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.
Piérard
L
L
Department of Applied Genetics, Université Libre de Bruxelles, Nivelles, Belgium.
García Quintana
L
L
Reff
M E
ME
Bollen
A
A
D000818:Animals; D002460:Cell Line; D002477:Cells / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D010960:Plasminogen Activators / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005337:Fibrin; D004247:DNA; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator