Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin.
Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.
Lyons
R M
RM
Department of Cell Biology, Vanderbilt School of Medicine, Nashville, Tennessee 37232.
Gentry
L E
LE
Purchio
A F
AF
Moses
H L
HL
D000818:Animals; D002460:Cell Line; D005341:Fibrinolysin / Q000378:metabolism*; D000882:Haplorhini; D015151:Immunoblotting; D008961:Models, Structural; D008970:Molecular Weight; D011498:Protein Precursors; D011506:Proteins / Q000235:genetics; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta; D015291:Transforming Growth Factors / Q000235:genetics* / Q000378:metabolism
D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D015291:Transforming Growth Factors; D005341:Fibrinolysin