RESUMO
Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.
Assuntos
Compostos Férricos/química , Ácido Hialurônico/metabolismo , Interleucina-8/química , Polissacarídeos/química , Técnicas Eletroquímicas , Compostos Férricos/imunologia , Humanos , Ácido Hialurônico/química , Interleucina-8/imunologia , Modelos Moleculares , Estrutura Molecular , Polissacarídeos/imunologiaRESUMO
The delivery of chemotactic signaling molecules via customized biomaterials can effectively guide the migration of cells to improve the regeneration of damaged or diseased tissues. Here, we present a novel biohybrid hydrogel system containing two different sulfated glycosaminoglycans (sGAG)/sGAG derivatives, namely either a mixture of short heparin polymers (Hep-Mal) or structurally defined nona-sulfated tetrahyaluronans (9s-HA4-SH), to precisely control the release of charged signaling molecules. The polymer networks are described in terms of their negative charge, i.e. the anionic sulfate groups on the saccharides, using two parameters, the integral density of negative charge and the local charge distribution (clustering) within the network. The modulation of both parameters was shown to govern the release characteristics of the chemotactic signaling molecule SDF-1 and allows for seamless transitions between burst and sustained release conditions as well as the precise control over the total amount of delivered protein. The obtained hydrogels with well-adjusted release profiles effectively promote MSC migration in vitro and emerge as promising candidates for new treatment modalities in the context of bone repair and wound healing.
Assuntos
Quimiocina CXCL12/metabolismo , Glicosaminoglicanos/metabolismo , Hidrogéis/metabolismo , Quimiocina CXCL12/química , Glicosaminoglicanos/química , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismo , Estrutura MolecularRESUMO
Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm-1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. Graphical abstract.
Assuntos
Glicosaminoglicanos/química , Oligossacarídeos/química , Espectrofotometria Infravermelho/métodos , Animais , Temperatura Baixa , Hélio/química , Humanos , Íons/química , Isomerismo , Espectrofotometria Infravermelho/instrumentação , Sulfatos/análiseRESUMO
Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g. fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.
Assuntos
Glicosaminoglicanos , Proteína 2 Glutamina gama-Glutamiltransferase , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Transglutaminases/metabolismoRESUMO
Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and sulfated GAG, at which the sulfation code-which means the number and positions of sulfate groups along the polysaccharide chain-plays an important role. GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues. We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode.
Assuntos
Heparina/análise , Heparina/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sulfatos/análise , Sulfatos/química , Oligossacarídeos/químicaRESUMO
Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF)165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF165-HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF165-mediated signaling and, thereby, elicit VEGF165-independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.