Systematic Mutational Analysis of the Receptor‐Binding Region of the Human Urokinase‐Type Plasminogen Activator

Abstract

The amino-terminal fragment of human uPA (ATF; amino acids I-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His, tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, Ile28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATE Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPARbinding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thrl8 by alanine or Asn32 by serine had hardly any effect, replacement of Am22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Va120, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated UPNUPAR interaction.Keywords : urokinase-type plasminogen activator ; urokinase-type-plasminogen-activator receptor ; sitedirected mutagenesis ; tumor invasion; ligand-receptor interaction.The serine protease urokinase-type plasminogen activator (uPA) is synthesized and secreted as a single-chain proenzyme, pro-uPA, by a variety of cells, such as monocytes, fibroblasts, and tumor cells. pro-uPA is activated by limited proteolysis into the enzymatically active high-molecular-mass two-chain form of Correspondence to V. Magdolen, Klinische Forschergruppe (DFG)

Cyclo19,31[D-Cys19]-uPA19-31 Is a Potent Competitive Antagonist of the Interaction of Urokinase-Type Plasminogen Activator with Its Receptor (CD87)

“…The application of large molecules to treat cancer patients appears rather difficult and depends on sophisticated, e. g. viral, delivery systems. Consequently, our aim was to develop synthetic, small peptide inhibitors derived from the continuous uPAR-binding sequence of uPA (Magdolen et al, 1996;Bürgle et al, 1997). Using the cyclic disulfide-bridged form of the minimal uPAR-binding region as lead structure (cyclo 19,31 uPA 19-31 ), we have identified cyclo 19,31 ]-uPA 19-31 as -to our knowledge -the most potent competitive uPA-antagonist presently known that displays only a 20-to 40-fold lower uPAR-binding capacity as compared to the natural uPAR ligands uPA or ATF.…”

“…The region between amino acids Thr18 and Asn32 of uPA consists of a flexible, seven-residue Ω-loop (Asn22 to Ile28) which by means of a double stranded, antiparallel ß-sheet (between Thr18 to Ser21 and His29 to Asn32) is forced into a ring-like structure (Hansen et al, 1994a, b;Magdolen et al, 1996;Schmitt et al, 2000). In uPA, Cys19 and Cys31, although in close proximity, form disulfide bonds with distinct cysteines (Cys11/Cys19 and Cys13/Cys31, respectively; Hansen et al, 1994a, b).…”

“…All of the reactive peptides (with the clone 20 peptide being most active) harbor relatively short common motifs, such as LWXXY and FXXYLW, which may represent a variation of the motif 24 YFXXIXW 30 within the uPA molecule (only the critical hydrophobic amino acids for uPAR-binding are depicted) (Magdolen et al, 1996;Bürgle et al, 1997). Recently, a non-competitive, allosteric antagonist of prouPA/uPAR-interaction (Å6-peptide) derived from a non-receptor binding region of uPA ) has been identified (Guo et al, 2000).…”

“…This peptide was found to inhibit binding of the amino-terminal fragment (ATF) of uPA (which binds to uPAR with the same affinity as uPA but lacks the uPA protease domain) to cell surface uPAR. Later, several linear peptides spanning uPA [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] were synthesised in which the naturally occurring amino acids were individually replaced by alanine (Ala scan) in order to identify the amino acids critical for binding to uPAR (Magdolen et al, 1996). The exchange of Cys19, Lys23, Tyr24, Phe25, Ile28, Trp30, and Cys31, respectively, by Ala resulted in peptides with strongly impaired uPAR-binding capacities, whereas replacement of the other amino acids had no or little effect on uPAR binding.…”

Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice

“…This interaction involves the EGF-like domain of ATF (residues 1-46) and it exhibits species restriction: this is the case between mice and humans for which critical residues 19-32 are not conserved. [34][35][36] Preclinical data indicate that ATF-based antagonists constitute potent anticancer molecules. For example, intraperitoneal bolus injections of a human ATF-based antagonist has been shown to inhibit lung metastases of human tumor cells grown in athymic mice, with no significant effect on primary tumor growth.…”