Abstract
This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix.
Highlights
This study describes the binding of plasminogen and addition, diverse biological activities have been attributed to tissue-type plasminogen activator +PA) to the extra- laminin including stimulation of cellular adhesion, growth, cellular matrix proteinsfibronectin and laminin
0.2 r gradient SDS-polyacrylamide gels, and transferred to Immobilon as described under "Experimental Procedures." A, Immobilonmembrane stained with Coomassie Blue; B, Immobilon incubated with anti-fibronectin antibody (1:5000); C, Immobilon incubated with 15 pg/ml Pg; D,Immobilon incubated with 3 pg/ml t-PA; E, Immobilon incubated with Pg in the presence of 100 mM 6-aminohexanoic acid (6-AHA);F, Immobilon incubated with t-PA in the presence of 100 mM 6-AHA
The extracellular matrix adhesive glycoproteins laminin and fibronectin had a differential effect on Pg activation by t-PA
Summary
Materials-Bovine serum albumin, murine Engelbreth-HolmSwarm sarcoma laminin, 6-aminohexanoic acid (6-AHA), Pg fragments Tyrm-ValW (kringle 1-3, lysine binding site l), Valw-AlaeB (kringle 4, lysinebinding site 2),benzylamine, anti-(goatIgG) alkaline phosphatase conjugate, anti-(rabbit IgG) alkaline phosphatase conjugate, dichloroisocoumarin, 5-bromo-4-chloro-3-indolylphosphate (BCIP), nitro blue tetrazolium (NBT) grade 111, and endoproteinase Glu-C from Staphylococcus aureusstrain V8 (endoproteinase V8) were purchased from Sigma. Ligand Blot Binding Assay-Intact and proteolyzed laminin, fibronectin, or Pg were electrophoresed on 5-15% gradient or 11%SDS-. Prior to the ligand binding studies, blots were incubated in 10 mM Tris-HC1, 0.15 M NaCl, 0.05% Nonidet P-40, 1%bovine serum albumin, pH 7.5 (TSN buffer), at room temperature for 30 min to block nonspecific sites. In studies examining the binding of Pg toimmobilizedextracellular matrix proteins, intact and proteolyzed laminin or fibronectin was electrophoresed on 5-15% gradient SDS-polyacrylamide gels and electroblotted onto Immobilon. Following blocking with TSN buffer, blots were incubated with either fibronectin (15pg/ ml) or laminin (15 pg/ml) in TSN buffer overnight at 5 “C.In separate experiments, laminin or fibronedin was incubated with immobilized Pg in the presence of 100 mM 6-AHA or benzylamine in TSN buffer. Pg binding to fibronectin and laminin coated wells was quantitated using ‘=I-Pg to determine association and dissociation rates. lZ5I-Pg(0.15,0.3, or 0.5 mM) was added to fibronectin or laminin
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