Orientation of Heparin-binding Sites in Native Vitronectin

Gibson, Angelia D.; Lamerdin, John A.; Zhuang, Pingping; Baburaj, Kunnumal; Serpersu, Engin H.; Peterson, Cynthia B.

doi:10.1074/jbc.274.10.6432

Cited by 32 publications

(15 citation statements)

References 58 publications

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“…This result suggests that these 12 amino-acids represent the major high affinity binding site for heparin in vitronectin, and it is consistent with data from Gibson et al (24) who have demonstrated through biophysical studies the existence of only one heparin binding domain in vitronectin; and data from Francois et al (23) who have shown by FRET (fluorescence resonance energy transfer) analysis that a peptide consisting of amino acids 347-361 exhibits heparin binding kinetics similar to intact vitronectin. Since our binding experiments used nanogram quantities of heparin, we cannot however, rule out the possibility that vitronectin may contain additional low affinity heparin binding sites.…”

Section: Discussionsupporting

confidence: 89%

“…Studies have shown that peptides derived from regions within vitronectin's heparin binding domain exhibit variable levels of heparin binding (14,24,39,43,77). A majority of two heparin binding consensus sequences (5) comprise a previously described 12 amino acid "basic domain" within vitronectin's heparin binding domain (84).…”

Section: Vitronectin's Basic Domain Is a Proteoglycan Binding And Hepmentioning

confidence: 99%

“…Mapping studies using peptides from both within the 40 amino acid heparin binding domain as well as regions within the connecting sequence and the first hemopexin repeat have been used to identify the sequences within vitronectin which mediate heparin binding (39,43,93). This approach has met with varying degrees of success, as peptide conformations often do not duplicate those seen within the context of the intact molecule, and aggregation and low solubility can result in nonspecific cellular effects (24). However, several studies using overlapping peptides have provided consistent evidence that aminoacids 341-370 may constitute a significant proportion of vitronectin's heparin binding activity (23,39,63,64).…”

Section: Introductionmentioning

confidence: 99%

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Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Wilkins-Port

Sanderson²,

Tominna-Sebald³

et al. 2003

Cell Communication & Adhesion

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During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptormediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectin's 12 amino acid "basic domain" which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVn 347-358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVn 347-358. Degradation of rVn 347-358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBD 347-358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in trans to regulate vitronectin degradation.

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Section: Discussionsupporting

confidence: 89%

Section: Vitronectin's Basic Domain Is a Proteoglycan Binding And Hepmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Wilkins-Port

Sanderson²,

Tominna-Sebald³

et al. 2003

Cell Communication & Adhesion

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“…Defining the binding sites for these various biomolecules, along with determining the molecular mechanism of regulation, constitutes an active area of research on the protein. Work to date has focused on binding sites for several ligands, including heparin, PAI-1, 1 and integrins; a working model representing the domain structure of vitronectin has been recently reported from this laboratory (5).…”

mentioning

confidence: 99%

New Insights into the Size and Stoichiometry of the Plasminogen Activator Inhibitor Type-1·Vitronectin Complex

Podor¹,

Shaughnessy²,

Blackburn³

et al. 2000

Journal of Biological Chemistry

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Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M r ϳ 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.Vitronectin is a versatile glycoprotein that is found in circulation, in the extracellular matrix of endothelial cells, in platelets, and within various tissues of the human body. Circulating at micromolar levels, vitronectin participates in the regulation of humoral responses such as coagulation, fibrinolysis, and the complement cascade (reviewed in Ref. [1][2][3][4]. Other functions of the protein that are confined to surfaces or tissues include cell-adhesion and regulation of pericellular proteolysis. Interactions with an assortment of biological molecules are responsible for the multiple functions exhibited by vitronectin. Defining the binding sites for these various biomolecules, along with determining the molecular mechanism of regulation, constitutes an active area of research on the protein. Work to date has focused on binding sites for several ligands, including heparin, PAI-1, 1 and integrins; a working model representing the domain structure of vitronectin has been recently reported from this laboratory (5).Arguably, one of the most important interactions known for vitronectin occurs with the serine protease inhibitor, PAI-1. PAI-1 is the physiological inhibitor of plasminogen activators, both tPA and uPA, the enzymes responsible for generating plasmin from...

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“…Purification and Labeling of Vitronectin-Monomeric vitronectin was purified from human plasma as described previously (38,39). For multiwavelength studies, vitronectin was labeled with fluorescein isothiocyanate using a FluoReporter® protein labeling kit (Molecular Probes, Inc., Eugene, OR) that labels primary amines.…”

Section: Methodsmentioning

confidence: 99%

A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimentation Velocity Analysis

Minor

Schar

Blouse

et al. 2005

Journal of Biological Chemistry

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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. Plasminogen activator inhibitor type 1 (PAI-1) 1 and vitronectin are biological cofactors that play a central role in extracellular matrix remodeling during pathological events such as tissue injury and cancer progression (1-3). The serpin PAI-1 is the primary inhibitor of tissue-and urokinase-type plasminogen activators and thus is the main regulator of plasminmediated vascular and pericellular proteolysis (4 -6). The circulating form of PAI-1 is found primarily in a complex with vitronectin, with the inhibitor bound at a high affinity site located within the amino-terminal SMB domain of vitronectin (7-9). Vitronectin, which is found both in blood and surrounding tissues, also contains binding sites for cell surface receptors and ECM components, both of which serve in the attachment of cells to their surrounding matrices (10 -17).In many instances of tissue injury, cell invasion, or tumor development, PAI-1 and vitronectin are co-localized to the areas of vascular damage, necrosis, angiogenesis, and inflammation (2, 18 -23). In contrast to PAI-1, which is locally produced and secreted by cells into the pericellular environment, the main source of vitronectin for tissue deposition is probably its recruitment from circulation. Incorporation of vitronectin into tissue ECM is linked to "activation" of the protein into an altered, adhesive form, which is both structurally and functionally distinct from its native, plasma counterpart (3). Adhesive functions adopted by this altered form of vitronectin work in parallel with the inhibitory functions of PAI-1, in a fashion that allows for co-regulation of cell adhesion and tissue degradation. The exact mechanism by which plasma vitronectin adopts the altered form, is relocalized, and becomes associated with PAI-1 in tissues under pathological scenarios is currently undefined.Interaction of PAI-1 and vitronectin affects the structural and functional properties of both proteins. The active structure of free PAI-1 is relatively unstable, and it readily converts into a latent, inactive form by spontaneous i...

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Orientation of Heparin-binding Sites in Native Vitronectin

Cited by 32 publications

References 58 publications

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

New Insights into the Size and Stoichiometry of the Plasminogen Activator Inhibitor Type-1·Vitronectin Complex

A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimentation Velocity Analysis

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