Abstract
Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed by N-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4-6-fold increase in Kd) as assessed by real time biomolecular interaction analysis.
Highlights
Association of urokinase-type plasminogen activator to cells via binding to its specific cellular receptor augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling
In the present study we have examined the glycosylation pattern of a human soluble uPA receptor (uPAR) expressed in CHO cells
A similar soluble uPAR protein is secreted in vivo from peripheral blood cells affected by the disease paroxysmal nocturnal hemoglobinuria (43)
Summary
7.3 (7) 9.9 (10) 4.7 (5) 13.8 (13) 0.4 (0) 7.4 (7) 2.1 (2) 7.9 (8)d 5.4 (6) 0.9 (1) 1.6 (2) 11.4 (11) 1.8 (2) 0.0 (0) 1.1 (1) 4.1 (4) 7.3 (7) NDf (1) 3.2 1–87. B These data represent two disulfide linked peptides (T4 ϩ T(7 ϩ 8)) examined in a previous study on the disulfide assignment of uPAR domain. G Values represent data corrected for decomposition of N-acetylglucosamine during acid hydrolysis, assuming an average loss of 40%. These values are not as accurately determined as those measured concomitantly for the amino acids due to a variable decomposition of N-acetylglucosamine. Processing of its glycan moieties (22, 27, 28) In accordance with this speculation is the finding that abolishment of the N-linked glycosylation of Asn[52] in uPAR domain I by site-directed mutagenesis (Asn[52 3] Gln) leads to an approximately 5-fold reduction in its binding affinity, when expressed in murine LB-6 cells (29). The present paper reports the first structural determination of the glycosylation pattern of uPAR and demonstrates in a purified system the direct influence of the carbohydrate moiety of uPAR domain I on real time receptor-ligand binding kinetics using surface plasmon resonance analysis
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