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FEMS Immunology and Medical Microbiology Nov 2002Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected...
Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.
Topics: Bacterial Adhesion; Extracellular Matrix; Glycosaminoglycans; Haemophilus influenzae; Heparin; Humans; Lactoferrin; Mannose; Virulence Factors; Vitronectin
PubMed: 12423774
DOI: 10.1111/j.1574-695X.2002.tb00627.x -
Biochemistry Dec 2019Small-angle neutron scattering (SANS) measurements were pursued to study human vitronectin, a protein found in tissues and the circulation that regulates cell...
Small-angle neutron scattering (SANS) measurements were pursued to study human vitronectin, a protein found in tissues and the circulation that regulates cell adhesion/migration and proteolytic cascades that govern hemostasis and pericellular proteolysis. Many of these functions occur via interactions with its binding partner, plasminogen activator inhibitor-1 (PAI-1), the chief inhibitor of proteases that lyse and activate plasminogen. We focused on a region of vitronectin that remains uncharacterized from previous X-ray scattering, nuclear magnetic resonance, and computational modeling approaches and which we propose is involved in binding to PAI-1. This region, which bridges the N-terminal somatomedin B (SMB) domain with a large central β-propeller domain of vitronectin, appears unstructured and has characteristics of an intrinsically disordered domain (IDD). The effect of osmolytes was evaluated using circular dichroism and SANS to explore the potential of the IDD to undergo a disorder-to-order transition. The results suggest that the IDD favors a more ordered structure under osmotic pressure; SANS shows a smaller radius of gyration () and a more compact fold of the IDD upon addition of osmolytes. To test whether PAI-1 binding is also coupled to folding within the IDD structure, a set of SANS experiments with contrast variation were performed on the complex of PAI-1 with a vitronectin fragment corresponding to the N-terminal 130 amino acids (denoted the SMB-IDD because it contains the SMB domain and IDD in linear sequence). Analysis of the SANS data using the Ensemble Optimization Method confirms that the SMB-IDD adopts a more compact configuration when bound to PAI-1. Calculated structures for the PAI-1:SMB-IDD complex suggest that the IDD provides an interaction surface outside of the primary PAI-1-binding site located within the SMB domain; this binding is proposed to lead to the assembly of higher-order structures of vitronectin and PAI-1 commonly found in tissues.
Topics: Intrinsically Disordered Proteins; Models, Molecular; Plasminogen Activator Inhibitor 1; Protein Binding; Protein Domains; Vitronectin
PubMed: 31793295
DOI: 10.1021/acs.biochem.9b00605 -
The Journal of Biological Chemistry May 2008The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion,...
The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.
Topics: Amino Acid Substitution; Cell Adhesion; Cell Line; Cell Movement; Cell Shape; Cytoskeleton; Humans; Integrins; Peptides; Protein Binding; Protein Structure, Tertiary; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Urokinase-Type Plasminogen Activator; Vitronectin
PubMed: 18362146
DOI: 10.1074/jbc.C700214200 -
Cancer Science Aug 2008We have previously demonstrated that pathophysiological shifts in the concentrations of extracellular Mg(2+) and Ca(2+) activate the alpha(2)beta(1) integrin-mediated...
We have previously demonstrated that pathophysiological shifts in the concentrations of extracellular Mg(2+) and Ca(2+) activate the alpha(2)beta(1) integrin-mediated malignant phenotype on type I collagen in pancreatic cancer cells, as evidenced by increased adhesion, migration and proliferation. In the present study, we examined the integrin and divalent cation specificity of pancreatic cancer cell interactions with other physiologically relevant extracellular matrix proteins, including fibronectin, type IV collagen, laminin and vitronectin. Our results indicate that, like alpha(2)beta(1) integrin-mediated interactions with type I collagen, beta(1) integrin-mediated adhesion to fibronectin, type IV collagen and laminin are promoted by Mg(2+) but not by Ca(2+). On vitronectin, cells attach via alpha(v)beta(5) and beta(1) integrins, and in the presence of either divalent cation. We also demonstrate that, like type I collagen, pancreatic cancer cell migration and proliferation on fibronectin, laminin and type IV collagen is maximal when Mg(2+) is present at concentrations that promote optimal adhesion and Ca(2+) is present at concentrations less than Mg(2+). On vitronectin, Panc-1 cell migration is maximal with decreased Mg(2+) and increased Ca(2+), but the reverse is true for BxPC-3 cells. Both cell lines exhibited maximal proliferation with increased Mg(2+) and decreased Ca(2+), however. Together with evidence indicating that the in vivo local tumor microenvironment contains increased Mg(2+) and decreased Ca(2+), our studies demonstrate that such divalent cation shifts could activate the integrin-mediated malignant phenotype in pancreatic cancer.
Topics: Calcium; Cations; Cell Adhesion; Cell Proliferation; Collagen Type IV; Fibronectins; Humans; Integrin alpha2beta1; Integrin beta1; Integrins; Laminin; Magnesium; Pancreatic Neoplasms; Phenotype; Tumor Cells, Cultured; Vitronectin
PubMed: 18754866
DOI: 10.1111/j.1349-7006.2008.00855.x -
BioTechniques Jun 2014The secreted adhesive glycoprotein vitronectin (VTN) is a multifunctional component of plasma and the extracellular matrix. A high-yielding, inexpensive, low endotoxin...
The secreted adhesive glycoprotein vitronectin (VTN) is a multifunctional component of plasma and the extracellular matrix. A high-yielding, inexpensive, low endotoxin source of bioactive recombinant human vitronectin (rhVTN) is highly desirable for in vitro use in diverse cell culture systems ranging from basic research settings to clinical-grade production of human cells. We describe modifications to a previously reported heparin-based affinity chromatography procedure that improve yield and achieve efficient removal of endotoxin from washed and urea-solubilized human VTN inclusion bodies following standard autoinduction of expression in Escherichia coli. This simple procedure makes accessible the low-cost expression and purification of large quantities of bioactive rhVTN using basic equipment and facilitates its use in a spectrum of endotoxin-sensitive applications.
Topics: Chromatography, Affinity; Endotoxins; Escherichia coli; Gene Expression; Human Umbilical Vein Endothelial Cells; Humans; Inclusion Bodies; Recombinant Proteins; Vitronectin
PubMed: 24924394
DOI: 10.2144/000114181 -
Gastroenterology Jan 2010Insulin-like growth factor-I (IGF-I) regulates human intestinal smooth muscle growth by stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is...
BACKGROUND & AIMS
Insulin-like growth factor-I (IGF-I) regulates human intestinal smooth muscle growth by stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is augmented when alphaVbeta3 integrin is occupied by its ligands, fibronectin and vitronectin. Increased IGF-I expression and muscle cell hyperplasia are features of stricturing Crohn's disease (CD); however, the role of IGF-I in stricture formation is unknown. The aim was to identify the functional role of endogenous IGF-I and alphaVbeta3 integrin ligands in regulating muscle cell hyperplasia in stricturing CD.
METHODS
Smooth muscle cells were isolated from muscularis propria of stricturing CD or normal margins. Quantitative polymerase chain reaction, immunoblot analysis, and enzyme-linked immunosorbent assay were used to measure fibronectin, vitronectin, alphaVbeta3 integrin, and IGF-I levels. Activation of the IGF-I receptor, Erk1/2, p70S6 kinase, and GSK-3beta was measured by immunoblot. Proliferation was quantified by Ki67 immunostaining and [(3)H]thymidine incorporation. Apoptosis was measured from caspase-3 cleavage and nucleosome accumulation.
RESULTS
IGF-I, vitronectin, and fibronectin RNA and protein levels were increased 1.8- to 3.4-fold in muscle cells from strictures over normal margins. Basal IGF-I receptor phosphorylation was increased 320% in strictured over normal muscle, and basal Erk1/2, p70S6 kinase, and GSK-3beta phosphorylation were increased 205%-292% in strictures. In muscle cells from strictures, Ki67 immunoreactivity and [(3)H]thymidine incorporation were increased and apoptosis was decreased compared with normal margins. Antagonists of the IGF-I receptor or alphaVbeta3 integrin reversed these changes.
CONCLUSIONS
Smooth muscle cell hyperplasia in stricturing CD is regulated by increased endogenous IGF-I and alphaVbeta3 integrin ligands that regulate augmented proliferation and diminished apoptosis.
Topics: Adult; Cell Division; Crohn Disease; Extracellular Signal-Regulated MAP Kinases; Female; Fibronectins; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hyperplasia; Insulin-Like Growth Factor I; Integrin alphaVbeta3; Ki-67 Antigen; Ligands; MAP Kinase Signaling System; Male; Middle Aged; Muscle, Smooth; Myocytes, Smooth Muscle; Phosphorylation; Vitronectin; Young Adult
PubMed: 19751734
DOI: 10.1053/j.gastro.2009.09.003 -
Journal of Biological Inorganic... Oct 2017Components of the fibrinolytic system are subjected to stringent control to maintain proper hemostasis. Central to this regulation is the serpin plasminogen activator...
Components of the fibrinolytic system are subjected to stringent control to maintain proper hemostasis. Central to this regulation is the serpin plasminogen activator inhibitor-1 (PAI-1), which is responsible for specific and rapid inhibition of fibrinolytic proteases. Active PAI-1 is inherently unstable and readily converts to a latent, inactive form. The binding of vitronectin and other ligands influences stability of active PAI-1. Our laboratory recently observed reciprocal effects on the stability of active PAI-1 in the presence of transition metals, such as copper, depending on the whether vitronectin was also present (Thompson et al. Protein Sci 20:353-365, 2011). To better understand the molecular basis for these copper effects on PAI-1, we have developed a gel-based copper sensitivity assay that can be used to assess the copper concentrations that accelerate the conversion of active PAI-1 to a latent form. The copper sensitivity of wild-type PAI-1 was compared with variants lacking N-terminal histidine residues hypothesized to be involved in copper binding. In these PAI-1 variants, we observed significant differences in copper sensitivity, and these data were corroborated by latency conversion kinetics and thermodynamics of copper binding by isothermal titration calorimetry. These studies identified a copper-binding site involving histidines at positions 2 and 3 that confers a remarkable stabilization of PAI-1 beyond what is observed with vitronectin alone. A second site, independent from the two histidines, binds metal and increases the rate of the latency conversion.
Topics: Binding Sites; Copper; Histidine; Humans; Kinetics; Models, Molecular; Plasminogen Activator Inhibitor 1; Protein Binding; Protein Conformation; Protein Stability; Vitronectin
PubMed: 28913669
DOI: 10.1007/s00775-017-1489-5 -
International Journal of Molecular... Jan 2024and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily...
Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of and Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.
and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of and . GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in and , respectively.
Topics: Humans; Candida albicans; Glyceraldehyde-3-Phosphate Dehydrogenases; Plasminogen; Vitronectin; Fungal Proteins
PubMed: 38256088
DOI: 10.3390/ijms25021013 -
Molecular Vision Oct 2006Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in...
PURPOSE
Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in wound healing, has been detected in PCO specimens. Here we set out to investigate the distribution of vitronectin in the lens and determine how it, and other ECM components, influence the lens epithelial phenotype.
METHODS
Rat lens epithelial explants were cultured on vitronectin, fibronectin, and laminin substrata. Explants were monitored for cell migration and the appearance of markers for epithelial mesenchymal transition (EMT), using phase contrast microscopy and immunohistochemistry, respectively. Explants were also monitored for evidence of Smad signaling. Vitronectin expression was analyzed in embryonic and postnatal rodent lens development by immunohistochemistry, western blotting, and in situ hybridization.
RESULTS
Vitronectin, like fibronectin and laminin, provided a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by a major phenotypic change. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped and were strongly reactive for filamentous alpha-smooth muscle actin. In these respects this transition was typical of the well known TGFbeta-induced EMT. In explants cultured on vitronectin and fibronectin, but not laminin, cell nuclei showed prominent reactivity for Smad 2/3. Vitronectin was also shown to be expressed during embryonic and postnatal development. Initially mRNA and protein were detected in all lens cells, however as development progressed, expression became restricted to cells of the epithelium and transition zone.
CONCLUSIONS
The results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect and that Smad 2/3 signaling is involved. Thus when considering strategies to slow or prevent PCO, these results highlight the need to take into account ECM molecules such as vitronectin that have the capacity to promote EMT.
Topics: Animals; Animals, Newborn; Embryo, Mammalian; Epithelial Cells; Fibronectins; In Vitro Techniques; Laminin; Lens Capsule, Crystalline; Mesoderm; Mice; Rats; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Vitronectin
PubMed: 17110906
DOI: No ID Found -
Proceedings of the National Academy of... Feb 2010Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not...
Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFbeta, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance.
Topics: Adipose Tissue; Animals; Chimera; Coculture Techniques; Fibroblast Growth Factors; Fibroblasts; Fibronectins; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Inbred C57BL; Transforming Growth Factor alpha; Vitronectin
PubMed: 20133714
DOI: 10.1073/pnas.0910172106