GLYCOSYLATION PROFILE OF A RECOMBINANT UROKINASE-TYPE PLASMINOGEN-ACTIVATOR RECEPTOR EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potenti al of these cells to support plasminogen activation, a process that ha s been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling, The uPA receptor is a gly colipid-anchored membrane protein belonging to the Ly-6/uPAR superfami ly and is the only multidomain member identified so far. We have now p urified the three individual domains of a recombinant soluble uPAR var iant, expressed in Chinese hamster ovary cells, after limited proteoly sis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ioni zation and electrospray ionization mass spectrometry, Of the five pote ntial attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III con taining Asn(233) was quantitatively recovered without carbohydrate, Th e remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosyl ation on Asn(52) (domain I) and Asn(172) (domain II) is dominated by t he smaller biantennary complex-type oligosaccharides, while Asn(162) ( domain II) and Asn(200) (domain III) predominantly carry tri- and tetr aantennary complex-type oligosaccharides, The carbohydrate moiety on A sn(52) in uPAR domain I could be selectively removed by N-glycanase tr eatment under nondenaturing conditions. This susceptibility was abroga ted when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proxim ity of the ligand-binding site to this particular carbohydrate moiety, uPAR preparations devoid of carbohydrate on domain I exhibited altere d binding kinetics toward uPA (a 4-6-fold increase in K-d) as assessed by real time biomolecular interaction analysis.