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

• Published in: The Journal of biological chemistry 2001-01, Vol.276 (1), p.179-186
• Main Authors: Hawley, Stephen B., Tamura, Taka-aki, Miles, Lindsey A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; ATPases Associated with Diverse Cellular Activities; Carboxypeptidase B; Carboxypeptidases - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cell Membrane - metabolism; Cloning, Molecular; DNA Helicases; e-aminocaproic acid; Electrophoresis, Gel, Two-Dimensional; Expressed Sequence Tags; Humans; Lysine - metabolism; Mass Spectrometry; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Molecular Sequence Data; Molecular Weight; Monocytes - cytology; Monocytes - metabolism; Plasminogen - metabolism; Plasminogen Activators - metabolism; plasminogen receptors; plasminogen-binding protein; Protein Binding; Rats; Recombinant Proteins; Sequence Alignment; TIP49a protein; U937 Cells
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M004919200

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 with125I-plasminogen. A 54-kDa protein lost the ability to bind 125I-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 125I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by ε-aminocaproic acid. A single class of binding sites was detected, and a Kd of 0.57 ± 0.14 μm 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.