Biochemical analysis of prostate specific antigen-proteolyzed insulin-like growth factor binding protein-3

P. J. Fielder, R. G. Rosenfeld, H. C.B. Graves, K. Grandbois, C. A. Maack, S. Sawamura, Y. Ogawa, A. Sommer, P. Cohen

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62 Scopus citations

Abstract

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 IGPBP-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 (α-BP-3 gl). 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 α-1-antichymotrypsin and chymostatin to block PSA cleavage of radiolabeled IGFBP-3. These data indicate that PSA proteolyzes IGFBP-3 with both 'kallikrein-like' and (chymotryptic-like' activity and that some of the fragments produced retain their ability to bind 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 reproductive tract and in PSA-secreting tumors.

Original languageEnglish (US)
Pages (from-to)164-172
Number of pages9
JournalGrowth regulation
Volume4
Issue number4
StatePublished - Dec 1 1994

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Keywords

  • IGFBP-3
  • IGFs
  • Male reproduction
  • Prostate specific antigen (PSA)
  • Proteolysis

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Fielder, P. J., Rosenfeld, R. G., Graves, H. C. B., Grandbois, K., Maack, C. A., Sawamura, S., Ogawa, Y., Sommer, A., & Cohen, P. (1994). Biochemical analysis of prostate specific antigen-proteolyzed insulin-like growth factor binding protein-3. Growth regulation, 4(4), 164-172.