Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites.
The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1-283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS-PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.
Gårdsvoll
Henrik
H
Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark. gvoll@finsenlab.dk
Werner
Finn
F
Søndergaard
Leif
L
Danø
Keld
K
Ploug
Michael
M
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004330:Drosophila / Q000235:genetics; D005260:Female; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator