Eduardo Vilanova

Distinct structures of the α-fucose branches in fucosylated chondroitin sulfates do not affect their anticoagulant activity

Fucosylated chondroitin sulfate (FCS) is a glycosaminoglycan found in sea cucumbers. It has a backbone like that of mammalian chondroitin sulfate (4-β-d-GlcA-1→3-β-d-GalNAc-1)n but substituted at the 3rd position of the β-d-glururonic acid residues with α-fucose branches. The structure of these branches varies among FCSs extracted from different species of sea cucumbers, as revealed by solution NMR spectroscopy. Some species (Isostichopus badionotus and Patalus mollis) contain branches formed by single α-fucose residues but with variable sulfation patterns (2,4-, 3,4- and 4-sulfation). FCS from Ludwigothurea grisea is distinguished because it contains preponderant branches formed by disaccharide units containing non-sulfated and 3-sulfated α-fucose units at the reducing and non-reducing ends, respectively. Despite the structural variability on their α-fucose branches, these FCSs have similar anticoagulant action on assays using purified reagents. They have serpin-dependent and serpin-independent effects. Pharmacological assays using experimental animals showed that the three types of FCSs have similar antithrombotic effect and bleeding tendency. They also activate factor XII on the same range of concentration. Based on these observations, we proposed that only few sulfated α-fucose branches along the FCS chain are enough to assure the binding of this glycosaminoglycan to proteins of the coagulation system. Substitution with additional sulfated α-fucose does not increase further the activity. Overall, the use of FCSs with marked variability on their branches of α-fucose allowed us to establish correlations between structures vs biological effects of these glycosaminoglycans on a more refined basis. It opens new avenues for therapeutic intervention using FCSs.

Sulfated polysaccharides from marine sponges (Porifera): an ancestor cell-cell adhesion event based on the carbohydrate-carbohydrate interaction

Marine sponges (Porifera) are ancient and simple eumetazoans. They constitute key organisms in the evolution from unicellular to multicellular animals. We now demonstrated that pure sulfated polysaccharides from marine sponges are responsible for the species-specific cell-cell interaction in these invertebrates. This conclusion was based on the following observations: (1) each species of marine sponge has a single population of sulfated polysaccharide, which differ among the species in their sugar composition and sulfate content; (2) sulfated polysaccharides from sponge interact with each other in a species-specific way, as indicated by an affinity chromatography assay, and this interaction requires calcium; (3) homologous, but not heterologous, sulfated polysaccharide inhibits aggregation of dissociated sponge cells; (4) we also observed a parallel between synthesis of the sulfated polysaccharide and formation of large aggregates of sponge cells, known as primmorphs. Once aggregation reached a plateau, the demand for the de novo synthesis of sulfated polysaccharides ceased. Heparin can mimic the homologous sulfated polysaccharide on the in vitro interaction and also as an inhibitor of aggregation of the dissociated sponge cells. However, this observation is not relevant for the biology of the sponge since heparin is not found in the invertebrate. In conclusion, marine sponges display an ancestor event of cell-cell adhesion, based on the calcium-dependent carbohydrate-carbohydrate interaction.

Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs

Heparins extracted from different animal sources have been conventionally considered effective anticoagulant and antithrombotic agents despite of their pharmacological dissimilarities. We performed herein a systematic analysis on the physicochemical properties, disaccharide composition, in vitro anticoagulant potency and in vivo antithrombotic and bleeding effects of several batches of pharmaceutical grade heparins obtained from porcine intestine, bovine intestine and bovine lung. Each of these three heparin types unambiguously presented differences in their chemical structures, physicochemical properties and/or haemostatic effects. We also prepared derivatives of these heparins with similar molecular weight differing exclusively in their disaccharide composition. The derivatives from porcine intestinal and bovine lung heparins were structurally more similar with each other and hence presented close anticoagulant activities whereas the derivative from bovine intestinal heparin had a higher proportion of 6-desulfated α-glucosamine units and about half anticoagulant activity. Our findings reasonably indicate that pharmaceutical preparations of heparin from different animal sources constitute distinct drugs, thus requiring specific regulatory rules and therapeutic evaluations.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans

Early metazoans had to evolve the first cell adhesion mechanism addressed to maintain a distinctive multicellular morphology. As the oldest extant animals, sponges are good candidates for possessing remnants of the molecules responsible for this crucial evolutionary innovation. Cell adhesion in sponges is mediated by the calcium-dependent multivalent self-interactions of sulfated polysaccharides components of extracellular membrane-bound proteoglycans, namely aggregation factors. Here, we used atomic force microscopy to demonstrate that the aggregation factor of the sponge Desmapsamma anchorata has a circular supramolecular structure and that it thus belongs to the spongican family. Its sulfated polysaccharide units, which were characterized via nuclear magnetic resonance analysis, consist preponderantly of a central backbone composed of 3-α-Glc1 units partially sulfated at 2- and 4-positions and branches of Pyr(4,6)α-Gal1→3-α-Fuc2(SO3)1→3-α-Glc4(SO3)1→3-α-Glc→4-linked to the central α-Glc units. Single-molecule force measurements of self-binding forces of this sulfated polysaccharide and their chemically desulfated and carboxyl-reduced derivatives revealed that the sulfate epitopes and extracellular calcium are essential for providing the strength and stability necessary to sustain cell adhesion in sponges. We further discuss these findings within the framework of the role of molecular structures in the early evolution of metazoans.

How Much Is Too Little to Detect Impacts? A Case Study of a Nuclear Power Plant

Several approaches have been proposed to assess impacts on natural assemblages. Ideally, the potentially impacted site and multiple reference sites are sampled through time, before and after the impact. Often, however, the lack of information regarding the potential overall impact, the lack of knowledge about the environment in many regions worldwide, budgets constraints and the increasing dimensions of human activities compromise the reliability of the impact assessment. We evaluated the impact, if any, and its extent of a nuclear power plant effluent on sessile epibiota assemblages using a suitable and feasible sampling design with no ‘before’ data and budget and logistic constraints. Assemblages were sampled at multiple times and at increasing distances from the point of the discharge of the effluent. There was a clear and localized effect of the power plant effluent (up to 100 m from the point of the discharge). However, depending on the time of the year, the impact reaches up to 600 m. We found a significantly lower richness of taxa in the Effluent site when compared to other sites. Furthermore, at all times, the variability of assemblages near the discharge was also smaller than in other sites. Although the sampling design used here (in particular the number of replicates) did not allow an unambiguously evaluation of the full extent of the impact in relation to its intensity and temporal variability, the multiple temporal and spatial scales used allowed the detection of some differences in the intensity of the impact, depending on the time of sampling. Our findings greatly contribute to increase the knowledge on the effects of multiple stressors caused by the effluent of a power plant and also have important implications for management strategies and conservation ecology, in general.

Sulfated polysaccharides from marine sponges: conspicuous distribution among different cell types and involvement on formation of in vitro cell aggregates

Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction. The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction, as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated molecules and as intracellular inclusions; (2) 35S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the culture of sponge cells is necessary for the production of molecules with biotechnological applications.

Unveiling the structure of sulfated fucose-rich polysaccharides via nuclear magnetic resonance spectroscopy

Sulfated fucans from marine invertebrates are composed of regular repetitive fucose building-blocks with sulfation patterns differing in a species-specific manner. These polysaccharides can act as mediators of the acrosome reaction of sea-urchins or play a structural role in the body-wall of sea-cucumbers. Other fucose-rich polysaccharides found in the body-wall of sea-cucumbers are the fucosylated chondroitin sulfates composed of a vertebrate-like chondroitin sulfate decorated with species-specific fucose branches. Fine-tuning structural determinations of these polysaccharides have been accomplished since the 1980s almost exclusively via high-resolution NMR. In this review, we present an overview on NMR-based structural and conformational analyses of these sulfated fucose-rich polysaccharides. This constitutes an outstanding example of the potential of NMR in studying the chemical aspects of complex carbohydrates.

Systematic Analysis of Pharmaceutical Preparations of Chondroitin Sulfate Combined with Glucosamine

Glycosaminoglycans are carbohydrate-based compounds widely employed as nutraceuticals or prescribed drugs. Oral formulations of chondroitin sulfate combined with glucosamine sulfate have been increasingly used to treat the symptoms of osteoarthritis and osteoarthrosis. The chondroitin sulfate of these combinations can be obtained from shark or bovine cartilages and hence presents differences regarding the proportions of 4- and 6-sulfated N-acetyl β-d-galactosamine units. Herein, we proposed a systematic protocol to assess pharmaceutical batches of this combination drug. Chemical analyses on the amounts of chondroitin sulfate and glucosamine in the batches were in accordance with those declared by the manufacturers. Anion-exchange chromatography has proven more effective than electrophoresis to determine the type of chondroitin sulfate present in the combinations and to detect the presence of keratan sulfate, a common contaminant found in batches prepared with shark chondroitin sulfate. 1D NMR spectra revealed the presence of non-sulfated instead of sulfated glucosamine in the formulations and thus in disagreement with the claims declared on the label. Moreover, 1D and 2D NMR analyses allowed a precise determination on the chemical structures of the chondroitin sulfate present in the formulations. The set of analytical tools suggested here could be useful as guidelines to improve the quality of this medication.

A color‐code for glycosaminoglycans identification by means of polyacrylamide gel electrophoresis stained with the cationic carbocyanine dye Stains‐all

Cationic dyes such as toluidin blue are commonly employed to visualize glycosaminoglycans (GAGs) on electrophoresis gels; however, the carbocyanine‐based dye Stains‐all have been increasingly used to stain the non‐sulfated hyaluronic acid and other GAGs in submicrogram quantities. In this short communication, we demonstrate that Stains‐all is able to stain the most common GAGs on polyacrylamide gels with distinct and contrasting colors in a reproducible manner. We also show that this staining method is useful to identify GAGs present both in mixtures and in submicrogram quantities. Therefore, Stains‐all has shown to be useful in identifying GAGs on polyacrylamide gels with basis on their specific colors, at least on screening level.

Update on Brazilian biosimilar enoxaparins

ABSTRACT Introduction: Brazil is among the first countries approving the commercialization and clinical use of biosimilar enoxaparins. Our research group has performed quality control assessments of these drugs over the last decade. Areas covered: We have not found noticeable differences between Brazilian biosimilar enoxaparins and the original product regarding their physicochemical properties, disaccharide composition, anticoagulant activity, bioavailability and safety. Expert commentary: In spite of clinical and pharmacological advantages of enoxaparin, subcutaneous formulations of unfractionated heparin are employed by the Brazilian public health system for prevention and treatment of thromboembolism. The underuse of both original and biosimilar enoxaparins in Brazil directly correlates with their high cost.