Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library.

The Journal of Biological Chemistry
I R HornA J van Zonneveld

Abstract

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immun...Continue Reading

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Citations

Jan 11, 2001·Trends in Cardiovascular Medicine·J G NeelsP J Lenting
Mar 10, 2001·The Biochemical Journal·T ChitlaruA Shafferman
May 28, 2010·The Journal of Biological Chemistry·Henry R MaunSarah G Hymowitz
Aug 17, 1999·Annual Review of Nutrition·M M HussainA Bakillah
Mar 18, 1997·Proceedings of the National Academy of Sciences of the United States of America·R A OrlandoM G Farquhar
Jan 30, 2007·Protein Expression and Purification·Daniel Iván BarreraLuis Fernando Arbeláez
Jul 26, 2003·Blood·Khalil BdeirDouglas B Cines
Sep 27, 2003·Blood·Taher NassarAbd Al-Roof Higazi
Aug 14, 1999·The Journal of Biological Chemistry·P J LentingA J van Zonneveld

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