Localization of vitronectin binding domain in plasminogen activator inhibitor-1.

D.A Lawrence,M.B Berkenpas,S Palaniappan,D Ginsburg

doi:10.1016/s0021-9258(17)36595-x

D.A Lawrence, M.B Berkenpas + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(17)36595-x

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Abstract

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.

Highlights

In order tocharacterize the PAI-1structural domain re- that Plasminogen activator inhibitor type 1 (PAI-1) functions in vivoto regulate hemostas(i6s).PAI-1, a sponsible for binding to vitronectin, the segment of the 50-kJla single-chain glycoprotein, is a rapid inhibitor of both
Amplification was performedin a Perkin-Elmer thermal cycler for 30 cycles of 1min at 94 "C, 1min at 55 "C, and 1min a t 72 "C.After PCR, 70 p1 (-1 pg) of the mutagenized product was precipitated with ethanol, resuspended, and incubated at Random Mutagenesis of PAI-1-Preliminary studies of chimeric Serpins containing varying segments of a,antitrypsin fused to PAI-1 suggested that the PAI-1 binding domain for vitronectin might be locatnedear the amino terminusI.n2 order
Little information is available to localize the corresponding PAI-1binding domain. vitronectin is known t o stabilize PAI-1 in theactive conformation,substitution of the entire strained loop had no effect on vitronectin binding [11]

Summary

RESULTS

DATP,0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, and 5 units of Taqpolymerase (AmpliTaq,Perkin-Elmer). A total of five single substitutions that decrease vitronectin binding relative to uPA inhibitory activity were identified: Gln-55 + Pro, Phe-109 4 Ser, Met-110 + Thr, Leu-116 + Pro, and Gln-123 + Lys (Fig. 3). Mutations Associatedwith Loss of Vitronectin Binding Define a Surface Domain on PAI-1-The locations of the individual mutants, identified above, are shown on the three-dimensional structure of latent wtPAI-1 (Fig. 4) [13](coordinatesgenerously provided by Dr Elizabeth Goldsmith). Both panels represent PAI-1in thesame orientation,with p-strand 1Aon the closest edge at thetop.

DISCUSSION

20.3 L116P Q123K

Methods