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 (*)
- Ivo R. Horn,
- SK. Moestrup(1),
- Birgit M. M. van den Berg,
- Hans Pannekoek,
- Morten S. Nielsen(1) and
- Anton-Jan van Zonneveld(§)
- From the (1) Department of Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands and
- the Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
- §To whom correspondence should be addressed: Academic Medical Center, Dept. of Biochemistry (K1-161), Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: 31-20-5665129; Fax: 31-20-6915519.
Abstract
The low density lipoprotein receptor-related protein/α
-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), α
-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the
receptor in a Ca
-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
-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 (IC
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 
I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [
I]u-PA
PAI-1 complexes (IC
= 1.1 nM) and completely inhibits the binding of [
I]pro-u-PA to the receptor (IC
= 2.2 nM). No inhibition was observed for the binding of 
I-labeled methylamine-activated α
-macroglobulin or [
I]t-PA
PAI-1 to LRP. Degradation of [
I]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 α
-macroglobulin interaction sites.
Footnotes
-
↵* This work was supported by Grant 902-26-128 from the Netherlands Organization for Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- LRP
-
low density lipoprotein receptor-related protein/α
-macroglobulin receptor
- α
M
-
methylamine-activated α
-macroglobulin
- BSA
-
bovine serum albumin
- ELISA
-
enzyme-linked immunosorbent assay
- LpL
-
lipoprotein lipase
- PAI-1
-
plasminogen activator inhibitor type 1
- u-PA
-
urokinase-type plasminogen activator or urokinase
- pro-u-PA
-
single-chain u-PA
- RAP
-
receptor-associated protein
- t-PA
-
tissue-type plasminogen activator
- TBS
-
Tris-buffered saline.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
