Native and non-glycosylated recombinant single-chain urokinase-type plasminogen activator are recognized by different receptor systems on rat parenchymal liver cells.
The recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding characteristics to freshly isolated rat parenchymal liver cells. The binding of 125I-HMW-scu-PA at 4 degrees C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding. Recombinant scu-PA also bound with high affinity (Kd = 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37 degrees C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems.(ABSTRACT TRUNCATED AT 250 WORDS)
van der Kaaden
M E
ME
Division of Biopharmaceutics, Sylvius Laboratory, Leiden/Amsterdam Center for Drug Research, University of Leiden, The Netherlands.
Rijken
D C
DC
Groeneveld
E
E
van Berkel
T J
TJ
Kuiper
J
J
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology; D019286:Endopeptidase K; D006031:Glycosylation; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008297:Male; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000494:pharmacology; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D026503:Low Density Lipoprotein Receptor-Related Protein-1; C524898:PLAUR protein, human; C524900:Plaur protein, rat; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium