Studies on the effect of fucosylated and non-fucosylated finger/growth-factor constructs on the clearance of tissue-type plasminogen activator mediated by the low-density-lipoprotein-receptor-related protein.
Previously we observed that the finger/growth factor (FG) region of tissue-type plasminogen activator (t-PA) blocked the low-density-lipoprotein-receptor-related protein (LRP)-mediated clearance of t-PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t-PA. The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t-PA by human hepatoma cells. At position 83 it had a serine, whereas human t-PA has a free cysteine and rodent t-PA an arginine at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2-Fc). Two variants were studied, one having Ser83, the other Arg83. The two fucosylated FG2-Fc chimeras were compared with each other, with non-fucosylated FG and with intact t-PA with regard to their effect on the clearance of t-PA and t-PA x plasminogen-activator inhibitor type 1 (PAI-1). For this comparison, LRP-specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF-1) the clearance of t-PA and of t-PA x PAI-1 was inhibited more than 95% by receptor-associated protein, an inhibitor of LRP-mediated clearance, whereas no t-PA or t-PA x PAI-1 clearance was observed in LRP-deficient PEA-13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG2-Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t-PA and t-PA x PAI-1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non-fucosylated FG (IC50 1950 nM and 1560 nM) and 75-fold lower than that of intact t-PA (IC50 9.9 nM and 21.1 nM). The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP-mediated clearance of t-PA.
Camani
C
C
Division of Angiology and Hemostasis, University Hospital Geneva, Switzerland.
Gavin
O
O
Bertossa
C
C
Samatani
E
E
Kruithof
E K
EK
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D005347:Fibroblasts; D005643:Fucose; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008657:Metabolic Clearance Rate; D051379:Mice; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005643:Fucose; D010959:Tissue Plasminogen Activator