Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 48, Pages 37638-37644Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M006107200
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- NIA NIH HHS [AGO23331] Funding Source: Medline
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Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions, understanding the chemical identity and regulation of this protease is important for understanding how IGF-I stimulates anabolic functions. The protease was purified from human fibroblast-conditioned medium by hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS polyacrylamide gel electrophoresis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Sequencing of the high pressure liquid chromatography-purified peptides yielded the complement components C1r and C1s. To confirm that C1r/C1s accounted for the proteolytic activity in the medium, immunoaffinity chromatography was performed. Most of the protease activity adhered to the column, and the eluant was fully active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa band contained only Clr and Cls. Clr in the fibroblast medium underwent autoactivation, and the activated form cleaved Cls. Cls purified from the conditioned medium cleaved C-4, a naturally occurring substrate. The purified protease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit activated Cls, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete Clr and Cls that actively cleave IGFBP-5. The findings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.
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