Mussel adhesive Protein-conjugated Vitronectin (fp-151-VT) Induces Anti-inflammatory Activity on LPS-stimulated Macrophages and UVB-irradiated Keratinocytes.

BACKGROUND: Skin inflammation and dermal injuries are a major clinical problem because current therapies are limited to treating established scars, and there is a poor understanding of healing mechanisms. Mussel adhesive proteins (MAPs) have great potential in many tissue engineering and biomedical applications. It has been successfully demonstrated that the redesigned hybrid type MAP (fp-151) can be utilized as a promising adhesive biomaterial. The aim of this study was to develop a novel recombinant protein using fp-151 and vitronectin (VT) and to elucidate the anti-inflammatory effects of this recombinant protein on macrophages and keratinocytes. METHODS: Lipopolysaccharide (LPS) was used to stimulate macrophages and UVB was used to stimulate keratinocytes. Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot. Inflammatory cytokines and NO and ROS production were analyzed. RESULT: In macrophages stimulated by LPS, expression of the inflammatory factors iNOS, COX-2, and NO production increased, while the r-fp-151-VT-treated groups had suppressed expression of iNOS, COX-2, and NO production in a dose-dependent manner. In addition, keratinocytes stimulated by UVB and treated with r-fp-151-VT had reduced expression of iNOS and COX-2. Interestingly, in UVB-irradiated keratinocytes, inflammatory cytokines, such as interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a, were significantly reduced by r-fp-151-VT treatment. CONCLUSIONS: These results suggest that the anti-inflammatory activity of r-fp-151-VT was more effective in keratinocytes, suggesting that it can be used as a therapeutic agent to treat skin inflammation.

Dynamic contact angle analysis of protein adsorption on polysaccharide multilayer's films for biomaterial reendothelialization.

Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte's multilayer (PEM) films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE) as polycation and dextran sulphate (DS) as polyanion. One film was composed with 4 bilayers of (DEAE-DS)4 and was labeled D-. The other film was the same as D- but with an added terminal layer of DEAE polycation: (DEAE-DS)4-DEAE (labeled D+). The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA) and atomic force microscopy (AFM). Human endothelial cell (HUVEC) adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA). Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS)4 films. (DEAE-DS)4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation.

Expression and purification of bioactive, low-endotoxin recombinant human vitronectin.

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.

The effect of elastomer chain flexibility on protein adsorption.

Cells are known to respond differently when grown on materials of varying stiffness. However, the mechanism by which a cell senses substrate stiffness is unknown. Lower crosslink density elastomers formed from acrylated star-poly(d,l lactide-co-ϵ-caprolactone) have previously been shown to support higher smooth muscle cell proliferation in in vitro culture. This difference in growth was hypothesized to be due to differences in protein adsorption that resulted from differences in polymer chain mobility at the surface. Therefore, layer mass and viscoelastic properties were measured for HSA, IgG, fibronectin, vitronectin, and serum supplemented media adsorbed to elastomers of two crosslink densities. Significantly more fibronectin adsorbed to the lower crosslink density surface while significantly more IgG adsorbed to the higher crosslink density surface. Furthermore, differences in fibronectin and IgG layer shear moduli were observed, suggesting that there was a difference in the conformation of the adsorbed protein. ATR-FTIR analysis showed that the lower crosslink density elastomer absorbed more surface water. The increased amount of water may cause greater entropic gains upon protein adsorption to the lower crosslink density surface, which increases total protein adsorption from serum and may cause differences in protein conformation and thus cell behavior.

Purification of vitronectin.

This unit describes the purification of extracellular vitronectin from plasma or serum by using heparin-affinity chromatography. First, the plasma is depleted of fibronectin plus other heparin- and Sepharose-binding proteins and treated with urea to activate the heparin-binding activity of vitronectin, which is subsequently bound to a heparin affinity column and eluted. The resulting vitronectin should be ∼ 98% pure.

Targeted mass spectrometric approach for biomarker discovery and validation with nonglycosylated tryptic peptides from N-linked glycoproteins in human plasma.

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.

Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading.

Vitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin alphavbeta3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.

One-step purification of vitronectin from human plasma by affinity chromatography on phage-displayed peptides.

A novel affinity purification method for rapid isolation of vitronectin (VN) from human plasma is described. Recently we have used phage display technology to obtain clones expressing peptides with high binding activity for VN. The isolated "strong VN binders" were covalently coupled to CNBr-activated Sepharose. Human plasma was applied to the column and bound VN was eluted using 0.5 M acetic acid, giving purity exceeding 90%. The developed method is a convenient alternative to conventional antibody-antigen affinity chromatography techniques for purification of VN, as it offers low ligand cost, is rapid and ensures good protein recovery from human plasma.

PAI1 stimulates assembly of the fibronectin matrix in osteosarcoma cells through crosstalk between the alphavbeta5 and alpha5beta1 integrins.

The plasminogen activation system regulates matrix remodeling through both proteolytic and non-proteolytic mechanisms. Studies were undertaken to determine the effects of the plasminogen activator inhibitor 1 (PAI1) on the assembly of the fibronectin matrix. The addition of PAI1 to MG-63 cells caused a 1.5- to threefold increase in the rate of fibronectin matrix assembly which was associated with an increase in beta integrin activation. PAI1 treatment led to a marked decrease in focal contacts and stress fibers, whereas tensin-containing matrix contacts remained unaffected. The effects of PAI1 on matrix assembly were independent of both urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), indicating that the stimulation of matrix assembly by PAI1 does not depend on its anti-proteolytic activity or on the association of uPAR with integrin receptors. Antagonists of the alphavbeta5 integrin mimicked the effect of PAI1 on cell morphology and fibronectin matrix deposition, indicating that stimulation of matrix assembly by PAI1 required disruption of the interaction between the alphavbeta5 integrin and vitronectin. Consistent with this conclusion, the Q123K PAI1 mutant which does not bind vitronectin had no effect on matrix assembly. Our data identify PAI1 as a novel regulator of fibronectin matrix assembly, and indicate that this regulation occurs through a previously undescribed crosstalk between the alphavbeta5 and alpha5beta1 integrins.

Goat vitronectin: characterization and binding to Staphylococcus aureus.

Vitronectin (Vn) is a multifunctional protein present in plasma and in the extracellular matrix. Previous studies have demonstrated binding of many bacteria to human Vn. In this study, we have characterized goat Vn and studied its interaction with S. aureus considering the importance of this bacterium in animal husbandry. Goat Vn possesses two RGD motifs, at positions 45 and 106, and two multimerization sites that were identified from the recombinant fragments of the protein. The first site was localized at the N-terminus of the protein and the second at the C-terminus that did not require a full heparin-binding region, as partial deletion of this site did not affect multimerization. The 40 kDa N-terminal fragment, Vn1-200, supported S. aureus binding. Similarly, two fragments representing the C-terminus of the protein (35 kDa Vn183-444 and the 22 kDa Vn323-444) with complete heparin-binding site also supported S. aureus binding whereas the 14 kDa fragment, Vn363-444, with truncated heparin-binding site did not. Thus, a complete heparin-binding site at the C-terminus of Vn is essential for S. aureus binding. Maximum S. aureus binding was observed with Vn isolated by immunoaffinity chromatography, which predominantly consisted of multimers. This observation is significant considering the fact that the multimeric Vn is a component of the matrix surrounding the cells and may play an important part in initial bacterial adhesion and subsequent colonization.

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells.

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.

A mechanism for assembly of complexes of vitronectin and plasminogen activator inhibitor-1 from sedimentation velocity analysis.

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes.

A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements.

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.

Characterization of goat plasma vitronectin.

Vitronectin (VN) was isolated and characterized from goat plasma in native and denatured state. Native VN consisted of 160 and >250 kDa polypeptides, whereas denatured VN showed bands of 81 and >250 kDa on SDS-gel. Storage of 81 kDa polypeptide for 3 days at 4 degrees C resulted in formation of 160 and >250 kDa proteins. Hence high molecular weight forms of VN may be dimer and multimeric forms of 81 kDa monomer. Both native as well as denatured VN showed cell adhesive activity. Cells bound to native VN were round, whereas cells adhered to denatured VN were fully spread, a characteristic also observed with 81 kDa polypeptide. The 81 kDa VN bound to Heparin, whereas the 160 kDa preparation did not bind to Heparin in presence of urea. Absence of EDTA resulted in the degradation of goat VN. Similarly, addition of excess Ca(2+) caused total degradation of VN polypeptides in buffers with EDTA, suggesting metalloprotease activity inthe protein.

Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization.

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.

Full-length and truncated forms of vitronectin provide insight into effects of proteolytic processing on function.

A genetic polymorphism in the vitronectin allele directs the production of two distinct forms of the 459 amino acid glycoprotein. A methionine present at position 381 favors production of the single-chain form of vitronectin, while threonine at this position increases the susceptibility of vitronectin to cleavage just beyond its heparin-binding domain at residue 379. This reaction gives rise to a disulfide-bonded, two-chain form of vitronectin. In order to investigate the functional significance of the vitronectin polymorphism, the baculovirus system has been used to express recombinant full-length vitronectin and a truncated form of the molecule that represents the 62-kDa fragment of two-chain vitronectin. Both forms of vitronectin bind and neutralize heparin anticoagulant activity. The proteins also bind PAI-1 and stabilize its active conformation. These experiments suggest that the C-terminal 80 amino acids do not confer a functional difference in the two allelic variants. Immunoassays and gel filtration experiments indicate that both full-length and truncated recombinant forms of vitronectin are multimeric. Together with other reports from this laboratory, these results provide information regarding the primary binding sites for two vitronectin ligands and further define regions that may be involved in multimerization of the protein.

Specific affinity depletion of cell adhesion molecules and growth factors from serum.

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.

Purification of vitronectin.

This unit describes the purification of extracellular vitronectin from plasma by a series of affinity chromatography steps. First plasma is depleted of fibronectin and other heparin-binding proteins; the sample is then treated with urea to activate the heparin-binding activity of vitronectin. The resulting vitronectin is approximately 98% pure.

Evidence showing that the two-chain form of vitronectin is produced in the liver by a selective furin cleavage.

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one-chain (Vn75) or a two-chain form (Vn65-10), and is produced by a specific cleavage (at Arg379-Ala380), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one-chain recombinant Vn, a specific protein kinase A phosphorylation at Ser378, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn75 to Vn65-10 conversion takes place in the liver (not in blood) and is carried out by furin.