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1.
PLoS One ; 15(10): e0240920, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33079966

RESUMO

As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.


Assuntos
Bacteroides/enzimologia , Heparina Liase/genética , Heparina Liase/metabolismo , Heparina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/genética , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Ativação Enzimática , Heparina Liase/química , Ligação de Hidrogênio , Magnésio/metabolismo , Manganês/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica
2.
Cell ; 183(4): 1043-1057.e15, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32970989

RESUMO

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.


Assuntos
Betacoronavirus/fisiologia , Heparitina Sulfato/metabolismo , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Linhagem Celular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Humanos , Rim/metabolismo , Pulmão/metabolismo , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
3.
Int J Biol Macromol ; 163: 1649-1658, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32979436

RESUMO

The SARS-CoV-2 spike glycoproteins (SGPs) and human angiotensin converting enzyme 2 (ACE2) are the two key targets for the prevention and treatment of COVID-19. Host cell surface heparan sulfate (HS) is believed to interact with SARS-CoV-2 SGPs to facilitate host cell entry. In the current study, a series of polysaccharides from Saccharina japonica were prepared to investigate the structure-activity relationship on the binding abilities of polysaccharides (oligosaccharides) to pseudotype particles, including SARS-CoV-2 SGPs, and ACE2 using surface plasmon resonance. Sulfated galactofucan (SJ-D-S-H) and glucuronomannan (Gn) displayed strongly inhibited interaction between SARS-CoV-2 SGPs and heparin while showing negligible inhibition of the interaction between SARS-CoV-2 SGPs and ACE2. The IC50 values of SJ-D-S-H and Gn in blocking heparin SGP binding were 27 and 231 nM, respectively. NMR analysis showed that the structure of SJ-D-S-H featured with a backbone of 1, 3-linked α-L-Fucp residues sulfated at C4 and C2/C4 and 1, 3-linked α-L-Fucp residues sulfated at C4 and branched with 1, 6-linked ß-D-galacto-biose; Gn had a backbone of alternating 1, 4-linked ß-D-GlcAp residues and 1, 2-linked α-D-Manp residues. The sulfated galactofucan and glucuronomannan showed strong binding ability to SARS-CoV-2 SGPs, suggesting that these polysaccharides might be good candidates for preventing and/or treating SARS-CoV-2.


Assuntos
Infecções por Coronavirus/virologia , Glucuronatos/metabolismo , Manose/análogos & derivados , Pneumonia Viral/virologia , Polissacarídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/química , Betacoronavirus/metabolismo , Sítios de Ligação , Glucuronatos/química , Heparina/química , Heparina/metabolismo , Humanos , Manose/química , Manose/metabolismo , Oligossacarídeos/química , Pandemias , Peptidil Dipeptidase A/metabolismo , Feófitas/química , Polissacarídeos/química , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Relação Estrutura-Atividade
4.
EBioMedicine ; 59: 102969, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32853989

RESUMO

Coronavirus disease-2019 (COVID-19) is associated with severe inflammation in mainly the lung, and kidney. Reports suggest a beneficial effect of the use of heparin/low molecular weight heparin (LMWH) on mortality in COVID-19. In part, this beneficial effect could be explained by the anticoagulant properties of heparin/LMWH. Here, we summarise potential beneficial, non-anticoagulant mechanisms underlying treatment of COVID-19 patients with heparin/LMWH, which include: (i) Inhibition of heparanase activity, responsible for endothelial leakage; (ii) Neutralisation of chemokines, and cytokines; (iii) Interference with leukocyte trafficking; (iv) Reducing viral cellular entry, and (v) Neutralisation of extracellular cytotoxic histones. Considering the multiple inflammatory and pathogenic mechanisms targeted by heparin/LMWH, it is warranted to conduct clinical studies that evaluate therapeutic doses of heparin/LMWH in COVID-19 patients. In addition, identification of specific heparin-derived sequences that are functional in targeting non-anticoagulant mechanisms may have even higher therapeutic potential for COVID-19 patients, and patients suffering from other inflammatory diseases.


Assuntos
Anti-Inflamatórios/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Heparina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina/metabolismo , Heparina/farmacologia , Heparina de Baixo Peso Molecular/metabolismo , Heparina de Baixo Peso Molecular/farmacologia , Heparina de Baixo Peso Molecular/uso terapêutico , Histonas/sangue , Histonas/metabolismo , Humanos , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Internalização do Vírus/efeitos dos fármacos
5.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1450-1458, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748603

RESUMO

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.


Assuntos
Carboidratos Epimerases/biossíntese , Carboidratos Epimerases/química , Heparina/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Animais , Carboidratos Epimerases/genética , Escherichia coli , Expressão Gênica , Heparitina Sulfato/metabolismo , Ácido Idurônico/metabolismo , Proteínas de Peixe-Zebra/genética
6.
Anal Chem ; 92(16): 10930-10934, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32678978

RESUMO

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over 6,000,000 cases and nearly 400,000 deaths reported worldwide by the end of May 2020. A rush to find a cure prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the noncovalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease in its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Heparina/metabolismo , Espectrometria de Massas/métodos , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Gases/química , Heparina/farmacologia , Heparina/uso terapêutico , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos
7.
Antiviral Res ; 181: 104873, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32653452

RESUMO

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 µM, 0.12 µM, and 26.4 µM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Heparina/metabolismo , Pandemias , Pneumonia Viral/virologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítios de Ligação , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Ressonância de Plasmônio de Superfície
8.
Mol Immunol ; 126: 8-13, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32717572

RESUMO

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.


Assuntos
Proteína Inibidora do Complemento C1/metabolismo , Complemento C1s/antagonistas & inibidores , Polímeros/metabolismo , Sítios de Ligação/genética , Proteína Inibidora do Complemento C1/genética , Proteína Inibidora do Complemento C1/isolamento & purificação , Complemento C1s/metabolismo , Heparina/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Polifosfatos/metabolismo , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
Nat Commun ; 11(1): 2694, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483155

RESUMO

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.


Assuntos
Toxinas Bacterianas/toxicidade , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Polissacarídeos/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Insetos/microbiologia , Antígenos CD15/química , Antígenos CD15/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Morganella morganii/patogenicidade , Photorhabdus/patogenicidade , Polissacarídeos/química , Xenorhabdus/patogenicidade
10.
PLoS One ; 15(4): e0231977, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352982

RESUMO

Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.


Assuntos
Poxviridae/fisiologia , Replicação Viral , Animais , Linhagem Celular , Heparina/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Poxviridae/metabolismo , Sulfotransferases/deficiência
11.
J Med Vasc ; 45(3): 147-157, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32402428

RESUMO

The recognized therapeutic effect of heparins is an anticoagulant activity (anti-Xa and anti-IIa) acting in an indirect manner (cofactor of antithrombin) but which is carried by only 20% at best of the glycan chains composing any commercial preparation of heparin, whether unfractionated or low molecular weight. However, the effects of glycan chains that participate in the therapeutic but also potentially adverse effects of heparin preparations must also be considered. These specific effects of glycans are potentially different for each commercial preparation of heparins and, in particular, low molecular weight heparins (LMWH) compared with unfractionated heparin (UFH) and LMWH between them. The glycanic nature of heparin is responsible for its very particular pharmacology: exchange with the glycocalyx of cells in particular endothelial. Exchanges which depend on the length and structure of the glycan chains therefore different between UFH and LMWH between the different heparin preparations between them but also according to the state of glycocalyx differently altered according to the underlying diseases and their degree of evolution. If the anticoagulant effects of heparins can potentially be replaced with those of new oral anticoagulants, the glycan effects of heparins cannot be replaced by synthetic non-glycan molecules. This replacement will undoubtedly limit certain risks such as heparin-induced thrombocytopenia (HIT) but other beneficial effects participating to the overall efficacy of heparin (whose relative importance remains to be ascertained), will also disappear: effects on surfaces, anti-inflammatory effects, antineoplastic and anti-metastatic effects, ancillary anticoagulant effects (not dependent on antithrombin), effect on endothelial dysfunction. This review will be focused on all of these related/pleiotropic effects of heparins that are in fact the effects of the glycan nature of heparin. Among the antithrombotic effects not dependent on antithrombin one has been more recently highlighted: the passivation/neutralization of the positively charged fibrils of Netosis, by the negatively charged glycan chains of heparin. This also has clinical implications: in the era of generics and biosimilars where biosimilar heparins begin to appear, it is important to know that accordingly to FDA and EMEA rules: their biosimilarity is judged only on the "classical" anticoagulation effect cofactor of antithrombin (anti-IIa/anti-Xa) but that all glycan effects that are potentially beneficial or potentially deleterious are not taken into consideration in their assessment.


Assuntos
Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Heparina/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Anticoagulantes/efeitos adversos , Anticoagulantes/química , Anticoagulantes/metabolismo , Antineoplásicos/uso terapêutico , Células Endoteliais/metabolismo , Glicocálix/metabolismo , Hemorragia/induzido quimicamente , Heparina/efeitos adversos , Heparina/química , Heparina/metabolismo , Humanos , Peso Molecular , Conformação Proteica , Medição de Risco , Fatores de Risco , Relação Estrutura-Atividade , Trombocitopenia/sangue , Trombocitopenia/induzido quimicamente
12.
Enzyme Microb Technol ; 137: 109549, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423676

RESUMO

Heparinase has attracted much attention because of its applications in pharmaceutical industry. Herein, the heparinases III from Flavobacterium heparinum (FhepIII) and Bacteroides thetaiotaomicron (BhepIII) were firstly comparatively characterized. BhepIII showed higher catalytic activity and thermostability toward heparin comparing to FhepIII. To further upgraded BhepIII, a protein engineering approach based on B-factor was performed. By site-saturated mutagenesis of the flexible residues within an 8 Šradius around the catalytic residue, Asp321 and Ser264 were identified as essential residues for catalytic efficiency and thermostability, respectively. D321Q mutation enhanced catalytic efficiency (kcat/Km) with a 68.4% increase by increasing the surface potential while S264 F mutation increased thermostability with a half-time at 50℃ (t1/250℃) of 3.8 h versus 2.7 h of the wild-type by increasing rigidity and interactions within the active pocket. Double mutation of S264 F and D321Q resulted in a 245% increase in kcat/Km but with a decreased t1/250℃ (2.0 h). E105R mutation that generated a 348% increase in kcat/Km was further identified by electric potential engineering of the pocket tunnel. Eventually, the variant E105R/S264 F that showed a 418% increase in kcat/Km without compromise of thermostability was constructed. The engineered E105R/S264 F has a great potential for the commercial production of low molecular weight heparin in the future.


Assuntos
Bacteroides thetaiotaomicron/enzimologia , Heparina/metabolismo , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Temperatura , Bacteroides thetaiotaomicron/genética , Sítios de Ligação , Catálise , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Pedobacter/enzimologia , Pedobacter/genética , Engenharia de Proteínas
13.
Proc Natl Acad Sci U S A ; 117(17): 9311-9317, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32277030

RESUMO

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 Transcrição
14.
Adv Exp Med Biol ; 1221: 169-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274710

RESUMO

Heparanase is the principal enzyme that degrades heparan sulfate (HS) in both physiological (HS turnover) and pathological (tumor metastasis, inflammation) cell conditions, catalysing the hydrolysis of the ß-1-4 glycosidic bond in -GlcUA-ß(1-4)-GlcNX-. Despite efforts to define the minimum trisaccharide sequence that allows glycans to be recognized by heparanase, a rigorous "molecular code" by which the enzyme reads and degrades HS chains has not been identified. The X-ray diffraction model of heparanase, resolved by Wu et al (2015), revealed a complex between the trisaccharide GlcNS6S-GlcUA-GlcNS6S and heparanase. Efforts are ongoing to better understand how HS mimetics longer than three residues are recognized by heparanase before being hydrolyzed or inhibit the enzyme. It is also important to consider the flexibility of the enzyme active site, a feature that opens up the development of heparanase inhibitors with structures significantly different from HS or heparin. This chapter reviews the state-of-the-art knowledge about structural aspects of heparanase activities in terms of substrate recognition, mechanism of hydrolysis, and inhibition.


Assuntos
Glucuronidase , Glicóis , Heparina , Heparitina Sulfato , Glucuronidase/antagonistas & inibidores , Glucuronidase/química , Glucuronidase/metabolismo , Glicóis/química , Glicóis/metabolismo , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Hidrólise , Especificidade por Substrato
15.
Carbohydr Polym ; 237: 116143, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32241440

RESUMO

A sulfated glucurono-xylo-rhamnan (EP-3-H) was purified from a green alga, Enteromorpha prolifera. EP-3-H and its oligomers were characterized by high performance liquid chromatography, mass spectrometry and one and two-dimensional nuclear magnetic resource spectroscopy. The structural analysis showed EP-3-H has a backbone of glucurono-xylo-rhamnan, branches with glucuronic acid and sulfated at C3 of rhamnose and/or C2 of xylose. The inhibition of EP-3-H on human lung cancer A549 cell proliferation in vitro and its therapeutic effects in BALB/c-nu mice in vivo were determined to evaluate the anti-lung cancer activity of EP-3-H. The tumor inhibition level was 59 %, suggesting that EP-3-H might be a good candidate for the treatment of lung cancer. Surface plasmon resonance (SPR) studies revealed the IC50 on the binding of fibroblast growth factors, (FGF1 and FGF2), to heparin were 0.85 and 1.47 mg/mL, respectively. These results suggest that EP-3-H inhibits cancer proliferation by interacting with these growth factors.


Assuntos
Antineoplásicos , Desoxiaçúcares , Neoplasias Pulmonares/tratamento farmacológico , Mananas , Células A549 , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Desoxiaçúcares/isolamento & purificação , Desoxiaçúcares/farmacologia , Desoxiaçúcares/uso terapêutico , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Mananas/isolamento & purificação , Mananas/farmacologia , Mananas/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Sulfatos , Ulva/química
16.
Proc Natl Acad Sci U S A ; 117(16): 8890-8899, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245806

RESUMO

Eastern equine encephalitis virus (EEEV), a mosquito-borne icosahedral alphavirus found mainly in North America, causes human and equine neurotropic infections. EEEV neurovirulence is influenced by the interaction of the viral envelope protein E2 with heparan sulfate (HS) proteoglycans from the host's plasma membrane during virus entry. Here, we present a 5.8-Å cryoelectron microscopy (cryo-EM) structure of EEEV complexed with the HS analog heparin. "Peripheral" HS binding sites were found to be associated with the base of each of the E2 glycoproteins that form the 60 quasi-threefold spikes (q3) and the 20 sites associated with the icosahedral threefold axes (i3). In addition, there is one HS site at the vertex of each q3 and i3 spike (the "axial" sites). Both the axial and peripheral sites are surrounded by basic residues, suggesting an electrostatic mechanism for HS binding. These residues are highly conserved among EEEV strains, and therefore a change in these residues might be linked to EEEV neurovirulence.


Assuntos
Desenho de Fármacos , Vírus da Encefalite Equina do Leste/ultraestrutura , Encefalomielite Equina/tratamento farmacológico , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/ultraestrutura , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Sulfatos de Condroitina/farmacologia , Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste/metabolismo , Encefalomielite Equina/virologia , Proteoglicanas de Heparan Sulfato/análogos & derivados , Heparina/metabolismo , Humanos , Mesocricetus , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/ultraestrutura , Ligação Viral/efeitos dos fármacos
17.
Artigo em Inglês | MEDLINE | ID: mdl-32289504

RESUMO

Low-density lipoprotein (LDL) binding to arterial proteoglycans initiates LDL retention and modification in the arterial wall, triggering atherosclerosis. The details of this binding, its effectors, and its ramifications are incompletely understood. We combined heparin affinity chromatography with biochemical, spectroscopic and electron microscopic techniques to show that brief binding to heparin initiates irreversible pro-atherogenic remodeling of human LDL. This involved decreased structural stability of LDL and increased susceptibility to hydrolysis, oxidation and fusion. Furthermore, phospholipid hydrolysis, mild oxidation and/or glycation of LDL in vitro increase the proteolytic susceptibility of apoB and its heparin binding affinity, perhaps by unmasking additional heparin-binding sites. For LDL from hyperglycemic type-2 diabetic patients, heparin binding was particularly destabilizing and caused apoB fragmentation and LDL fusion. However, for similar patients whose glycemic control was restored upon therapy, LDL-heparin binding affinity was rectified and LDL structural stability was partially restored. These results complement previous studies of LDL binding to arterial proteoglycans and suggest that such interactions may produce a particularly pro-atherogenic subclass of electronegative LDL. In summary, binding to heparin alters apoB conformation, perhaps by partially peeling it off the lipid, and triggers pro-atherogenic LDL modifications including hydrolysis, oxidation, and destabilization. Furthermore, phospholipid lipolysis, mild oxidation and glycation of LDL in vitro strengthen its binding to heparin, which helps explain stronger binding observed in hyperglycemic LDL. Combined effects of hyperglycemia and heparin binding are especially deleterious but are largely rectified upon diabetes therapy. These findings help establish a mechanistic link between diabetes and atherosclerosis.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Heparina/metabolismo , Hiperglicemia/metabolismo , Lipoproteínas LDL/metabolismo , Sítios de Ligação , Humanos , Hidrólise , Lipoproteínas LDL/sangue , Lipoproteínas LDL/química , Tamanho da Partícula , Agregados Proteicos , Conformação Proteica , Propriedades de Superfície
18.
Sci Rep ; 10(1): 5225, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251304

RESUMO

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer's disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.


Assuntos
Ribossomos/metabolismo , Proteínas tau/metabolismo , Ânions/química , Ânions/metabolismo , Células HeLa , Heparina/metabolismo , Humanos , Microtúbulos/metabolismo , Doenças Neurodegenerativas/metabolismo , Domínios Proteicos , RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas tau/química , Proteínas tau/genética
19.
Mikrochim Acta ; 187(4): 226, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170394

RESUMO

Heparin was employed as the stabilizing agent in the synthesis of peroxidase-mimicking Pd nanoparticles. The heparin-capped Pd nanozyme can act as both the signal amplifier and the selective binder of protamine. The most efficient nanozyme with the mean size of 3.5 nm consists of 70.8% metallic Pd0 and 29.2% Pd2+ species. Enzyme kinetic studies show that the Km values are 0.036 mM for 3,3',5,5'-tetramethylbenzidine and 78 mM for H2O2. Protamine shows strong affinity to the heparin-capped Pd nanozyme, and induces an apparent aggregation of the nanoparticles. This results in a significant inhibition of the peroxidase-mimicking activities. Hence, the oxidation of TMB by H2O2 to a blue product with a maximum absorption at 652 nm is suppressed. Based on this finding, a photometric assay is developed for the determination of protamine. The linear response is in the concentration range 0.02 ~ 0.8 µg mL-1, and the limit of detection is 0.014 µg mL-1. This assay presents high selectivity toward other biological substances. Graphical abstract Highly active and selective Pd nanozyme was synthesized through adopting heparin as the capping agent for quantitative determination of protamine.


Assuntos
Heparina/química , Nanopartículas/química , Paládio/química , Peroxidase/química , Fotometria , Protaminas/análise , Heparina/metabolismo , Nanopartículas/metabolismo , Paládio/metabolismo , Peroxidase/metabolismo , Protaminas/metabolismo
20.
Mediators Inflamm ; 2020: 8098439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184702

RESUMO

Neuroinflammation contributes to or even causes central nervous system (CNS) diseases, and its regulation is thus crucial for brain disorders. Mast cells (MCs) and microglia, two resident immune cells in the brain, together with astrocytes, play critical roles in the progression of neuroinflammation-related diseases. MCs have been demonstrated as one of the fastest responders, and they release prestored and newly synthesized mediators including histamine, ß-tryptase, and heparin. However, temporal changes in MC activation in this inflammation process remain unclear. This study demonstrated that MC activation began at 2 h and peaked at 4 h after lipopolysaccharide (LPS) administration. The number of activated MCs remained elevated until 24 h after LPS administration. In addition, the levels of histamine and ß-tryptase in the hippocampus markedly and rapidly increased within 6 h and remained higher than the baseline level within 24 h after LPS challenge. Furthermore, mast cell-deficient KitW-sh/W-sh mice were used to investigate the effects of MCs on microglial and astrocytic activation and blood-brain barrier (BBB) permeability at 4 h after LPS stimulation. Notably, LPS-induced proinflammatory cytokine secretion, microglial activation, and BBB damage were inhibited in KitW-sh/W-sh mice. However, no detectable astrocytic changes were found in WT and KitW-sh/W-sh mice at 4 h after LPS stimulation. Our findings indicate that MC activation precedes CNS inflammation and suggest that MCs are among the earliest participants in the neuroinflammation-initiating events.


Assuntos
Barreira Hematoencefálica/metabolismo , Lipopolissacarídeos/farmacologia , Mastócitos/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Heparina/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Histamina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Triptases/metabolismo
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