RESUMO
Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of "heparanome" and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin-spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described.
RESUMO
The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Heparitina Sulfato/biossíntese , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Complexo de Golgi/química , Complexo de Golgi/efeitos dos fármacos , Heparina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.
Assuntos
Anticoagulantes/farmacologia , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Enoxaparina/farmacologia , Heparina/farmacologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticoagulantes/uso terapêutico , Antivirais/uso terapêutico , Chlorocebus aethiops , Enoxaparina/uso terapêutico , Heparina/uso terapêutico , Humanos , Simulação de Dinâmica Molecular , Nebulizadores e Vaporizadores , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Relação Estrutura-Atividade , Células Vero , Internalização do VírusRESUMO
Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.
Assuntos
Anticoagulantes/isolamento & purificação , Sulfatos de Condroitina/isolamento & purificação , Contaminação de Medicamentos , Heparina/isolamento & purificação , Animais , Anticoagulantes/química , Bovinos , Sulfatos de Condroitina/química , Heparina/química , Heparina Liase/química , Humanos , Hidrólise , Mucosa Intestinal/química , Espectroscopia de Ressonância Magnética , Polissacarídeo-Liases/química , Controle de Qualidade , SuínosRESUMO
Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of 19F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing 19F NMR spectroscopy is followed. Furthermore, the ability of 19F labelled GAGs to be imaged using CARS microscopy is demonstrated. 19F labelled GAGs enable both 19F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.
Assuntos
Imagem por Ressonância Magnética de Flúor-19/métodos , Flúor/química , Glicosaminoglicanos/química , Sondas Moleculares/química , Coloração e Rotulagem/métodos , Acetilação , Antitrombinas/química , Glicosaminoglicanos/análise , Halogenação , Espectroscopia de Ressonância Magnética/métodos , Sondas Moleculares/análise , Ligação Proteica , Soluções , Análise Espectral Raman/métodosRESUMO
Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure-activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure-activity relationships and regulation will be discussed.