Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity.
We present evidence that over-expression of human plasminogen, the precursor to the serine protease plasmin, can be cytotoxic to mammalian cells. When an expression vector containing plasminogen cDNA is transfected into baby hamster kidney cells, the number of drug-resistant colonies as well as the levels of plasminogen secreted by those colonies is lower than observed in similar transfections of other protease precursor genes. The recombinant plasminogen accumulates intracellularly as degraded NH2-terminal fragments. In contrast, a mutant of plasminogen that produces inactive plasmin (active site Ser740 changed to Ala) is synthesized by these cells as a full-length plasminogen molecule, and the colony numbers and expression levels are normal. Thus, the generation of plasmin activity is responsible for the cytotoxic phenomena and the degradation associated with plasminogen expression. In addition, experiments using a plasminogen mutant that cannot be activated to plasmin (activation cleavage site Arg560 to Gly) or using coexpression of antisense urokinase RNA indicate that an endogenous plasminogen activator is responsible for converting newly synthesized plasminogen to plasmin. Finally, coexpression of plasminogen with alpha 2-plasmin inhibitor, a serpin which is the physiologic inhibitor of plasmin, prevents the toxic effects of intracellular plasmin activity and allows the synthesis and secretion of native human plasminogen.
Busby
S J
SJ
ZymoGenetics Corporation, Seattle, Washington 98105.
Mulvihill
E
E
Rao
D
D
Kumar
A A
AA
Lioubin
P
P
Heipel
M
M
Sprecher
C
C
Halfpap
L
L
Prunkard
D
D
Gambee
J
J
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D010958:Plasminogen / Q000096:biosynthesis* / Q000235:genetics; D011233:Precipitin Tests; D016372:RNA, Antisense / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D016372:RNA, Antisense; D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator