophosaurus collagen sequenced by mass spectrometry robustly support the bird-dinosaur clade, consistent with an endogenous source for these collagen peptides. These data complement earlier results from Tyrannosaurus rex (MOR 1125) and confirm that molecular preservation in Cretaceous dinosaurs is not a unique event.
Assuntos
Colágeno/química , Dinossauros , Fêmur/química , Fósseis , Proteínas/química , Sequência de Aminoácidos , Animais , Aves/classificação , Técnica de Desmineralização Óssea , Matriz Óssea/química , Colágeno/análise , Colágeno/isolamento & purificação , Dinossauros/classificação , Elastina/análise , Fêmur/irrigação sanguínea , Fêmur/ultraestrutura , Hemoglobinas/análise , Técnicas Imunológicas , Laminina/análise , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Osteócitos/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Filogenia , Proteínas/análise , Proteínas/isolamento & purificação , Alinhamento de SequênciaRESUMO
Activated fibroblasts are key contributors to the fibrotic extracellular matrix accumulation during liver fibrosis. The origin of such fibroblasts is still debated, although several studies point to stellate cells as the principal source. The role of adult hepatocytes as contributors to the accumulation of fibroblasts in the fibrotic liver is yet undetermined. Here, we provide evidence that the pro-fibrotic growth factor, TGF-beta1, induces adult mouse hepatocytes to undergo phenotypic and functional changes typical of epithelial to mesenchymal transition (EMT). We perform lineage-tracing experiments using AlbCre. R26RstoplacZ double transgenic mice to demonstrate that hepatocytes which undergo EMT contribute substantially to the population of FSP1-positive fibroblasts in CCL(4)-induced liver fibrosis. Furthermore, we demonstrate that bone morphogenic protein-7 (BMP7), a member of the TGFbeta superfamily, which is known to antagonize TGFbeta signaling, significantly inhibits progression of liver fibrosis in these mice. BMP7 treatment abolishes EMT-derived fibroblasts, suggesting that the therapeutic effect of BMP7 was at least partially due to the inhibition of EMT. These results provide direct evidence for the functional involvement of adult hepatocytes in the accumulation of activated fibroblasts in the fibrotic liver. Furthermore, our findings suggest that EMT is a promising therapeutic target for the attenuation of liver fibrosis.
Assuntos
Epitélio/patologia , Fibroblastos/citologia , Fibrose/patologia , Hepatócitos/citologia , Fígado/patologia , Mesoderma/patologia , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Linhagem da Célula , Fibroblastos/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Heparin and heparan sulfate are linear sulfated polysaccharides that exert a multitude of biological functions. Heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase isoform 2 (NDST-2), a key enzyme in the biosynthesis of heparin, contains two distinct activities. This bifunctional enzyme removes the acetyl group from N-acetylated glucosamine (N-deacetylase activity) and transfers a sulfuryl group to the unsubstituted amino position (N-sulfotransferase activity). The N-sulfotransferase activity of NDST has been unambiguously localized to the C-terminal domain of NDST. Here, we report that the N-terminal domain of NDST-2 retains N-deacetylase activity. The N-terminal domain (A66-P604) of human NDST-2, designated as N-deacetylase (NDase), was cloned as a (His)(6)-fusion protein, and protein expression was carried out in Escherichia coli. Heparosan treated with NDase contains N-unsubstituted glucosamine and is highly susceptible to N-sulfation by N-sulfotransferase. Our results conclude that the N-terminal domain of NDST-2 contains functional N-deacetylase activity. This finding helps further elucidate the mechanism of action of heparan sulfate N-deacetylase/N-sulfotransferases and the biosynthesis of heparan sulfate in general.
Assuntos
Escherichia coli/enzimologia , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Sulfotransferases/química , Sulfotransferases/metabolismo , Sítios de Ligação , Ativação Enzimática , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Niemann-Pick C1 (NPC1) is a late endosomal/lysosomal membrane protein originally reported on for its role in cholesterol trafficking in mammalian cells. NPC1 has been shown recently to share significant structural homology with a family of prokaryotic permeases and was proposed to play a role in intracellular drug transport; however, the mechanism for this has not been fully understood. We provide evidence here that is consistent with NPC1's involvement in a vesicle-mediated clearance of the anticancer agent daunorubicin from cells. In experiments with human fibroblasts, we demonstrate that lysosomal efflux of daunorubicin, as well as dextran molecules, are significantly reduced in cells with mutated and dysfunctional NPC1 compared with wild-type fibroblasts. Furthermore, we show that NPC1 is implicated in a lysosomal drug sequestration phenotype exhibited by the multidrug-resistant (MDR) human leukemic HL-60 cancer cell line. Evaluations of cholesterol trafficking, NPC1 mRNA levels, and protein expression are all consistent with a loss of NPC1 activity that is associated with the emergence of the MDR phenotype in this cell line. Collectively, this work proposes a novel role for NPC1 in a vesicle-mediated pathway responsible for the clearance of drugs from cells and provides an explanation for a drug sequestration phenotype exhibited by the MDR HL-60 cell line.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Proteínas de Transporte/metabolismo , Daunorrubicina/metabolismo , Glicoproteínas de Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Colesterol/metabolismo , Dextranos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes MDR/genética , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Bicamadas Lipídicas , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microscopia de Fluorescência , Proteína C1 de Niemann-Pick , Nocodazol/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vesículas Transportadoras/efeitos dos fármacosRESUMO
Membrane fusion induced by herpes simplex virus (HSV) is required for both entry and cell-to-cell spread. It is mediated by the viral glycoprotein gB, gD, gH-gL and gD receptors. Although 3-O-sulfated heparan sulfate (3-OS HS) is a receptor for HSV-1 entry, the requirement for heparan sulfate in the fusion process has been ruled out. Here, it is demonstrated that cells expressing 3-OS HS, generated by D-glucosaminyl 3-O-sulfotransferase isoforms-3 and/or -5 (3-OST-3 and 3-OST-5), fused with cells expressing the four glycoproteins. The cell fusion observed exhibited similar requirements but was independent of protein receptors, HVEM or nectin-1. Additionally, removal of 3-OS HS from the cell surface by heparinase-I treatment and, in separate experiments, the presence of soluble 3-OST-3- and 3-OST-5-modified HS, significantly inhibited fusion. Taken together, these results indicate that 3-OS HS can play a crucial role in virus entry and cell fusion.
Assuntos
Heparitina Sulfato/fisiologia , Herpesvirus Humano 1/patogenicidade , Fusão de Membrana/fisiologia , Animais , Células CHO , Cricetinae , Heparitina Sulfato/química , Isoenzimas/genética , Isoenzimas/metabolismo , Receptores Virais/química , Receptores Virais/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , TransfecçãoRESUMO
Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.
Assuntos
Heparitina Sulfato/biossíntese , Sulfotransferases/metabolismo , Animais , Antitrombinas/metabolismo , Linhagem Celular , Heparitina Sulfato/química , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sulfotransferases/genéticaRESUMO
Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate d-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gD-binding site is an octasaccharide, and has a binding affinity to gD around 18 microm, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is DeltaUA-GlcNS-IdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH(2)3S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry.