Purification, Cloning, and Characterization of a Profibrinolytic Plasminogen-binding Protein, TIP49a

Stephen B Hawley,Taka-Aki Tamura,Lindsey A Miles

doi:10.1074/jbc.m004919200

Stephen B Hawley, Taka-Aki Tamura + Show 1 more

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https://doi.org/10.1074/jbc.m004919200

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Abstract

The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with (125)I-plasminogen. A 54-kDa protein lost the ability to bind (125)I-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human (125)I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by epsilon-aminocaproic acid. A single class of binding sites was detected, and a K(d) of 0.57 +/- 0.14 microm was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.

Highlights

Of fibrinolytic molecules on the cell surface promotes plasminogen activation and the association of plasmin with cell surfaces
Exposure of Carboxyl-terminal Lysines on the U937 Cell Surface—We examined U937 membrane-associated proteins for the presence of COOH-terminal lysines exposed to the extracellular environment
Cytoplasmic proteins did not show changes in plasminogen binding following carboxypeptidase B (CpB) treatment of intact cells (Fig. 1, compare panels B and D), suggesting that proteolysis by CpB did not occur in the interior of intact cells

Summary

Introduction

Of fibrinolytic molecules on the cell surface promotes plasminogen activation and the association of plasmin with cell surfaces (reviewed by Plow et al in Ref. 1). We have utilized susceptibility to CpB treatment as a means to identify a previously unrecognized plasminogen-binding protein that exposes a COOH-terminal lysine in an accessible orientation on the cell surface. Comparison of ligand blots of membranes of untreated intact cells with membranes of CpBtreated intact cells revealed the plasminogen-binding proteins that exposed COOH-terminal lysines on the cell surface, i.e. the class of plasminogen-binding proteins that is predominantly responsible for the cell surface-dependent enhancement of plasminogen activation. Using this methodology we have purified and identified a previously unrecognized plasminogen-binding protein present on the surface of U937 monocytoid cells. TIP49a Is a Cell Surface Plasminogen-binding Protein demonstrate that TIP49a binds plasminogen and enhances plasminogen activation

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