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 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 immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine-activated alpha 2-macroglobulin interaction sites.
Horn
I R
IR
Department of Biochemistry, University of Amsterdam, The Netherlands.
Moestrup
S K
SK
van den Berg
B M
BM
Pannekoek
H
H
Nielsen
M S
MS
van Zonneveld
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D003165:Complement System Proteins / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D015723:Gene Library; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D003165:Complement System Proteins; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium; C067551:saruplase