Functional topology of human tissue-type plasminogen activator: characterization of two deletion derivatives and of a duplication derivative.
Three types of permanent Chinese hamster ovary (CHO) cell lines with different amplified expression constructs that abundantly secrete derivatives of human tissue-type plasminogen activator (t-PA) were established. The first one expresses a deletion derivative in which the kringle 2 domain (K2) has been removed (FGK1L). In the second derivative, the growth-factor-homologous domain (G) has also been deleted (FK1L); a third line expresses a duplication derivative of K2 (FK2K2L) lacking the (G) and kringle 1 (K1). All deletion derivatives were constructed according to the exon-intron organization of the gene. We have analyzed the secreted proteins and the fibrinogen-stimulated plasminogenolytic activity as a function of different culturing conditions (fetal calf serum, aprotinin) of the cells. The specific activities of the two deletion derivatives (FGK1L and FK1L) were only 10-20% of the specific activity of t-PA. Surprisingly, the specific activity of the K2-duplication derivative, FK2K2L, was three times higher than that of t-PA. These data were correlated with the morphological properties of CHO cells constitutively secreting the described derivatives under different culturing conditions. CHO cells secreting the deletion derivatives (FGK1L and FK1L) remained attached to the surface of the petri dishes. Cell lines secreting the duplication derivative FK2K2L detached from the surface even in the presence of the protease inhibitor aprotinin.
Stern
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A
Boehringer Mannheim GmbH, Department of Genetics, Penzberg, F.R.G.
Mattes
R
R
Buckel
P
P
Weidle
U H
UH
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D005810:Multigene Family; D010957:Plasmids; D011487:Protein Conformation; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004247:DNA; D007611:Aprotinin; D010959:Tissue Plasminogen Activator