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.
Abstract
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
.
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•HS-modifying enzymes are actively engaged in both anterograde and retrograde Golgi transport.•HS-modifying enzymes are sorted and trafficked by similar mechanisms.•BFA treatment changed HS-modifying ...enzymes vesicular trafficking.•HS-modifying enzymes moved from cis to trans-Golgi during heparin stimulus.•Changes in the localization of HS-modifying enzymes correlate with changes in HS structure.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
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
19
F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing
19
F NMR spectroscopy is followed. Furthermore, the ability of
19
F labelled GAGs to be imaged using CARS microscopy is demonstrated.
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F labelled GAGs enable both
19
F 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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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
F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing
F NMR spectroscopy is followed. Furthermore, the ability of
F labelled GAGs to be imaged using CARS microscopy is demonstrated.
F labelled GAGs enable both
F 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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Context:
Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is observed ...even after complete surgical removal without evidence of a recurrent or persistent tumor.
Objective:
To investigate the presence of assay interference in the serum CT of MTC patients who are apparently without a structural disease.
Patients and Methods:
We studied three index MTC cases for CT assay interference and 14 patients with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening for assay interference was performed by determination of CT levels before and after serum treatment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-performance liquid chromatography and protein A-Sepharose.
Results:
Patients with biochemical and structural disease showed CT mean recovery of 84.1% after polyethylene glycol treatment, whereas patients suspected of interference showed recovery from 2–7%. The elution profile on UPLC showed that the immunometric CT from these three patients behaved like a high molecular mass aggregate (>300 kDa). Additionally, when these samples were applied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only released after lowering the pH.
Conclusions:
For the first time, our results show the presence of a novel pitfall in the CT immunoassay: “macrocalcitonin.” Its etiology, frequency, and meaning remain to be defined, but its recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and treatment during the follow-up of MTC.
•Syagrus coronata (Mart.) Becc. (SC) and Acrocomia aculeate (AA) were studied.•Their potential for the biodiesel and thermo cracked bio-oil production was evaluated.•Physico-chemical properties of ...the oils, biodiesel and thermo cracked bio-oil were studied.•Biodiesel produced are in acceptable range for use in diesel engines.•Bio-oils from SC and AA showed good potential to produce renewable fuel.
This work examines the potential of two species of palm-tree, Syagrus coronata (Mart.) Becc. and Acrocomia aculeate, for biodiesel and thermo cracked bio-oil production. These species can offer the opportunity to obtain alternative fats and oils from perennial sources, which permits their combination with other crops or cattle production, as well as the recovery of degraded areas. The physico-chemical properties of the oils, biodiesel and bio-oil obtained from the two palms were evaluated. For biodiesel production, the high acid value of the Acrocomia aculeate crude vegetable oil required a previous acid esterification step to avoid the formation of soaps and stable emulsions, which was not necessary in the case of Syagrus coronata (Mart.) Becc. Concerning their fatty acid composition, SC exhibits mainly short, saturated chains, while AA exhibits long unsaturated chains. The short saturated chains make Syagrus coronata (Mart.) Becc. biodiesel less viscous and more stable to oxidation than Acrocomia aculeate biodiesel. However, both Syagrus coronata (Mart.) Becc. and Acrocomia aculeate biodiesel are in the acceptable range for use as biodiesel in diesel engines, showing promise for the economic exploitation of these raw materials, requiring only the use of an antioxidant additive in the case of Acrocomia aculeate. The bio-oils obtained from both Syagrus coronata (Mart.) Becc. and Acrocomia aculeate were not completely deoxygenated, but the physico-chemical properties showed good potential for producing renewable fuel acceptable for use in diesel engines. However, further studies are needed to achieve their complete deoxygenation and improve their properties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK