Biochemical analysis of prostate specific antigen-proteolyzed insulin-like growth factor binding protein-3.
Prostate specific antigen (PSA) has been shown to proteolyze IGFBP-3. However, the cleavage sites and mechanism of proteolysis are unknown. In this study, we proteolyzed recombinant human IGFBP-3 with PSA bound to a solid phase support. The reaction mixture was separated by centrifugation, with PSA remaining in the solid phase and the proteolyzed IGFBP-3 in the aqueous phase. The IGFBP-3 fragments were functionally analyzed by affinity labeling and Western ligand blotting (WLB). Further biochemical analyses were provided by silver staining of total protein and Western immunoblotting (WIB) of immunoreactive fragments with an IGFBP-3 specific antiserum (alpha-BP-3 g1). N-terminal sequence analysis was performed on filter-immobilized IGFBP-3 fragments, following size separation by SDS-polyacrylamide electrophoresis. PSA proteolyzed IGFBP-3 into at least 7 fragments (M(r) of 26 kDa to 13 kDa) as identified by silver staining and WIB. At least 3 fragments were visible by affinity labeling with radiolabeled IGF-I or IGF-II and 4 were weakly visible by WLB. These data indicate that some IGFBP-3 fragments retain their ability to bind IGF. N-terminal sequence analysis revealed at least 5 different proteolytic recognition sites for PSA in IGFBP-3. Three of the 5 sites were consistent with a 'kallikrein-like' enzymatic activity and 2 sites were consistent with a 'chymotryptic-like' enzymatic activity. The chymotryptic activity of PSA was further confirmed by the ability of alpha-1-antichymotrypsin and chymostatin to block PSA cleavage of radiolabeled IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)
Fielder
P J
PJ
Department of Pediatrics, Stanford University School of Medicine, CA 94305, USA.
Rosenfeld
R G
RG
Graves
H C
HC
Grandbois
K
K
Maack
C A
CA
Sawamura
S
S
Ogawa
Y
Y
Sommer
A
A
Cohen
P
P
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016622:Silver Staining
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017430:Prostate-Specific Antigen