RESUMO
Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Glipicanas , Heparitina Sulfato , Animais , Proteínas de Drosophila/metabolismo , Glipicanas/genética , Glipicanas/química , Glipicanas/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
Mast cells are innate immune cells strategically positioned around blood vessels near body surfaces. Their primary weapons are bioactive amines, mast cell-specific proteases, and cytokines stored in preformed granules. Mast cells granules constituents are packaged efficiently with the help of the highly negatively charged Heparan sulfate-derivative, Heparin. Heparin is one of the most widely used drugs to treat coagulation disorders, yet, it is not found in the circulation at a steady state, casting doubt that the prevention of blood clotting is its physiological function. Early studies using Ndst2 -/- mice have shown that Heparin is essential for mast cells granules formation. However, these mice could still produce less sulfated Heparan sulfate that could potentially replace Heparin. Here, we have created and validated a novel genetic model for Heparin deficiency, specifically in connective tissue mast cells, to address the physiological role of this molecule. Using this model, we have demonstrated that Heparin is required for mast cell granules formation; without it, mast cells are reduced in the peritoneal cavity and the skin. The absence of Heparin impaired the response to passive cutaneous anaphylaxis but, surprisingly, enhanced ear swelling in an irritant dermatitis model and reduced the lesion size and bacterial burden in a Staphylococcus aureus necrotizing dermatitis model. The altered function of Heparin-deficient mast cells in the latter two models was not mediated through enhanced Histamine or TNFα release. However, the Mrgprb2 receptor was up-regulated in knock-out mast cells, potentially explaining the enhanced response of mutant mice to irritant and necrotizing dermatitis. Altogether our results expand our current understanding of the physiological role of Heparin and provide unique tools to further dissect its importance.
Assuntos
Dermatite , Heparina , Camundongos , Animais , Heparina/farmacologia , Mastócitos , Heparitina Sulfato/genética , Tecido ConjuntivoRESUMO
Hereditary multiple exostoses (HME) is a rare, pediatric disorder characterized by osteochondromas that form along growth plates and provoke significant musculoskeletal problems. HME is caused by mutations in heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. Seemingly paradoxically, osteochondromas were found to contain excessive extracellular heparanase (Hpse) that could further reduce HS levels and exacerbate pathogenesis. To test Hpse roles, we asked whether its ablation would protect against osteochondroma formation in a conditional HME model consisting of mice bearing floxed Ext1 alleles in Agr-CreER background (Ext1f/f ;Agr-CreER mice). Mice were crossed with a new global Hpse-null (Hpse-/- ) mice to produce compound Hpse-/- ;Ext1f/f ;Agr-CreER mice. Tamoxifen injection of standard juvenile Ext1f/f ;Agr-CreER mice elicited stochastic Ext1 ablation in growth plate and perichondrium, followed by osteochondroma formation, as revealed by microcomputed tomography and histochemistry. When we examined companion conditional Ext1-deficient mice lacking Hpse also, we detected no major decreases in osteochondroma number, skeletal distribution, and overall structure by the analytical criteria above. The Ext1 mutants used here closely mimic human HME pathogenesis, but have not been previously tested for responsiveness to treatments. To exclude some innate therapeutic resistance in this stochastic model, tamoxifen-injected Ext1f/f ;Agr-CreER mice were administered daily doses of the retinoid Palovarotene, previously shown to prevent ectopic cartilage and bone formation in other mouse disease models. This treatment did inhibit osteochondroma formation compared with vehicle-treated mice. Our data indicate that heparanase is not a major factor in osteochondroma initiation and accumulation in mice. Possible roles of heparanase upregulation in disease severity in patients are discussed.
Assuntos
Neoplasias Ósseas , Exostose Múltipla Hereditária , Glucuronidase , N-Acetilglucosaminiltransferases , Osteocondroma , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Criança , Modelos Animais de Doenças , Exostose Múltipla Hereditária/genética , Exostose Múltipla Hereditária/metabolismo , Exostose Múltipla Hereditária/patologia , Glucuronidase/genética , Glucuronidase/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Osteocondroma/genética , Osteocondroma/metabolismo , Osteocondroma/patologia , Retinoides , Tamoxifeno , Microtomografia por Raio-XRESUMO
Hepatic failure is an important risk factor for poor outcome in septic patients. Using a chemical tagging workflow and high-resolution mass spectrometry, we demonstrate that rapid proteome remodeling of the vascular surfaces precedes hepatic damage in a murine model of Staphylococcus aureus sepsis. These early changes include vascular deposition of neutrophil-derived proteins, shedding of vascular receptors, and altered levels of heparin/heparan sulfate-binding factors. Modification of endothelial heparan sulfate, a major component of the vascular glycocalyx, diminishes neutrophil trafficking to the liver and reduces hepatic coagulopathy and organ damage during the systemic inflammatory response to infection. Modifying endothelial heparan sulfate likewise reduces neutrophil trafficking in sterile hepatic injury, reflecting a more general role of heparan sulfate contribution to the modulation of leukocyte behavior during inflammation. IMPORTANCE Vascular glycocalyx remodeling is critical to sepsis pathology, but the glycocalyx components that contribute to this process remain poorly characterized. This article shows that during Staphylococcus aureus sepsis, the liver vascular glycocalyx undergoes dramatic changes in protein composition associated with neutrophilic activity and heparin/heparan sulfate binding, all before organ damage is detectable by standard circulating liver damage markers or histology. Targeted manipulation of endothelial heparan sulfate modulates S. aureus sepsis-induced hepatotoxicity by controlling the magnitude of neutrophilic infiltration into the liver in both nonsterile and sterile injury. These data identify an important vascular glycocalyx component that impacts hepatic failure during nonsterile and sterile injury.
Assuntos
Células Endoteliais/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Ativação de Neutrófilo , Neutrófilos/patologia , Sepse/microbiologia , Staphylococcus aureus/imunologia , Animais , Modelos Animais de Doenças , Células Endoteliais/imunologia , Feminino , Glicocálix/metabolismo , Glicocálix/patologia , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/patogenicidadeRESUMO
A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Hipóxia Celular/fisiologia , Heparitina Sulfato/metabolismo , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/fisiologia , Sindecana-1/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica/efeitos dos fármacos , Heparitina Sulfato/genética , Humanos , Neuropilina-1/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sindecana-1/genética , Células Vero , Ligação Viral/efeitos dos fármacosRESUMO
The biosynthesis of heparan sulfate (HS) proteoglycans occurs in the Golgi compartment of cells and will determine the sulfation pattern of HS chains, which in turn will have a large impact on the biological activity of the proteoglycans. Earlier studies in mice have demonstrated the importance of HS for embryonic development. In this review, the enzymes participating in zebrafish HS biosynthesis, along with a description of enzyme mutants available for functional studies, are presented. The consequences of the zebrafish genome duplication and maternal transcript contribution are briefly discussed as are the possibilities of CRISPR/Cas9 methodologies to use the zebrafish model system for studies of biosynthesis as well as proteoglycan biology.
Assuntos
Vias Biossintéticas , Heparitina Sulfato/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sistemas CRISPR-Cas , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Heparitina Sulfato/genética , Mutação , Sulfotransferases/genética , Sulfotransferases/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.
Assuntos
Arilsulfatases/metabolismo , Sulfatos de Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Mucopolissacaridoses/metabolismo , Animais , Arilsulfatases/genética , Sulfatos de Condroitina/genética , Ativação Enzimática , Heparitina Sulfato/genética , Camundongos , Camundongos Knockout , Mucopolissacaridoses/genéticaRESUMO
Exostosin-like 3 (EXTL3) encodes the glycosyltransferases responsible for the biosynthesis of the backbone structure of heparan sulfate (HS), a sulfated polysaccharide that is ubiquitously distributed on the animal cell surface and in the extracellular matrix. A lack of EXTL3 reduces HS levels and causes embryonic lethality, indicating its indispensable role in the biosynthesis of HS. EXTL3 has also been identified as a receptor molecule for regenerating islet-derived (REG) protein ligands, which have been shown to stimulate islet ß-cell growth. REG proteins also play roles in keratinocyte proliferation and/or differentiation, tissue regeneration and immune defenses in the gut as well as neurite outgrowth in the central nervous system. Compared with the established function of EXTL3 as a glycosyltransferase in HS biosynthesis, the REG-receptor function of EXTL3 is not conclusive. Genetic diseases caused by biallelic mutations in the EXTL3 gene were recently reported to result in a neuro-immuno-skeletal dysplasia syndrome. EXTL3 is a key molecule for the biosynthesis of HS and may be involved in the signal transduction of REG proteins.
Assuntos
N-Acetilglucosaminiltransferases/genética , Animais , Matriz Extracelular/genética , Heparitina Sulfato/genética , Humanos , Células Secretoras de Insulina/metabolismo , Crescimento Neuronal/genética , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear. METHODS: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system. RESULTS: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells. SIGNIFICANCE: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.
Assuntos
Heparitina Sulfato/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/genética , Camundongos , Atividade Motora , Músculo Esquelético/citologia , Mioblastos/metabolismoRESUMO
Type V collagen (ColV) is a component of the endothelial basement membrane zone. During angiogenesis, extracellular matrix remodelling results in the release of active protein fragments that display pro- or anti-angiogenic properties. The latter often exert their activity through their heparin-binding site. We previously characterized a ColVα1-derived fragment called HEPV that contains a high affinity-binding site for heparin and heparan sulphate chains. Here we show that HEPV binds to FGF2 through its heparin-binding site. Using in vitro and in vivo angiogenesis assays, we show that HEPV but not the HEPV mutant at the heparin-binding site, inhibits FGF2-dependant angiogenesis. On the opposite, HEPV does not bind to VEGFA and has no effect on VEGFA-mediated angiogenesis. In 3D collagen gels, the addition of HEPV abrogates endothelial cell invasion and sprouting induced by FGF2. Interestingly, in vivo experiments reveal that HEPV anti-angiogenic activity is associated with the appearance of endothelial to mesenchymal transition (EndMT) markers. Together, these findings indicate that the ColVα1-derived fragment HEPV functions as an anti-angiogenic factor that represses FGF2-mediated angiogenesis through the regulation of endothelial cell plasticity. Previous observations showing that ColV overexpression negatively regulates pathological angiogenesis were left unexplained. Our data provide insights into the possible molecular mechanisms.
Assuntos
Colágeno Tipo V/genética , Fator 2 de Crescimento de Fibroblastos/genética , Morfogênese/genética , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Sequência de Aminoácidos/genética , Inibidores da Angiogênese/farmacologia , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Plasticidade Celular/genética , Células Endoteliais/efeitos dos fármacos , Heparina/genética , Heparitina Sulfato/genética , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Morfogênese/efeitos dos fármacos , Neovascularização Patológica/patologia , Ligação Proteica/genéticaRESUMO
Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Heparina/metabolismo , Heparitina Sulfato/biossíntese , Animais , Anticoagulantes , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica/genética , Células HeLa , Heparina/biossíntese , Heparina/genética , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mastócitos/metabolismo , Sulfotransferases/metabolismo , Suínos , Fatores de TranscriçãoRESUMO
Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson's Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors.
Assuntos
Autofagia , Proteínas de Drosophila/genética , Heparitina Sulfato/biossíntese , Longevidade , Estresse Oxidativo , Ubiquitina-Proteína Ligases/genética , Animais , Encéfalo/metabolismo , Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Heparitina Sulfato/genética , Músculos/metabolismo , Mutação , Presenilinas/genética , Presenilinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Increasing knowledge of how peptides bind saccharides, and of how saccharides bind peptides, is starting to revolutionize understanding of cell-extracellular matrix relationships. Here, a historical perspective is taken of the relationship between heparan sulfate glycosaminoglycans and how they interact with peptide growth factors in order to both drive and modulate signaling through the appropriate cognate receptors. Such knowledge is guiding the preparation of targeted sugar mimetics that will impact the treatment of many different kinds of diseases, including cancer.
Assuntos
Glicômica , Heparitina Sulfato/genética , Peptídeos/genética , Proteômica , Matriz Extracelular/genética , Glicosaminoglicanos/genética , Humanos , Neoplasias/genética , Ligação Proteica/genética , Transdução de Sinais/genéticaRESUMO
The Mucopolysaccharidoses (MPS) are group of inherited metabolic diseases caused by the deficiency of enzymes required to degrade glycosaminoglycans (GAGs) in the lysosomes. GAGs are sulfated polysaccharides involving repeating disaccharides, uronic acid and hexosamines including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS). Hyaluronan is excluded in terms of being non-sulfated in the GAG family. Different types of mutations have been identified as the causative agent in all types of MPS. Herein, we planned to investigate the pathogenic mutations in different types of MPS including type I (IDUA gene), IIIA (SGSH) and IIIB (NAGLU) in the eight Iranian patients. Autozygosity mapping was performed to identify the potential pathogenic variants in these 8 patients indirectly with the clinical diagnosis of MPSs. so three panels of STR (Short Tandem Repeat) markres flanking IDUA, SGSH and NAGLU genes were selected for multiplex PCR amplification. Then in each family candidate gene was sequenced to identify the pathogenic mutation. Our study showed two novel mutations c.469 T > C and c.903C > G in the IDUA gene, four recurrent mutations: c.1A > C in IDUA, c.220C > T, c.1298G > A in SGSH gene and c.457G > A in the NAGLU gene. The c.1A > C in IDUA was the most common mutation in our study. In silico analysis were performed as well to predict the pathogenicity of the novel variants.
Assuntos
Análise Mutacional de DNA/métodos , Testes Genéticos/métodos , Mucopolissacaridoses/genética , Mutação , Adolescente , Criança , Pré-Escolar , Sulfatos de Condroitina/genética , Dermatan Sulfato/genética , Feminino , Heparitina Sulfato/genética , Humanos , Lactente , Sulfato de Queratano/genética , Masculino , Reação em Cadeia da Polimerase MultiplexRESUMO
Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.
Assuntos
Haploidia , Heparitina Sulfato/genética , Heparitina Sulfato/isolamento & purificação , Pinocitose/fisiologia , Vaccinia virus/genética , Vaccinia virus/metabolismo , Vacínia/virologia , Proteínas de Transporte Vesicular/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Vírus da Varíola Bovina/genética , Vírus de DNA , Técnicas de Inativação de Genes , Testes Genéticos , Complexo de Golgi , Células HEK293 , Células HeLa , Heparitina Sulfato/metabolismo , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana , Monkeypox virus/genética , N-Acetilglucosaminiltransferases , Fosfatidilserinas/metabolismo , Poxviridae/genética , Ligação ViralRESUMO
PROBLEM: Mast cells (MC) have been known to play an important role in inflammation and angiogenesis by secreting numerous mediators, such as proteases, gelatinases, and proteoglycans. Three different MC subtypes were found in the endometrial layers of the uterus. In this study, we aim to investigate the role of endometrial MCs in recurrent pregnancy losses (RPL). METHOD OF STUDY: Endometrial biopsy was performed 5-7 days post-ovulation (implantation window) in women with a history of two or more RPL (n = 46) and normal fertile women (n = 10). Quantitative RT-PCR was performed to detect the expression of various mast cell mediators. Endometrial samples were evaluated using immunohistochemistry for c-kit receptor (CD117) and tryptase (MC activation marker). RESULTS: Mast cells were present throughout the entire layers of the endometrium; their count was elevated in RPL patients as compared to controls. The gene expression of c-Kit receptor was not different between the study groups. There are significant increases in the mRNA expression of various mediators, that is, stem cell factor (P = 0.029), tryptase (P = 0.024), heparan sulfate (P = 0.0005), and MMP-2 (P < 0.0001) in women with RPL as compared to normal controls. Chymase gene expression was not detected in most of the endometrial samples. CONCLUSION: This study has shown that MCs are overactive in RPL patients by creating a pro-inflammatory milieu, suggesting a novel role in the immunopathology of RPL. Future studies are needed to better understand the role of MC in implantation and placental angiogenesis.
Assuntos
Aborto Habitual/imunologia , Endométrio/citologia , Mastócitos/imunologia , Aborto Habitual/genética , Adulto , Endométrio/imunologia , Feminino , Expressão Gênica , Heparitina Sulfato/genética , Humanos , Metaloproteinase 2 da Matriz/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Fator de Células-Tronco/genética , Triptases/genéticaRESUMO
The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.
Assuntos
Substituição de Aminoácidos/genética , Proteínas do Capsídeo/genética , Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Animais , Células CHO , Cricetulus , Heparitina Sulfato/genética , Camundongos , Fases de Leitura Aberta/genética , Sorogrupo , Vírion/genéticaRESUMO
Vaccinia virus (VACV), like many other viruses, binds to cell surface heparan sulfate (HS) prior to infecting cells. Since HS is ubiquitously expressed extracellularly, it seemed likely that VACV-HS interaction may impede virus spread, with host heparanase, the only known mammalian endoglycosidase that can degrade HS, potentially overcoming this problem. In support of this hypothesis, we found that, compared to wild type, mice deficient in heparanase showed a 1-3 days delay in the spread of VACV to distant organs, such as ovaries, following intranasal inoculation, or to ovaries and spleen following intramuscular inoculation. These delays in spread occurred despite heparanase deficiency having no effect on VACV replication at inoculation sites. Subsequent in vitro studies revealed that heparanase treatment released VACV from HS expressing, but not HS deficient, infected cell monolayers. Collectively these data suggest that VACV relies on host heparanase to degrade HS in order to spread to distant sites.
Assuntos
Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Vaccinia virus/metabolismo , Animais , Glucuronidase/genética , Heparitina Sulfato/genética , Humanos , Camundongos , Camundongos Knockout , Replicação ViralRESUMO
The attachment of a variety of Lactobacilli to the mucosal surfaces is accomplished through the interaction of OppA, a superficial bacterial protein also involved in oligopeptide internalization, and the glycosaminoglycan moiety of the proteoglycans that form the epithelial cell glycocalyx. Upon the interaction of the vaginal isolate Lactobacillus salivarius Lv72 and HeLa cell cultures, the expression of oppA increased more than 50-fold over the following 30 min, with the overexpression enduring, albeit at a lower rate, for up to 24 h. Conversely, transcriptional analysis of 62 genes involved in proteoglycan biosynthesis revealed generalized repression of genes whose products catalyze different steps of the whole pathway. This led to decreases in the superficial concentration of heparan (60%) and chondroitin sulfate (40%), although the molecular masses of these glycosaminoglycans were higher than those of the control cultures. Despite this lowering in the concentration of the receptor, attachment of the Lactobacilli proceeded, and completely overlaid the underlying HeLa cell culture.
Assuntos
Infecções Bacterianas/genética , Proteínas de Bactérias/genética , Ligilactobacillus salivarius/genética , Biossíntese de Proteínas/genética , Linhagem Celular Tumoral , Sulfatos de Condroitina/genética , Glicosaminoglicanos/genética , Células HeLa , Heparitina Sulfato/genética , Humanos , Ligantes , Proteoglicanas/genética , Transcrição Gênica/genéticaRESUMO
Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2-FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.