RESUMEN
Hydrolysis of VLDL triacylglycerol (TG) by lipoprotein lipase (LpL) is a major step in energy metabolism and VLDL-to-LDL maturation. Most functional LpL is anchored to the vascular endothelium, yet a small amount circulates on TG-rich lipoproteins. As circulating LpL has low catalytic activity, its role in VLDL remodeling is unclear. We use pre-heparin plasma and heparin-sepharose affinity chromatography to isolate VLDL fractions from normolipidemic, hypertriglyceridemic, or type-2 diabetic subjects. LpL is detected only in the heparin-bound fraction. Transient binding to heparin activates this VLDL-associated LpL, which hydrolyses TG, leading to gradual VLDL remodeling into IDL/LDL and HDL-size particles. The products and the timeframe of this remodeling closely resemble VLDL-to-LDL maturation in vivo. Importantly, the VLDL fraction that does not bind heparin is not remodeled. This relatively inert LpL-free VLDL is rich in TG and apoC-III, poor in apoE and apoC-II, shows impaired functionality as a substrate for the exogenous LpL or CETP, and likely has prolonged residence time in blood, which is expected to promote atherogenesis. This non-bound VLDL fraction increases in hypertriglyceridemia and in type-2 diabetes but decreases upon diabetes treatment that restores the glycemic control. In stark contrast, heparin binding by LDL increases in type-2 diabetes triggering pro-atherogenic LDL modifications. Therefore, the effects of heparin binding are associated negatively with atherogenesis for VLDL but positively for LDL. Collectively, the results reveal that binding to glycosaminoglycans initiates VLDL remodeling by circulating LpL, and suggest heparin binding as a marker of VLDL functionality and a readout for treatment of metabolic disorders.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Hipertrigliceridemia/genética , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/genética , Diabetes Mellitus Tipo 2/metabolismo , Endotelio Vascular/metabolismo , Metabolismo Energético/genética , Heparina/genética , Heparina/metabolismo , Humanos , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/patología , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Lipoproteínas LDL/genética , Triglicéridos/genéticaRESUMEN
Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.
Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Proteoglicanos de Heparán Sulfato/farmacología , Degeneración Macular/tratamiento farmacológico , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/química , Animales , Proliferación Celular/efectos de los fármacos , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Cristalografía por Rayos X , Células Endoteliales/efectos de los fármacos , Ojo/efectos de los fármacos , Ojo/patología , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/inmunología , Heparina/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/ultraestructura , Inyecciones Intravítreas , Degeneración Macular/genética , Degeneración Macular/patología , Ratones , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/ultraestructura , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Cuerpo Vítreo/efectos de los fármacosRESUMEN
Type V collagen (ColV) is a component of the endothelial basement membrane zone. During angiogenesis, extracellular matrix remodelling results in the release of active protein fragments that display pro- or anti-angiogenic properties. The latter often exert their activity through their heparin-binding site. We previously characterized a ColVα1-derived fragment called HEPV that contains a high affinity-binding site for heparin and heparan sulphate chains. Here we show that HEPV binds to FGF2 through its heparin-binding site. Using in vitro and in vivo angiogenesis assays, we show that HEPV but not the HEPV mutant at the heparin-binding site, inhibits FGF2-dependant angiogenesis. On the opposite, HEPV does not bind to VEGFA and has no effect on VEGFA-mediated angiogenesis. In 3D collagen gels, the addition of HEPV abrogates endothelial cell invasion and sprouting induced by FGF2. Interestingly, in vivo experiments reveal that HEPV anti-angiogenic activity is associated with the appearance of endothelial to mesenchymal transition (EndMT) markers. Together, these findings indicate that the ColVα1-derived fragment HEPV functions as an anti-angiogenic factor that represses FGF2-mediated angiogenesis through the regulation of endothelial cell plasticity. Previous observations showing that ColV overexpression negatively regulates pathological angiogenesis were left unexplained. Our data provide insights into the possible molecular mechanisms.
Asunto(s)
Colágeno Tipo V/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Morfogénesis/genética , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/genética , Secuencia de Aminoácidos/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Plasticidad de la Célula/genética , Células Endoteliales/efectos de los fármacos , Heparina/genética , Heparitina Sulfato/genética , Xenoinjertos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Morfogénesis/efectos de los fármacos , Neovascularización Patológica/patología , Unión Proteica/genéticaRESUMEN
Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Heparina/metabolismo , Heparitina Sulfato/biosíntesis , Animales , Anticoagulantes , Línea Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Regulación de la Expresión Génica/genética , Células HeLa , Heparina/biosíntesis , Heparina/genética , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mastocitos/metabolismo , Sulfotransferasas/metabolismo , Porcinos , Factores de TranscripciónRESUMEN
Heparin-induced thrombocytopenia is a relatively common drug-induced immune disorder that can have life-threatening consequences for affected patients. Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive Abs are central to the pathogenesis of heparin-induced thrombocytopenia. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that play a key role in regulating immune responses, but their role in controlling PF4/heparin-specific Ab production is unknown. In the studies described in this article, we found that Foxp3-deficient mice lacking functional Treg cells spontaneously produced PF4/heparin-specific Abs. Following transplantation with bone marrow cells from Foxp3-deficient but not wild-type mice, Rag1-deficient recipients also produced PF4/heparin-specific Abs spontaneously. Adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific Abs in Foxp3-deficient mice and inhibited PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune responses mainly through releasing anti-inflammatory cytokines, such as IL-10. IL-10-deficient mice spontaneously produced PF4/heparin-specific Abs. Moreover, bone marrow chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production upon PF4/heparin complex challenge. Short-term IL-10 administration suppresses PF4/heparin-specific IgG production in wild-type mice. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific Ab production.
Asunto(s)
Formación de Anticuerpos , Heparina/inmunología , Inmunoglobulina G/inmunología , Factor Plaquetario 4/inmunología , Linfocitos T Reguladores/inmunología , Animales , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/inmunología , Heparina/genética , Inmunoglobulina G/genética , Interleucina-10/deficiencia , Interleucina-10/inmunología , Ratones , Ratones Noqueados , Factor Plaquetario 4/genética , Linfocitos T Reguladores/citologíaRESUMEN
Polydeoxyadenosine (poly (dA)) has been extensively applied for detecting many drug molecules. Herein, we developed a sensitive method for detecting coralyne and heparin using a modified DNA probe with poly (dA) at one end. In the absence of coralyne, the DNA probe was digested by the Exonuclease I (Exo I), and therefore the SYBR Green I (SG I) emitted an extremely low fluorescent signal. While coralyne specifically binding to poly (dA) with strong propensity could remarkably restrain the disintegration of the DNA probe, through which as a template the second strand of DNA sequence was formed with the introduction of DNA polymerase. Therefore, the fluorescent signal of SG I was intensified to quantify coralyne. Based on this method, heparin can be determined due to its strong affinity towards coralyne. This method showed a linear range from 2 to 500â¯nM for coralyne with a low detection limit of 0.98â¯nM, and the linear range of heparin was from 1 to 100â¯nM when 1.25â¯nm was the detection limit. The proposed method was also implemented successfully in biological samples and showed a potential application for screening potential therapeutic molecules.
Asunto(s)
Alcaloides de Berberina/aislamiento & purificación , Técnicas Biosensibles , Exodesoxirribonucleasas/genética , Heparina/aislamiento & purificación , Benzotiazoles , Alcaloides de Berberina/química , ADN/química , Sondas de ADN/química , Sondas de ADN/genética , Desoxiadenosinas/química , Desoxiadenosinas/genética , Diaminas , Exodesoxirribonucleasas/química , Heparina/química , Heparina/genética , Humanos , Límite de Detección , Compuestos Orgánicos/química , QuinolinasRESUMEN
CXCL14, chemokine (C-X-C motif) ligand 14, is a novel highly conserved chemokine with unique features. Despite exhibiting the typical chemokine fold, it has a very short N-terminus of just two amino acid residues responsible for chemokine receptor activation. CXCL14 actively participates in homeostatic immune surveillance of skin and mucosae, is linked to metabolic disorders and fibrotic lung diseases and possesses strong anti-angiogenic properties in early tumor development. In this work, we investigated the interaction of CXCL14 with various glycosaminoglycans (GAGs) by nuclear magnetic resonance spectroscopy, microscale thermophoresis, analytical heparin (HE) affinity chromatography and in silico approaches to understand the molecular basis of GAG-binding. We observed different GAG-binding modes specific for the GAG type used in the study. In particular, the CXCL14 epitope for HE suggests a binding pose distinguishable from the ones of the other GAGs investigated (hyaluronic acid, chondroitin sulfate-A/C, -D, dermatan sulfate). This observation is also supported by computational methods that included molecular docking, molecular dynamics and free energy calculations. Based on our results, we suggest that distinct GAG sulfation patterns confer specificity beyond simple electrostatic interactions usually considered to represent the driving forces in protein-GAG interactions. The CXCL14-GAG system represents a promising approach to investigate the specificity of GAG-protein interactions, which represents an important topic for developing the rational approaches to novel strategies in regenerative medicine.
Asunto(s)
Quimiocinas CXC/metabolismo , Epítopos/genética , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Sitios de Unión/genética , Quimiocinas CXC/química , Quimiocinas CXC/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Dermatán Sulfato/química , Dermatán Sulfato/genética , Epítopos/química , Glicosaminoglicanos/química , Glicosaminoglicanos/genética , Heparina/genética , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica/genética , Pliegue de ProteínaRESUMEN
Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, driven by changes in communication between cells. Heparin-binding proteins (HBPs) play a central role in health and diseases. Therefore, we used heparin affinity proteomics to identify extracellular HBPs in pancreas and plasma of normal mice and in a caerulein mouse model of AP. Many new extracellular HBPs (360) were discovered in the pancreas, taking the total number of HBPs known to 786. Extracellular pancreas HBPs form highly interconnected protein-protein interaction networks in both normal pancreas (NP) and AP. Thus, HBPs represent an important set of extracellular proteins with significant regulatory potential in the pancreas. HBPs in NP are associated with biological functions such as molecular transport and cellular movement that underlie pancreatic homeostasis. However, in AP HBPs are associated with additional inflammatory processes such as acute phase response signalling, complement activation and mitochondrial dysfunction, which has a central role in the development of AP. Plasma HBPs in AP included known AP biomarkers such as serum amyloid A, as well as emerging targets such as histone H2A. Other HBPs such as alpha 2-HS glycoprotein (AHSG) and histidine-rich glycoprotein (HRG) need further investigation for potential applications in the management of AP. Pancreas HBPs are extracellular and so easily accessible and are potential drug targets in AP, whereas plasma HBPs represent potential biomarkers for AP. Thus, their identification paves the way to determine which HBPs may have potential applications in the management of AP.
Asunto(s)
Biomarcadores/sangre , Pancreatitis/genética , Proteoma/genética , alfa-2-Glicoproteína-HS/genética , Animales , Modelos Animales de Enfermedad , Heparina/genética , Homeostasis , Humanos , Ratones , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/sangre , Pancreatitis/patología , Unión Proteica/genética , Proteínas/genética , Proteómica/métodos , Proteína Amiloide A Sérica/metabolismoRESUMEN
Advanced fluorescence techniques, commonly known as the F-techniques, measure the kinetics and the interactions of biomolecules with high sensitivity and spatiotemporal resolution. Applications of the F-techniques, which were initially limited to cells, were further extended to study in vivo protein organization and dynamics in whole organisms. The integration of F-techniques with multi-photon microscopy and light-sheet microscopy widened their applications in the field of developmental biology. It became possible to penetrate the thick tissues of living organisms and obtain good signal-to-noise ratio with reduced photo-induced toxicity. In this review, we discuss the principle and the applications of the three most commonly used F-techniques in developmental biology: Fluorescence Recovery After Photo-bleaching (FRAP), Fo¨ rster Resonance Energy Transfer (FRET), and Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS).
Asunto(s)
Caenorhabditis elegans/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Espectrometría de Fluorescencia/métodos , Xenopus laevis/metabolismo , Pez Cebra/metabolismo , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/ultraestructura , Biología Evolutiva/instrumentación , Biología Evolutiva/métodos , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/ultraestructura , Embrión no Mamífero/ultraestructura , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Transferencia Resonante de Energía de Fluorescencia , Regulación del Desarrollo de la Expresión Génica , Heparina/análogos & derivados , Heparina/genética , Heparina/metabolismo , Proteoglicanos/genética , Proteoglicanos/metabolismo , Espectrometría de Fluorescencia/instrumentación , Xenopus laevis/crecimiento & desarrollo , Pez Cebra/crecimiento & desarrolloRESUMEN
Neurons sometimes completely fill available space in their receptive fields with evenly spaced dendrites to uniformly sample sensory or synaptic information. The mechanisms that enable neurons to sense and innervate all space in their target tissues are poorly understood. Using Drosophila somatosensory neurons as a model, we show that heparan sulfate proteoglycans (HSPGs) Dally and Syndecan on the surface of epidermal cells act as local permissive signals for the dendritic growth and maintenance of space-filling nociceptive C4da neurons, allowing them to innervate the entire skin. Using long-term time-lapse imaging with intact Drosophila larvae, we found that dendrites grow into HSPG-deficient areas but fail to stay there. HSPGs are necessary to stabilize microtubules in newly formed high-order dendrites. In contrast to C4da neurons, non-space-filling sensory neurons that develop in the same microenvironment do not rely on HSPGs for their dendritic growth. Furthermore, HSPGs do not act by transporting extracellular diffusible ligands or require leukocyte antigen-related (Lar), a receptor protein tyrosine phosphatase (RPTP) and the only known Drosophila HSPG receptor, for promoting dendritic growth of space-filling neurons. Interestingly, another RPTP, Ptp69D, promotes dendritic growth of C4da neurons in parallel to HSPGs. Together, our data reveal an HSPG-dependent pathway that specifically allows dendrites of space-filling neurons to innervate all target tissues in Drosophila.
Asunto(s)
Dendritas/metabolismo , Proteínas de Drosophila/metabolismo , Heparina/análogos & derivados , Nociceptores/metabolismo , Proteoglicanos/metabolismo , Proteínas Tirosina Fosfatasas Similares a Receptores/metabolismo , Transducción de Señal , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Heparina/genética , Heparina/metabolismo , Nociceptores/citología , Proteoglicanos/genética , Proteínas Tirosina Fosfatasas Similares a Receptores/genéticaRESUMEN
Heparin has been used clinically as an anti-coagulant for more than 100 y and the major source of this therapeutic is still animal tissues. Contamination issues in some batches of heparin over 10 y ago have highlighted the need to develop alternative methods of production of this essential drug. 1 Bioengineering heparin by expressing serglycin in mammalian cells is a promising approach that was recently reported by the authors. 2 This addendum explores the approaches that the authors are taking to increase the yield of recombinantly expressed serglycin decorated with heparin/heparan sulfate focusing on cell culture and bioreactor conditions and proposes that the cell microenvironment is a key modulator of heparin biosynthesis.
Asunto(s)
Mejoramiento Genético/métodos , Glucosa/metabolismo , Heparina/biosíntesis , Heparitina Sulfato/biosíntesis , Proteoglicanos/genética , Proteínas de Transporte Vesicular/genética , Células HEK293 , Heparina/genética , Heparina/aislamiento & purificación , Heparitina Sulfato/genética , Heparitina Sulfato/aislamiento & purificación , Humanos , Proteoglicanos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Å resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of â¼10 nm involving â¼8 monosaccharides and â¼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m-1 s-1, its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates.
Asunto(s)
Angiotensinas/química , Proteínas de Peces/química , Heparina/química , Lampreas , Trombina/química , Angiotensinas/genética , Angiotensinas/metabolismo , Animales , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Heparina/genética , Heparina/metabolismo , Mutación , Dominios Proteicos , Estructura Secundaria de Proteína , Trombina/genética , Trombina/metabolismoRESUMEN
Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Ingeniería Genética/métodos , Heparina/análogos & derivados , Proteoglicanos/administración & dosificación , Proteoglicanos/biosíntesis , Proteínas de Transporte Vesicular/administración & dosificación , Proteínas de Transporte Vesicular/biosíntesis , Anticoagulantes/administración & dosificación , Anticoagulantes/metabolismo , Células HEK293 , Heparina/administración & dosificación , Heparina/biosíntesis , Heparina/genética , Humanos , Ingeniería Metabólica , Proteoglicanos/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
PRELP (proline/arginine-rich end leucine-rich repeat protein) is a member of the leucine-rich repeat (LRR) family of extracellular matrix proteins in connective tissue. In contrast with other members of the family, the N-terminal domain of PRELP has a high content of proline and positively charged amino acids. This domain has previously been shown to bind chondrocytes and to inhibit osteoclast differentiation. In the present study, we show that PRELP mediates cell adhesion by binding to cell-surface glycosaminoglycans (GAGs). Thus, rat skin fibroblasts (RSFs) bound to full-length PRELP and to the N-terminal part of PRELP alone, but not to truncated PRELP lacking the positively charged N-terminal region. Cell attachment to PRELP was inhibited by addition of soluble heparin or heparan sulfate (HS), by blocking sulfation of the fibroblasts or by treating the cells with a combination of chondroitinase and heparinase. Using affinity chromatography, we identified syndecan-1, syndecan-4 and glypican-1 as cell-surface proteoglycans (PGs) binding to the N-terminal part of PRELP. Finally, we show that the N-terminal domain of PRELP in combination with the integrin-binding domain of fibronectin, but neither of the fragments alone, induced fibroblast focal adhesion formation. These findings provide support for a role of the N-terminal region of PRELP as an important regulator of cell adhesion and behaviour, which may be of importance in pathological conditions.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Glicoproteínas/metabolismo , Proteoglicanos/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas de la Matriz Extracelular/genética , Adhesiones Focales/genética , Glicoproteínas/genética , Células HEK293 , Heparina/genética , Heparina/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Dominios Proteicos , Proteoglicanos/genética , RatasRESUMEN
BACKGROUND: Antithrombin (AT) is a key regulator of the coagulation. In type II deficiency, the heparin-binding-site defect (type II HBS) is considered to be relatively low thrombosis risk. OBJECTIVES: Our aims were to search for SERPINC1 mutation(s) and to describe the clinical and laboratory phenotype of a large number of AT Budapest3 (ATBp3, p.Leu131Phe) carriers and confirm the presence of a founder effect. PATIENTS/METHODS: AT-deficient patients were recruited and carriers of ATBp3, n = 102 (63 families) were selected. To investigate the founder effect, eight intragenic single nucleotide polymorphisms, a 5'-length dimorphism, and five microsatellite markers were detected. Clinical and laboratory data of the patients were collected and analyzed. RESULTS: In AT deficiency, 16 different causative mutations were found, and the great majority of patients were of type II HBS subtype. Most of them (n = 102/118, 86.5%) carried the ATBp3 mutation. The ATBp3 mutant allele was associated with one single haplotype, while different haplotypes were detected in the case of normal allele. The anti-factor Xa-based AT activity assay that we used could detect all ATBp3 patients with high sensitivity in our cohort. ATBp3 homozygosity (n = 26) was associated with thrombosis at a young age and conferred a high thrombotic risk. Half of the heterozygotes (n = 41/76, 53.9%) also had venous and/or arterial thrombosis, and pregnancy complications were also recorded. CONCLUSION: In Hungary, the founder mutation, ATBp3, is the most common AT deficiency. Our study is the first in which the clinical characterization of ATBp3 mutation was executed in a large population.
Asunto(s)
Antitrombinas/química , Efecto Fundador , Heparina/genética , Leucina/genética , Mutación , Fenilalanina/genética , Adolescente , Adulto , Anciano , Arterias/fisiopatología , Sitios de Unión , Niño , Preescolar , Estudios de Cohortes , Factor Xa/genética , Femenino , Estudios de Asociación Genética , Heterocigoto , Humanos , Hungría , Repeticiones de Microsatélite , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple , Embarazo , Complicaciones Cardiovasculares del Embarazo , Sensibilidad y Especificidad , Trombosis/fisiopatología , Adulto JovenRESUMEN
AIMS: One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large-scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. METHODS AND RESULTS: The 6-O-sulfotransferase isoform-3 (6-OST-3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria-Bertani medium in shake flasks and inducing with isopropyl ß-D-1-thiogalactopyranoside at an optical density of 0·6-0·8. The production of larger amounts of 6-OST-3 required fed-batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5-20 mg g-cell-dry weight(-1)) of active 6-OST-3 with excellent productivity (2-5 mg l(-1) h(-1)). CONCLUSIONS: The production of 6-OST-3 in a fed-batch fermentation on an inexpensive carbon source has been demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to scale-up the production of heparin biosynthetic enzymes, such as 6-OST-3, is critical for scaling-up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal-sourced anticoagulant product currently on the market.
Asunto(s)
Anticoagulantes/metabolismo , Escherichia coli/genética , Heparina/biosíntesis , Sulfotransferasas/metabolismo , Técnicas de Cultivo Celular por Lotes/métodos , Escherichia coli/metabolismo , Fermentación , Heparina/genética , Proteínas Recombinantes de Fusión/metabolismo , Sulfotransferasas/genéticaRESUMEN
NMR and density functional theory (DFT) have afforded detailed information on the molecular geometry and spin-spin coupling constants of a trisaccharide from the heparin repeating-sequence. The fully optimized molecular structures of two trisaccharide conformations (differing from each other in the form of the central iduronic acid residue) were obtained using the B3LYP/6-311+G(d,p) level of theory in the presence of solvent, the latter included as either explicit water molecules or via a continuum solvent model. NMR spin-spin coupling constants were also computed using various basis sets and functionals and then compared with measured experimental values. Optimized structures for both conformers showed differences in geometry at the glycosidic linkages and in the formation of intramolecular hydrogen bonds. Three-bond proton-proton coupling constants ((3)JH-C-C-H), based on fully optimized geometry computed using the B3LYP/6-311+G(d,p)/UFF level of theory and hydrated with 57 water molecules, showed that the best agreement with experiment was obtained with the 6-311+G(d,p) basis set and a weighted average of 55:45 ((1)C4:(2)S0) of the IdoA2S forms. Other basis sets, DGDZVP and TZVP, also gave acceptable data for most coupling constants, with DGDZVP outperforming the TZVP. Detailed analysis of Fermi-contact contributions to (3)JH-C-C-H showed that important contributions arise from oxygen at both glycosidic linkages, as well as from oxygen atoms on the neighboring monosaccharide units. Their contribution to the Fermi term cannot be neglected and must be taken into account for a correct description of coupling constants. The analysis also showed that the magnitude of paramagnetic (PSO) and diamagnetic (DSO) spin-orbit contributions is comparable to the magnitude of the Fermi-contact contribution in some coupling constants in the IdoA2S residue. Calculations of the localized molecular orbital contributions to the DSO terms from separate conformational residues showed that the contribution from adjacent residues is not negligible and can be important for the spin-spin coupling constants between protons located close to the geometrical center of the molecule. These contributions should be taken into account when interpreting DSO terms in spin-spin coupling constants especially in large molecules.
Asunto(s)
Heparina/química , Trisacáridos/química , Heparina/genética , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Químicos , Estructura Molecular , Protones , Sodio/química , Solventes/química , Trisacáridos/genética , Agua/químicaRESUMEN
Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd=26.47±5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd=24.66±5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Heparina/genética , Heparitina Sulfato/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Tropoelastina/metabolismo , Animales , Células Cultivadas , Tejido Elástico/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/genética , Humanos , Ratones , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Tropoelastina/genéticaRESUMEN
We have previously shown that following traumatic brain injury (TBI) the presence of the amyloid precursor protein (APP) may be neuroprotective. APP knockout mice have increased neuronal death and worse cognitive and motor outcomes following TBI, which is rescued by treatment with exogenous sAPPα (the secreted ectodomain of APP generated by α-secretase cleavage). Two neuroprotective regions were identified in sAPPα, the N and C-terminal domains D1 and D6a/E2 respectively. As both D1 and D6a/E2 contain heparin binding activity it was hypothesized that this is responsible for the neuroprotective activity. In this study, we focused on the heparin binding site, encompassed by residues 96-110 in D1, which has previously been shown to have neurotrophic properties. We found that treatment with APP96-110 rescued motor and cognitive deficits in APP-/- mice following focal TBI. APP96-110 also provided neuroprotection in Sprague-Dawley rats following diffuse TBI. Treatment with APP96-110 significantly improved functional outcome as well as preserve histological cellular morphology in APP-/- mice following focal controlled cortical impact injury. Furthermore, following administration of APP96-110 in rats after diffuse impact acceleration TBI, motor and cognitive outcomes were significantly improved and axonal injury reduced. These data define the heparin binding site in the D1 domain of sAPPα, represented by the sequence APP96-110, as the neuroprotective site to confer neuroprotection following TBI. The product of α-secretase cleavage of the amyloid precursor protein, sAPPα is neuroprotective following traumatic brain injury (TBI). Of interest was whether this neuroprotective activity could be further delineated to a heparin binding region within sAPPα, corresponding to the region APP96-110 (see diagram demonstrating the domain structure of sAPPα). Indeed treatment with APP96-110 improved functional outcome following TBI, an effect that was not seen with a mutated version of the peptide that had reduced heparin binding affinity.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/prevención & control , Heparina/metabolismo , Fármacos Neuroprotectores/metabolismo , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/uso terapéutico , Animales , Sitios de Unión/fisiología , Heparina/química , Heparina/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fármacos Neuroprotectores/uso terapéutico , Estructura Terciaria de Proteína , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.