One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry

Jacobsen, B.; Gårdsvoll, Henrik; Funch, Gitte Juhl; Østergaard, Søren; Barkholt, Vibeke; Ploug, Michael

doi:10.1016/j.pep.2006.08.011

Cited by 27 publications

(24 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The presence of a large solvent-exposed hydrophobic surface on the one side of uPAR domain I facing the ligand-binding cavity (17)(18)(19) presumably confers some instability to the native, unoccupied uPAR. In accordance, we have previously shown that purified, monomeric, and recombinant uPAR WT does indeed slowly form dimers in solution when stored at 4°C (45). In addition, the binding isotherms shown in Fig.…”

Section: Discussionsupporting

confidence: 90%

Mapping of the Vitronectin-binding Site on the Urokinase Receptor

Gårdsvoll

Ploug

2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central four- The urokinase-type plasminogen activator receptor (uPAR 2 / CD87) is a modular, glycolipid-anchored membrane protein (1, 2). Through the specific binding of the urokinase-type plasminogen activator (uPA), uPAR assists in regulating and focalizing cell surface-associated plasminogen activation as demonstrated both in vitro (3, 4) and in vivo (5-7). The high affinity interaction between uPAR and uPA (K D Ͻ 1 nM) has been extensively characterized (2,8). Although all determinants required for this tight binding are contained within the small growth factor-like domain of uPA (GFD residues 1-48) (9), it critically depends on maintenance of a three-domain, modular structure of uPAR (10). Accordingly, site-directed mutagenesis and photoaffinity labeling studies have shown that elements located in distinct domains of uPAR are involved in the interactions with both uPA (8, 11-13) and a potent 9-mer peptide antagonist (9, 14 -16). Two crystal structures solved for uPAR in complex with either a linear peptide antagonist (17) or the amino-terminal fragment (ATF) of uPA (18, 19) have provided the structural basis for the existence of a composite ligandbinding site. The assembly of the three homologous domains in uPAR creates a large and deep central ligand-binding cavity, where aliphatic side chains, provided by uPAR domain I, establish a hydrophobic binding site on one side of the cavity. The high affinity for both uPA and the linear peptide antagonists is achieved by an intimate interaction with this cavity (8,17,18), providing a well defined target site for rational drug design (20).As opposed to this, the binding sites mediating the interactions between uPAR and its auxiliary binding partners, e.g. vitronectin (21, 22) and integrins (23, 24), must reside outside this cavity, but the molecular mechanisms underlying these interactions are largely unknown. As these int...

show abstract

Section: Discussionsupporting

confidence: 90%

Mapping of the Vitronectin-binding Site on the Urokinase Receptor

Gårdsvoll

Ploug

2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The versatility and specificity of this small uPAR-binding peptide is illustrated by the fact that it has been used to establish a specific immunoassay for various uPAR forms in plasma (39), that it forms the cornerstone in a newly developed one-step affinity purification protocol for human uPAR (17), that it can target uPAR covalently by photoaffinity labeling (40,41), and finally that it was instrumental for solving the first crystal structure of uPAR (23). In this study, we synthesized a 64 Cu-DOTA-conjugated uPAR-binding peptide AE105 and we have provided direct proof-of-principle evidence for its specific targeting of uPAR-expressing tumors using microPET imaging in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…The peptide antagonist AE105 (D-Cha-F-s-r-Y-L-W-S) and a nonbinding variant of AE105 (AE105 mut , D-Cha-F-s-r-Y-L-E-S) were synthesized by Peptides International. Human recombinant uPAR (residues 1-283) was produced by Drosophila melanogaster S2 cells (15) after deletion of the COOH-terminal signal sequence (residues 284-313) responsible for its membrane attachment by a glycolipid anchor (16), and the secreted protein was purified from the supernatant by a one-step affinity chromatography using an immobilized peptide antagonist (17). Activesite mutated, human pro-uPA S356A was produced recombinantly by S2 cells and affinity purified using a monoclonal anti-uPA antibody (18).…”

Section: Methodsmentioning

confidence: 99%

Imaging of Urokinase-Type Plasminogen Activator Receptor Expression Using a 64Cu-Labeled Linear Peptide Antagonist by microPET

Niu

Wang

et al. 2008

Clinical Cancer Research

Self Cite

View full text Add to dashboard Cite

Purpose: Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules in one of the major protease systems involved in extracellular matrix degradation. Noninvasive imaging of this receptor in vivo with radiolabeled peptides that specifically target uPAR may therefore be useful to decipher the potential invasiveness of malignant lesions. Experimental Design: In this study, we developed a 64 Cu-labeled uPAR-binding peptide for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N ¶,N 00 ,NØ-tetraacetic acid (DOTA) and labeled with 64 Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435 human breast cancer (uPAR negative). Results: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the a-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of Cu-DOTA-AE105 is specifically inhibited by a nonlabeled antagonist. Conclusion: The successful demonstration of the ability of a 64 Cu labeled uPAR-specific probe to visualize uPAR expression in vivo may allow clinical translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR systembased cancer therapy.

show abstract

“…Protein Preparations-Soluble, recombinant human uPAR (huPAR, residues 1-283), human pro-uPA S356A (residues 1-411), and human ATF (residues 1-143) were produced and affinity-purified as described before (29,33). Recombinant human SMB expressed in Pichia pastoris was purified and characterized as described (34).…”

Section: Methodsmentioning

confidence: 99%

Structure-based Engineering of Species Selectivity in the Interaction between Urokinase and Its Receptor

Lin

Gårdsvoll

Huai

et al. 2010

Journal of Biological Chemistry

Self Cite

131

View full text Add to dashboard Cite

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) is decisive for cell surface-associated plasminogen activation. Because plasmin activity controls fibrinolysis in a variety of pathological conditions, including cancer and wound healing, several intervention studies have focused on targeting the uPA⅐uPAR interaction in vivo. Evaluations of such studies in xenotransplanted tumor models are, however, complicated by the pronounced species selectivity in this interaction. We now report the molecular basis underlying this difference by solving the crystal structure for the murine uPA⅐uPAR complex and demonstrate by extensive surface plasmon resonance studies that the kinetic rate constants for this interaction can be swapped completely between these orthologs by exchanging only two residues. This study not only discloses the structural basis required for a successful rational design of the species selectivity in the uPA⅐uPAR interaction, which is highly relevant for functional studies in mouse models, but it also suggests the possible development of general inhibitors that will target the uPA⅐uPAR interaction across species barriers.The urokinase-type plasminogen activator receptor (uPAR) 3 is a glycolipid-anchored membrane protein (1) that recognizes the serine protease urokinase-type plasminogen activator (uPA) and its zymogen (pro-uPA) with very high affinity and specificity (2). This interaction is exclusively mediated by the N-terminal growth factor-like domain (GFD 1-48 ) of uPA, and it is indispensable for the focalized uPA-mediated plasminogen activation that occurs at cell surfaces both in vitro (3, 4) and in vivo (5-7). Several independent studies have correlated uPAR expression in vivo to e.g. neutrophil infiltration (8 -10) and to various pathological conditions such as cancer invasion and metastasis (11-13), hepatic fibrin deposition (14, 15), and kidney barrier function (16). Accordingly, uPAR has been proposed as a promising molecular target for intervention and/or cytotoxin-based cancer therapies (14,17,18). With the goal of future monitoring efficacy of such treatment modalities, we have developed a high affinity peptide antagonist of the human uPA⅐uPAR interaction (19), which has proven well suited for non-invasive in vivo imaging of uPAR by positron emission tomography (20).The extracellular domains of uPAR comprise three homologous Ly-6/uPAR (LU)-type modules, all of which adopt the three-fingered folding topology that is defined by the structures of the single domain snake venom ␣-neurotoxins (21-23). Crystal structures recently solved for human uPAR in complex with the abovementioned peptide antagonist (24), the N-terminal fragment (ATF) of uPA (25), or the somatomedin B (SMB) domain of vitronectin (26) all confirm this topology, and more importantly, they consistently reveal the presence of a large hydrophobic uPA-binding cavity in uPAR that requires all three LU domains for its assembly. In total, Ͼ2000 Å 2 of solve...

show abstract

One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry

Cited by 27 publications

References 51 publications

Mapping of the Vitronectin-binding Site on the Urokinase Receptor

Mapping of the Vitronectin-binding Site on the Urokinase Receptor

Imaging of Urokinase-Type Plasminogen Activator Receptor Expression Using a 64Cu-Labeled Linear Peptide Antagonist by microPET

Structure-based Engineering of Species Selectivity in the Interaction between Urokinase and Its Receptor

Contact Info

Product

Resources

About