Production in Eukaryotic Cells and Characterization of Four Hybrids of Tissue-Type and Urokinase-Type Plasminogen Activators

To investigate the structure–function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1–262 of the A-chain of t-PA is fused to AA 139–411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173–262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1–50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10–411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.