Purification and characterization of A61. An angiostatin-like plasminogen fragment produced by plasmin autodigestion in the absence of sulfhydryl donors.
Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000)Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys(78)-Lys(468). To avoid confusion with the plasmin(ogen) fragment, angiostatin(R) (Lys(78)-Ala(440)), we have named this protein A(61). Unlike angiostatin, A(61) was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A(61) bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A(61) was composed of 21% beta-strand, 14% beta-turn, 18% 3(1)-helix and 8% 3(10)-helix. A(61) was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A(61) by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A(61) was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a nonpathological setting.
Kassam
G
G
Cancer Biology Research Group, Department of Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Kwon
M
M
Yoon
C S
CS
Graham
K S
KS
Young
M K
MK
Gluck
S
S
Waisman
D M
DM
D043168:Angiostatins; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification*; D010958:Plasminogen / Q000737:chemistry* / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D043168:Angiostatins; D010958:Plasminogen; D005341:Fibrinolysin