Purification, cloning, and characterization of a profibrinolytic plasminogen-binding protein, TIP49a.
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.
Hawley
S B
SB
Department of Vascular Biology (CVN-26), Scripps Research Institute, La Jolla, California 92037, USA.
Tamura
T
T
Miles
L A
LA
D000074183:ATPases Associated with Diverse Cellular Activities; D000595:Amino Acid Sequence; D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004265:DNA Helicases; D015180:Electrophoresis, Gel, Two-Dimensional; D020224:Expressed Sequence Tags; D006801:Humans; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins; D016415:Sequence Alignment; D020298:U937 Cells
D002352:Carrier Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D010958:Plasminogen; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D010960:Plasminogen Activators; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486549:RUVBL1 protein, human; C486550:Ruvbl1 protein, rat; D008239:Lysine