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 centr ifugation, with PSA remaining in the solid phase and the proteolyzed I GFBP-3 in the aqueous phase. The IGFBP-3 fragments were functionally a nalyzed by affinity labeling and Western ligand blotting (WLB). Furthe r biochemical analyses were provided by silver staining of total prote in and Western immunoblotting (WIB) of immunoreactive fragments with a n IGFBP-3 specific antiserum (alpha-BP-3 gl). N-terminal sequence anal ysis 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(f) of 26 kDa to 13 kDa) as ident ified 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 retai n their ability to bind IGF. N-terminal sequence analysis revealed at least 5 different proteolytic recognition sites for PSA in IGFBP-3. Th ree of the 5 sites were consistent with a 'kallikrein-like' enzymatic activity and 2 sites were consistent with a 'chymotryptic-like' enzyma tic activity, The chymotryptic activity of PSA was further confirmed b y the ability of alpha-1-antichymotrypsin and chymostatin to block PSA cleavage of radiolabeled IGFBP-3. These data indicate that PSA proteo lyzes IGFBP-3 with both 'kallikrein-like' and 'chymotryptic-like' acti vity and that some of the fragments produced retain their ability to b ind IGFs with an apparent reduction in affinity. These results provide a mechanism whereby PSA may alter the biological activity of IGFBP-3 and possibly the bioavailability of IGFs, both within the male reprodu ctive tract and in PSA-secreting tumors.