Generation of angiostatin by reduction and proteolysis of plasmin. Catalysis by a plasmin reductase secreted by cultured cells.
Extracellular manipulation of protein disulfide bonds has been implied in diverse biological processes, including penetration of viruses and endotoxin into cells and activation of certain cytokine receptors. We now demonstrate reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary or HT1080 cells. Reduction of plasmin disulfide bond(s) triggered proteolysis of the enzyme, generating fragments with the domain structure of the angiogenesis inhibitor, angiostatin. Two of the known reductases secreted by cultured cells are protein disulfide isomerase and thioredoxin, and incubation of plasmin with these purified reductases resulted in angiostatin fragments comparable with those generated from plasmin in cell culture. Thioredoxin-derived angiostatin inhibited proliferation of human dermal microvascular endothelial cells with half-maximal effect at approximately 0.2 microg/ml. Angiostatin made by cells and by purified reductases contained free sulfhydryl group(s), and S-carbamidomethylation of these thiol group(s) ablated biological activity. Neither protein disulfide isomerase nor thioredoxin were the reductases used by cultured cells, because immunodepletion of conditioned medium of these proteins did not affect angiostatin generating activity. The plasmin reductase secreted by HT1080 cells required a small cofactor for activity, and physiologically relevant concentrations of reduced glutathione fulfilled this role. These results have consequences for plasmin activity and angiogenesis, particularly in the context of tumor growth and metastasis. Moreover, this is the first demonstration of extracellular reduction of a protein disulfide bond, which has general implications for cell biology.
Stathakis
P
P
Centre for Thrombosis and Vascular Research, School of Pathology and Department of Haematology, Prince of Wales Hospital, University of New South Wales, Sydney NSW 2052, Australia.
Fitzgerald
M
M
Matthias
L J
LJ
Chesterman
C N
CN
Hogg
P J
PJ
D043168:Angiostatins; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis*; D010958:Plasminogen / Q000096:biosynthesis*; D013879:Thioredoxins / Q000378:metabolism
D010446:Peptide Fragments; D013879:Thioredoxins; D043168:Angiostatins; D010958:Plasminogen; D010088:Oxidoreductases; D005341:Fibrinolysin