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1.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674659

RESUMO

Heparan sulfate is a ubiquitous, variably sulfated interactive glycosaminoglycan that consists of repeating disaccharides of glucuronic acid and glucosamine that are subject to a number of modifications (acetylation, de-acetylation, epimerization, sulfation). Variable heparan sulfate chain lengths and sequences within the heparan sulfate chains provide structural diversity generating interactive oligosaccharide binding motifs with a diverse range of extracellular ligands and cellular receptors providing instructional cues over cellular behaviour and tissue homeostasis through the regulation of essential physiological processes in development, health, and disease. heparan sulfate and heparan sulfate-PGs are integral components of the specialized glycocalyx surrounding cells. Heparan sulfate is the most heterogeneous glycosaminoglycan, in terms of its sequence and biosynthetic modifications making it a difficult molecule to fully characterize, multiple ligands also make an elucidation of heparan sulfate functional properties complicated. Spatio-temporal presentation of heparan sulfate sulfate groups is an important functional determinant in tissue development and in cellular control of wound healing and extracellular remodelling in pathological tissues. The regulatory properties of heparan sulfate are mediated via interactions with chemokines, chemokine receptors, growth factors and morphogens in cell proliferation, differentiation, development, tissue remodelling, wound healing, immune regulation, inflammation, and tumour development. A greater understanding of these HS interactive processes will improve therapeutic procedures and prognoses. Advances in glycosaminoglycan synthesis and sequencing, computational analytical carbohydrate algorithms and advanced software for the evaluation of molecular docking of heparan sulfate with its molecular partners are now available. These advanced analytic techniques and artificial intelligence offer predictive capability in the elucidation of heparan sulfate conformational effects on heparan sulfate-ligand interactions significantly aiding heparan sulfate therapeutics development.


Assuntos
Glicosaminoglicanos , Proteoglicanas , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Simulação de Acoplamento Molecular , Inteligência Artificial , Ligantes , Heparitina Sulfato/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(4): e2209528120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36649428

RESUMO

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.


Assuntos
Endotoxemia , Proteína HMGB1 , Sepse , Camundongos , Animais , Proteína HMGB1/metabolismo , Apolipoproteína A-I , Sepse/tratamento farmacológico , Sepse/metabolismo , Lipopolissacarídeos , Heparitina Sulfato , Modelos Animais de Doenças
3.
Viruses ; 15(1)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36680276

RESUMO

Heparan sulfate proteoglycans (HSPGs) are a major constituent of the extracellular matrix (ECM) and are found to be implicated in viral infections, where they play a role in both cell entry and release for many viruses. The enzyme heparanase-1 is the only known endo-beta-D-glucuronidase capable of degrading heparan sulphate (HS) chains of HSPGs and is thus important for regulating ECM homeostasis. Heparanase-1 expression is tightly regulated as the uncontrolled cleavage of HS may result in abnormal cell activation and significant tissue damage. The overexpression of heparanase-1 correlates with pathological scenarios and is observed in different human malignancies, such as lymphoma, breast, colon, lung, and hepatocellular carcinomas. Interestingly, heparanase-1 has also been documented to be involved in numerous viral infections, e.g., HSV-1, HPV, DENV. Moreover, very recent reports have demonstrated a role of heparanase-1 in HCV and SARS-CoV-2 infections. Due to the undenied pro-carcinogenic role of heparanase-1, multiple inhibitors have been developed, some reaching phase II and III in clinical studies. However, the use of heparanase inhibitors as antivirals has not yet been proposed. If it can be assumed that heparanase-1 is implicated in numerous viral life cycles, its inhibition by specific heparanase-acting compounds should result in a blockage of viral infection. This review addresses the perspectives of using heparanase inhibitors, not only for cancer treatment, but also as antivirals. Eventually, the development of a novel class antivirals targeting a cellular protein could help to alleviate the resistance problems seen with some current antiretroviral therapies.


Assuntos
COVID-19 , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , SARS-CoV-2/metabolismo , Glucuronidase/genética , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/metabolismo , Biologia
4.
Methods Mol Biol ; 2619: 71-90, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662463

RESUMO

Glycosaminoglycans (GAGs) are built up of repeating disaccharide units resulting in long, linear polysaccharide chains. In most classes of GAGs, sulfation and epimerization complicate the structure of the chain and influence biochemical functions. The most widespread way of their investigation by instrumental analytical techniques is to degrade them into the constituent disaccharide building blocks, followed by capillary electrophoresis or high-performance liquid chromatography (HPLC) separation. The analysis of GAG disaccharides with varying sulfation degrees poses a real challenge both from chromatographic and mass spectrometric (MS) points of view. This necessitates the constant improvement of their analytical methodology. In this chapter, an optimized workflow will be discussed for the sample preparation and subsequent HPLC-MS characterization of tissue-derived chondroitin sulfate and heparan sulfate.


Assuntos
Sulfatos de Condroitina , Heparitina Sulfato , Sulfatos de Condroitina/química , Cromatografia Líquida de Alta Pressão/métodos , Heparitina Sulfato/química , Glicosaminoglicanos/química , Dissacarídeos/química
5.
Methods Mol Biol ; 2619: 249-256, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662475

RESUMO

Heparin/heparan sulfate (HP/HS) is a class of acidic polysaccharides with many potential medical applications, especially HP, and its derivatives, low molecular weight heparins (LMWHs), have been widely used as anticoagulants to treat thrombosis for decades. However, the complex structure endows HP/HS a variety of biological functions and hinders the structural and functional studies of HP/HS. Heparinases derived from bacteria are useful tools for the structural studies of HP/HS as well as the preparation of LMWHs. The enzymatic method for the structural analysis of HP/HS chains is easy to operate, requires less samples, and is low cost. Here, we describe an enzymatic approach to investigate the primary sequences of the HP/HS oligosaccharides using a recently discovered exotype heparinase.


Assuntos
Heparina , Heparitina Sulfato , Heparina/química , Heparitina Sulfato/química , Heparina Liase , Anticoagulantes , Oligossacarídeos/química
6.
Methods Mol Biol ; 2619: 227-238, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662473

RESUMO

The enzyme heparanase cleaves heparan sulfate and is involved in a range of human diseases including cancer, inflammation, diabetes, and viral infection. There is a need for a simple and reliable enzymatic assay to allow for the screening of compounds to find inhibitors of heparanase. We have developed an assay that uses the heparinoid fondaparinux as enzyme substrate and detects one of the products of catalysis, which contains a newly formed reducing terminus, with the tetrazolium salt WST-1. Due to the homogenous substrate and single point of cleavage therein, this assay allows for more systematic kinetic analysis of heparanase inhibitors. Here, we provide a detailed method for conducting this assay and also provide information to assist researchers in evaluating whether the assay is performing properly in their laboratories.


Assuntos
Glucuronidase , Heparitina Sulfato , Humanos , Cinética , Glucuronidase/metabolismo , Heparitina Sulfato/química , Ensaios Enzimáticos/métodos
7.
FASEB J ; 37(1): e22717, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36563024

RESUMO

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.


Assuntos
Proteína Morfogenética Óssea 7 , Glicosaminoglicanos , Proteína Morfogenética Óssea 7/metabolismo , Heparina/metabolismo , Fibrilina-1/metabolismo , Simulação de Acoplamento Molecular , Proteínas Morfogenéticas Ósseas/metabolismo , Heparitina Sulfato/metabolismo , Ligação Proteica , Proteína Morfogenética Óssea 2/metabolismo
8.
Biochem J ; 480(1): 41-56, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36511224

RESUMO

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.


Assuntos
Sulfatos de Condroitina , Glicosaminoglicanos , Humanos , Camundongos , Animais , Triptases/metabolismo , Imuno-Histoquímica , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Heparina , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Ligação ao Cálcio , Biomarcadores Tumorais
9.
J Chromatogr A ; 1689: 463748, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36586283

RESUMO

Glycosaminoglycans (GAGs), which are one of the major components of proteoglycans, play a pivotal role in physiological processes such as signal transduction, cell adhesion, growth, and differentiation. Characterization of GAGs is challenging due to the tremendous structural diversity of heteropolysaccharides with numerous sulfate or carboxyl groups. In this present study, we examined the analysis of 2-aminobenzamide (2-AB) labeled GAG disaccharides by high-performance liquid chromatography (HPLC) using a reverse-phase (RP)-column with adamantyl groups. Under the analytical conditions, 17 types of 2-AB labeled GAG disaccharides derived from heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan were sequentially separated in a single analysis. The analysis time was fast with high retention time reproducibility. Moreover, the RP-HPLC column with adamantyl groups allowed the quantification of GAGs in various biological samples, such as serum, cultured cells, and culture medium.


Assuntos
Sulfatos de Condroitina , Glicosaminoglicanos , Glicosaminoglicanos/química , Sulfatos de Condroitina/química , Ácido Hialurônico/análise , Ácido Hialurônico/química , Dermatan Sulfato/análise , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Dissacarídeos/química , Reprodutibilidade dos Testes , Heparitina Sulfato/análise
10.
Eur J Med Chem ; 247: 115005, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36563498

RESUMO

Synthesis of a series of l-iduronic acid (IdoA)- and imino-IdoA-typed C-glycosides for modulating α-l-iduronidase (IDUA) activity is described. In an enzyme inhibition study, IdoA-typed C-glycosides were more potent than imino-IdoA analogs, with the most potent IdoA-typed C-glycoside 27c showing an IC50 value of 1 µM. On the other hand, co-treatment of 12 with rh-α-IDUA in mucopolysaccharidosis type I (MPS I) fibroblasts exhibited a nearly 3-fold increase of the IDUA activity, resulting in a clear reduction of the accumulated heparan sulfate (HS) compared to the exogenous enzyme treatment alone. This is the first report of small molecules facilitating IDUA stabilization, enhancing enzyme activity, and reducing accumulated HS in MPS I cell-based assays, which reveals that small molecules as rh-α-IDUA stabilizers to improve enzyme replacement therapy (ERT) efficacy toward MPS I is feasible and promising.


Assuntos
Mucopolissacaridose I , Humanos , Mucopolissacaridose I/tratamento farmacológico , Mucopolissacaridose I/metabolismo , Iduronidase/farmacologia , Iduronidase/metabolismo , Heparitina Sulfato/farmacologia , Fibroblastos/metabolismo , Glicosídeos
11.
Viruses ; 14(12)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36560700

RESUMO

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Filogenia , SARS-CoV-2/genética , Heparina , Heparitina Sulfato , Comunicação Celular , Ligação Proteica
12.
BMC Mol Cell Biol ; 23(1): 61, 2022 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-36564747

RESUMO

BACKGROUND: Considering the high correlation between the functional decline in Alzheimer's disease (AD) and the propagation of aggregated tau protein, many research efforts are focused on determining the underlying molecular mechanisms of tau spreading. Heparan sulfate proteoglycans (HSPGs) were reported to mediate cellular uptake of tau aggregates. Specifically, the heparan sulfates (HS) sulfation plays a critical role in the interaction of HSPGs with aggregated tau. HS can be N-/2-O/6-O- or 3-O-sulfated, some of which have been reported to take part in the interaction with tau aggregates. However, the role of the 3-O sulfation remains enigmatic. RESULTS: Here, we studied the contribution of HS 3-O sulfation in the binding and cellular uptake of tau aggregates. We observed reduced tau aggregates uptake in absence of 3-O sulfation or when outcompeting available cellular 3-O sulfated HS (3S-HS) with antithrombin III. The lack of HS3ST1-generated HS products in the HS3ST1-/- cells was further corroborated with an LC-MS/MS using 13C-labeled HS calibrants. Here, we showed that these functional changes can be explained by a higher affinity of aggregated tau to 3S-HS. When targeting tau aggregates with 3-O sulfation-containing HS, we observed an increase in inhibition of tau aggregates uptake. CONCLUSIONS: These data indicate that HS 3-O sulfation plays a role in the binding of tau aggregates and, thus, contributes to their cellular uptake, highlighting a potential target value to modulate tau pathogenesis.


Assuntos
Proteoglicanas de Heparan Sulfato , Proteínas tau , Proteoglicanas de Heparan Sulfato/metabolismo , Proteínas tau/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Heparitina Sulfato/metabolismo , Heparitina Sulfato/farmacologia
13.
Sci Rep ; 12(1): 21479, 2022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36509864

RESUMO

The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.


Assuntos
Barreira Hematoencefálica , Encéfalo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Anticorpos/metabolismo , Heparitina Sulfato/metabolismo , Sítios de Ligação
14.
Nat Commun ; 13(1): 7110, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402845

RESUMO

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.


Assuntos
Heparitina Sulfato , N-Acetilglucosaminiltransferases , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Microscopia Crioeletrônica , Heparitina Sulfato/metabolismo , Proteínas/metabolismo , Nucleotidiltransferases , Glicosiltransferases/metabolismo
15.
Biomolecules ; 12(11)2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36358923

RESUMO

(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer's disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau-heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*-Arixtra interaction had a much stronger binding affinity (0.37-0.67 mM) than that of tau R2*-Arixtra (1.90-5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau-heparin and tau-HS interaction.


Assuntos
Heparina , Heparitina Sulfato , Ligação Proteica , Fondaparinux , Sítios de Ligação , Heparitina Sulfato/química , Heparina/química , Prolina/metabolismo , Proteínas tau/metabolismo
16.
Cell Rep ; 41(9): 111721, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36450248

RESUMO

Influenza infection is substantially worsened by the onset of secondary pneumonia caused by bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). The bidirectional interaction between the influenza-injured lung microenvironment and MRSA is poorly understood. By conditioning MRSA ex vivo in bronchoalveolar lavage fluid collected from mice at various time points of influenza infection, we found that the influenza-injured lung microenvironment dynamically induces MRSA to increase cytotoxin expression while decreasing metabolic pathways. LukAB, a SaeRS two-component system-dependent cytotoxin, is particularly important to the severity of post-influenza MRSA pneumonia. LukAB's activity is likely shaped by the post-influenza lung microenvironment, as LukAB binds to (and is activated by) heparan sulfate (HS) oligosaccharide sequences shed from the epithelial glycocalyx after influenza. Our findings indicate that post-influenza MRSA pneumonia is shaped by bidirectional host-pathogen interactions: host injury triggers changes in bacterial expression of toxins, the activity of which may be shaped by host-derived HS fragments.


Assuntos
Coinfecção , Influenza Humana , Staphylococcus aureus Resistente à Meticilina , Pneumonia Bacteriana , Animais , Camundongos , Humanos , Influenza Humana/complicações , Virulência , Pneumonia Bacteriana/complicações , Citotoxinas , Heparitina Sulfato , Pulmão
17.
Front Immunol ; 13: 1000405, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36439118

RESUMO

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 Conjuntivo
18.
Cells ; 11(22)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36428962

RESUMO

In this study, we examined the roles of heparanase and IGFBP-3 in regulating A549 and H1299 non-small-cell lung cancer (NSCLC) survival. We found that H1299 cells, known to be p53-null with no expression of IGFBP-3, had higher heparanase levels and activity and higher levels of heparan sulfate (HS) in the media compared to the media of A549 cells. Inhibiting heparanase activity or its expression using siRNA had no effect on the levels of IGFBP-3 in the media of A549 cells, reduced the levels of soluble HS fragments, and led to decreased interactions between IGFBP-3 and HS in the media. HS competed with HA for binding to IGFBP-3 or IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant peptide (K228AR230A). HS abolished the cytotoxic effects of IGFBP-3 but not upon blocking HA-CD44 signaling with the anti-CD44 antibody (5F12). Blocking HA-CD44 signaling decreased the levels of heparanase in the media of both A549 and H1299 cell lines and increased p53 activity and the levels of IGFBP-3 in A549 cell media. Knockdown of p53 led to increased heparanase levels and reduced IGFBP-3 levels in A549 cell media while knockdown of IGFBP-3 in A549 cells blocked p53 activity and increased heparanase levels in the media.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Células A549 , Neoplasias Pulmonares/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteína Supressora de Tumor p53 , Sobrevivência Celular , Heparitina Sulfato/metabolismo , Peptídeos/metabolismo
19.
Int J Mol Sci ; 23(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36361973

RESUMO

Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar 23Na nucleus, we herein develop a 23Na NMR-based competition assay and monitor the binding of divalent Ca2+ and Mg2+ and trivalent Al3+ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The 23Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al3+ ion, the complementary approach of 27Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca2+ > Mg2+ > Al3+ is obtained, in which even the weakly binding Al3+ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding.


Assuntos
Heparina , Heparitina Sulfato , Heparina/química , Espectroscopia de Ressonância Magnética/métodos , Heparitina Sulfato/metabolismo , Metais/metabolismo , Íons , Sódio/metabolismo , Sítios de Ligação
20.
J Biol Chem ; 298(11): 102546, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36181793

RESUMO

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4 cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.


Assuntos
Heparitina Sulfato , Neoplasias Gástricas , Humanos , Heparitina Sulfato/metabolismo , Neoplasias Gástricas/genética , Glicosiltransferases/genética , Proteoglicanas de Heparan Sulfato , Movimento Celular , Microambiente Tumoral , N-Acetilglucosaminiltransferases/genética , Proteínas de Membrana
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