RESUMO
The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HCâ¢HA) complexes. While limited assessments of HCâ¢HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HCâ¢HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HCâ¢HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HCâ¢HA and total HA in an equine model of synovitis.
Assuntos
Ácido Hialurônico , Nanoporos , Humanos , Animais , Cavalos , Ácido Hialurônico/química , alfa-Globulinas/metabolismo , Inflamação , Líquido SinovialRESUMO
We used heparosan (HEP) polysaccharides for controlling nanoparticle delivery to innate immune cells. Our results show that HEP-coated nanoparticles were endocytosed in a time-dependent manner by innate immune cells via both clathrin-mediated and macropinocytosis pathways. Upon endocytosis, we observed HEP-coated nanoparticles in intracellular vesicles and the cytoplasm, demonstrating the potential for nanoparticle escape from intracellular vesicles. Competition with other glycosaminoglycan types inhibited the endocytosis of HEP-coated nanoparticles only partially. We further found that nanoparticle uptake into innate immune cells can be controlled by more than 3 orders of magnitude via systematically varying the HEP surface density. Our results suggest a substantial potential for HEP-coated nanoparticles to target innate immune cells for efficient intracellular delivery, including into the cytoplasm. This HEP nanoparticle surface engineering technology may be broadly used to develop efficient nanoscale devices for drug and gene delivery as well as possibly for gene editing and immuno-engineering applications.
Assuntos
Nanopartículas , Clatrina/metabolismo , Dissacarídeos , Endocitose , Imunidade Inata , PolissacarídeosRESUMO
Nanoparticle modification with poly(ethylene glycol) (PEG) is a widely used surface engineering strategy in nanomedicine. However, since the artificial PEG polymer may adversely impact nanomedicine safety and efficacy, alternative surface modifications are needed. Here, we explored the "self" polysaccharide heparosan (HEP) to prepare colloidally stable HEP-coated nanoparticles, including gold and silver nanoparticles and liposomes. We found that the HEP-coating reduced the nanoparticle protein corona formation as efficiently as PEG coatings upon serum incubation. Liquid chromatography-mass spectrometry revealed the protein corona profiles. Heparosan-coated nanoparticles exhibited up to 230-fold higher uptake in certain innate immune cells, but not in other tested cell types, than PEGylated nanoparticles. No noticeable cytotoxicity was observed. Serum proteins did not mediate the high cell uptake of HEP-coated nanoparticles. Our work suggests that HEP polymers may be an effective surface modification technology for nanomedicines to safely and efficiently target certain innate immune cells.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Coroa de Proteína , Adsorção , Proteínas Sanguíneas , Dissacarídeos , Nanopartículas/química , Polietilenoglicóis/química , Polímeros , Polissacarídeos , PrataRESUMO
A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through FGF receptor (FGFR) 1c. The HS polysaccharide tested contained both undersulfated (NA) domains and highly sulfated (NS) domains as well as very well defined non-reducing termini. This study examines differences in the HS selectivity of the positive canyons of the FGF12-FGFR1c2 and FGF22-FGFR1c2 HS binding sites of the symmetric FGF2-FGFR2-HS2 signal transduction complex. The results suggest that FGF12-FGFR1c2 binding site prefers a longer NS domain at the non-reducing terminus than FGF22-FGFR1c2 In addition, FGF22-FGFR1c2 can tolerate an HS chain having an N-acetylglucosamine residue at its non-reducing end. These results clearly demonstrate the different specificity of FGF12-FGFR1c2 and FGF22-FGFR1c2 for well defined HS structures and suggest that it is now possible to chemoenzymatically synthesize precise HS polysaccharides that can selectively mediate growth factor signaling. These HS polysaccharides might be useful in both understanding and controlling the growth, proliferation, and differentiation of cells in stem cell therapies, wound healing, and the treatment of cancer.
Assuntos
Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Transformada , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , CamundongosRESUMO
Glycosaminoglycans (GAGs) are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria Pasteurella multocida Type F and Escherichia coli K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, Chlorobium phaeobacteroides DSM 266, which was very similar (~62% identical) to the open reading frames of the known bifunctional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant E. coli-derived Chlorobium enzyme preparations were found to possess bona fide chondroitin synthase activity in vitro. This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in C. phaeobacteroides is unexpected for several reasons including (a) a free-living nonpathogenic organism should not "need" an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5-3 billion years ago and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.
Assuntos
Chlorobium/enzimologia , Glicosaminoglicanos/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/isolamento & purificação , Sequência de Aminoácidos/genética , Sulfatos de Condroitina/química , Escherichia coli/genética , Glicosaminoglicanos/genética , N-Acetilgalactosaminiltransferases/metabolismo , Especificidade por SubstratoRESUMO
Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60-70 kDa) and/or clearance by the body's components (e.g., proteases, antibodies, high-efficiency receptors) in their native form. FDA-approved polyethylene glycol (PEG) is a vehicle for improving therapeutics, but artificial polymers have potential biocompatibility and immunogenicity liabilities. Here, we utilized a natural vertebrate carbohydrate, heparosan (HEP), the biosynthetic precursor of heparan sulfate and heparin, to enhance performance of a biologic drug. The HEP polysaccharide was stable with a long half-life (~8 days for 99-kDa chain) in the nonhuman primate bloodstream, but was efficiently degraded to very short oligosaccharides when internalized by cells, and then excreted into urine and feces. Several HEP-modified human granulocyte-colony stimulating factor (G-CSF) conjugates were synthesized with defined quasi-monodisperse HEP polysaccharide chains. Single dosing of 55- or 99-kDa HEP-G-CSF in rats increased blood neutrophil levels comparable to PEG-G-CSF conjugates. Repeated dosing of HEP-G-CSF or HEP alone for 2 weeks did not cause HEP-specific toxic effects in rats. HEP did not possess the anticoagulant behavior of its daughter, heparin, based on testing in rats or clinical diagnostic assays with human plasma. Neither anti-HEP IgG nor IgM antibodies were detected in a long-term (9 doses over 7 months) immunogenicity study of the HEP-drug conjugate with rats. These proof-of-concept experiments with HEP-G-CSF indicate that it is a valid drug candidate for neutropenia and suggest the potential of this HEP-based platform as a safe alternative delivery vehicle for other therapeutics.
Assuntos
Dissacarídeos/farmacocinética , Portadores de Fármacos/farmacocinética , Fator Estimulador de Colônias de Granulócitos/farmacocinética , Neutropenia/tratamento farmacológico , Animais , Células Sanguíneas/efeitos dos fármacos , Dissacarídeos/efeitos adversos , Dissacarídeos/química , Portadores de Fármacos/efeitos adversos , Portadores de Fármacos/química , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/química , Humanos , Macaca , Masculino , RatosRESUMO
Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.
Assuntos
Glicosaminoglicanos/biossíntese , Nucleotidiltransferases/metabolismo , Biocatálise , Configuração de Carboidratos , Glicosaminoglicanos/química , Nucleotidiltransferases/química , Açúcares de Uridina Difosfato/biossíntese , Açúcares de Uridina Difosfato/químicaRESUMO
Unnatural chemically modified nucleotide sugars UDP-4-N3-GlcNAc and UDP-4-N3-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or chondroitin using polysaccharide synthases. UDP-4-N3-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N3-GlcNAc-terminated hyaluronan and heparosan were then successfully conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is generally applicable for labeling and detection of suitable glycosaminoglycans.
Assuntos
Glicosaminoglicanos/análise , Hexosaminas/síntese química , Difosfato de Uridina/síntese química , Química Click , Hexosaminas/química , Conformação Molecular , Difosfato de Uridina/químicaRESUMO
We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.
Assuntos
Condroitina/química , Ácido Hialurônico/química , Oligossacarídeos/química , Alginatos/química , alfa-Globulinas/química , Moléculas de Adesão Celular/química , Sulfatos de Condroitina/química , Dissacarídeos/química , Ácido Glucurônico/química , Heparitina Sulfato/química , Ácidos Hexurônicos/química , Humanos , Cinética , Ligação ProteicaRESUMO
Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was (13)C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.
Assuntos
Moléculas de Adesão Celular/química , Ácido Hialurônico/química , Modelos Moleculares , Oligossacarídeos/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/genética , Ácido Hialurônico/metabolismo , Inflamação/genética , Inflamação/metabolismo , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Ovulação/genética , Ovulação/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.
Assuntos
Receptores ErbB , Fator 2 de Crescimento de Fibroblastos , Heparitina Sulfato/química , Modelos Biológicos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Transdução de Sinais , Animais , Linhagem Celular Transformada , Receptores ErbB/química , Receptores ErbB/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (â¼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.
Assuntos
Proteínas de Bactérias/química , Glicosiltransferases/química , Pasteurella multocida/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Pasteurella multocida/genética , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/genética , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/genética , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
Testosteronan, an unusual glycosaminoglycan (GAG) first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on ß-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the GAG, heparan sulfate. Studies using (35) S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from (34) S-labeled 6-O-sulfo-N-sulfotestosteroran using (34) S-labeled PAPS because testosteronan was resistant to all of the tested GAG-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of (34) S into â¼14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to â¼20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin (AT) III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to AT III.
Assuntos
Glicosaminoglicanos , Heparitina Sulfato , Glicosaminoglicanos/química , Heparitina Sulfato/química , Espectrometria de Massas , Oligossacarídeos , Ligação Proteica , Especificidade por SubstratoRESUMO
Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope. By exploiting the nanoparticles' light scattering properties, we demonstrated quantitative label-free imaging of intracellular nanoparticles with ultrastructural context. We confirmed the compatibility of two expansion microscopy protocols, protein retention and pan-expansion microscopy, with nanoparticle uptake studies. We validated relative differences between nanoparticle cellular accumulation for various surface modifications using mass spectrometry and determined the intracellular nanoparticle spatial distribution in 3D for entire single cells. This super-resolution imaging platform technology may be broadly used to understand the nanoparticle intracellular fate in fundamental and applied studies to potentially inform the engineering of safer and more effective nanomedicines.
Assuntos
Nanopartículas Metálicas , Animais , Nanopartículas Metálicas/química , Microscopia Eletrônica , Nanomedicina , Espectrometria de Massas , Imageamento Tridimensional , MamíferosRESUMO
Eight N-acetylglucosamine-1-phosphate and N-acetylgalactosamine-1-phosphate analogs have been synthesized chemically and were tested for their recognition by the GlmU uridyltransferase enzyme. Among these, only substrates that have an amide linkage to the C-2 nitrogen were transferred by GlmU to afford their corresponding uridine diphosphate(UDP)-sugar nucleotides. Resin-immobilized GlmU showed comparable activity to nonimmobilized GlmU and provides a more facile final step in the synthesis of an unnatural UDP-donor. The synthesized unnatural UDP-donors were tested for their activity as substrates for glycosyltransferases in the preparation of unnatural glycosaminoglycans in vitro. A subset of these analogs was useful as donors, increasing the synthetic repertoire for these medically important polysaccharides.
Assuntos
Técnicas de Química Sintética/métodos , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Uridina Difosfato N-Acetilglicosamina/análogos & derivados , Uridina Difosfato N-Acetilglicosamina/síntese química , PolimerizaçãoRESUMO
Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond ('S-link') between sugar residues in place of a natural 'O-linked' bond. S-linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S-link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions.
Assuntos
Polímeros , Açúcares , Humanos , Glucuronidase , Enxofre , Compostos de SulfidrilaRESUMO
Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (- Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ - Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.
Assuntos
Glicosaminoglicanos , Ácido Hialurônico , Adesão Celular , Sulfatos de Condroitina , Glicosaminoglicanos/química , Ácido Hialurônico/química , Técnicas de Microbalança de Cristal de QuartzoRESUMO
The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of â¼10-15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.
RESUMO
An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5'-diphosphoiduronic acid (UDP-IdoA) and uridine 5'-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5'-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.
Assuntos
Glucuronosiltransferase/metabolismo , Heparina/síntese química , Ácido Idurônico/análogos & derivados , Ácido Idurônico/química , Açúcares de Uridina Difosfato/síntese química , Heparina/metabolismo , Ácidos Hexurônicos , Ácido Idurônico/síntese química , Metabolismo , Uridina Difosfato Ácido GlucurônicoRESUMO
There is much controversy in the literature regarding the role of p53 status response on hypoxia inducible factor (HIF) signaling in response to chronic relative hypoxia (CRH). The goal of this paper was to methodically examine this response in isogenically matched tumor cells. We report that p53-mutant (MUT) cells, versus p53-wild-type (WT) cells, showed decreased apoptosis, increased cell proliferation with higher basal HIF-1alpha levels in response to CRH. In addition, we found increased HIF-mediated transactivation and increased VEGF release with decreased HIF-1alpha/p53 and HIF-1alpha/MDM-2 partnering in p53-MUT versus p53-WT cells in response to CRH.