Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

Gårdsvoll, Henrik; Werner, Finn; Søndergaard, Leif; Danø, Keld; Ploug, Michael

doi:10.1016/j.pep.2003.12.002

Cited by 60 publications

(71 citation statements)

References 39 publications

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“…Previous studies identified N-linked glycans on residues 52, 162, 172, and 200 in huPAR expressed by Drosophila S2 or Chinese hamster ovary cells (25,36,47). The sequence for muPAR harbors 7 potential N-linked glycosylation sites at positions 9, 52, 160, 170, 198, 231, and 259 (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…muPAR and a selected number of single site mutants thereof were purified by immunoaffinity chromatography using an immobilized mouse monoclonal anti-uPAR antibody (KOR-1) raised against purified huPAR in a uPAR-deficient transgenic mouse (35). Studies by mass spectrometry using a protocol developed for huPAR (36) revealed that our purified muPAR preparation was heterogeneously glycosylated with roughly 25% carrying bi-antennary glycans on three sites, 50% on four sites, and 25% on five sites. Subsequent peptide mass mapping by MALDI-tandem mass spectrometry (MS) revealed that the recombinant muPAR carried N-linked glycans on Asn 52 , Asn 160 , Asn 170 , Asn 198 , and Asn 259 , whereas the potential sites Asn 9 and Asn 231 remained unmodified (data not shown).…”

Section: Methodsmentioning

confidence: 99%

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

Lin

Gårdsvoll

Huai

et al. 2010

Journal of Biological Chemistry

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131

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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...

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Section: Resultsmentioning

confidence: 92%

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

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“…Once again, this finding was not altogether surprising, but neither was it predictable. In the case of UPAR, eliminating all of the glycosylation sites in a truncated soluble version of the protein largely abolished the secretion of the protein (15). Very different results were obtained with Thy-1, a GPI-anchored Ly-6 protein with three N-glycosylation sites, all of which are utilized (16).…”

Section: Discussionmentioning

confidence: 98%

Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase

Beigneux

Gin

Davies

et al. 2008

Journal of Lipid Research

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GPIHBP1 is a glycosylphosphatidylinositolanchored protein in the lymphocyte antigen 6 (Ly-6) family that recently was identified as a platform for the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 binds both LPL and chylomicrons and is expressed on the luminal face of microvascular endothelial cells. Here, we show that mouse GPIHBP1 is N-glycosylated at Asn-76 within the Ly-6 domain. Human GPIHBP1 is also glycosylated. The N-linked glycan could be released from mouse GPIHBP1 with N-glycosidase F, endoglycosidase H, or endoglycosidase F1. The glycan was marginally sensitive to endoglycosidase F2 digestion but resistant to endoglycosidase F3 digestion, suggesting that the glycan on GPIHBP1 is of the oligomannose type. Mutating the N-glycosylation site in mouse GPIHBP1 results in an accumulation of GPIHBP1 in the endoplasmic reticulum and a markedly reduced amount of the protein on the cell surface. Consistent with this finding, cells expressing a nonglycosylated GPIHBP1 lack the ability to bind LPL or chylomicrons. Eliminating the N-glycosylation site in a truncated soluble version of GPIHBP1 causes a modest reduction in the secretion of the protein. These studies demonstrate that N-glycosylation of GPIHBP1 is important for the trafficking of GPIHBP1 to the cell surface.-Beigneux, A. P., P.

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“…Purified Protein Preparations-Soluble forms of recombinant human uPAR (residues 1-283) were expressed by stably transfected Drosophila melanogaster S2 cells (33), and a library of Ͼ300 purified uPAR mutants carrying single-site substitutions was prepared and characterized as described (34). Recombinant human pro-uPA S356A (residues 1-411) without catalytic activity due to the active-site mutation, pro-uPA ⌬GFD (residues 45-411), and the N-terminal fragment (ATF) of uPA (residues 1-143) were all expressed by D. melanogaster S2 cells and affinity-purified using the immobilized anti-uPA monoclonal antibody, clone-6 (34).…”

Section: Methodsmentioning

confidence: 99%

Conformational Regulation of Urokinase Receptor Function

Gårdsvoll

Jacobsen

Kriegbaum

et al. 2011

Journal of Biological Chemistry

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The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the ␤-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the ␤-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.Timely controlled cell migration is a decisive factor for a plethora of important biological processes that occur during development and adulthood. Controlled cell migration is thus intimately involved in both maintenance and dynamic remodeling of tissue architectures during, e.g. wound healing and mammary gland development (1). These processes are executed and tightly regulated via a complicated cross-talk between specific cell surface receptors (e.g. integrins) and insoluble protein components deposited in the extracellular matrix. The extracellular matrix is nonetheless thought to play a dual role in regulating cell migration, as it provides both the focal adhesion sites required for cellular traction and opposes migration by generating physical barriers (2, 3). Cell migration in vivo, therefore, requires a coordinated regulation of extracellular matrix proteolysis, adhesion, and signaling (4). The urokinase-type plasminogen activator receptor (uPAR) 2 may allegedly assist a rendezvous between these functions, as it has the potential to exert control at all three levels. Besides being responsible for focalizing uPA-mediated plasminogen activation on cell surfaces (5-7), uPAR also facilitates adherence to vitronectin embedded in the extracellular matrix (8 -10), and as a consequence, it promotes intracellular signaling (4, 11).The glycolipid-anchored uPAR is a modular glycoprotein composed of three homologous Ly6/uPAR-type (LU) protein domain repeats (5, 12). The far majority of proteins belonging to this domain family contain only a single copy of the LU module, as exemplified by the glycolipid-anchored CD59, the extracellular ligand binding domain in the TGF-␤ receptors, and the diverse group of secreted snake venom ␣-neurotoxins (13). In the human genome, five genes are recognized so far to encode proteins with multiple LU domains, and these are all confined to a small ...

show abstract

Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

Cited by 60 publications

References 39 publications

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

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

Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase

Conformational Regulation of Urokinase Receptor Function

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