The complement component C1s is the protease that accounts for cleavage ofinsulin-like growth factor-binding protein-5 in fibroblast medium

Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin -like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been s hown to regulate IGF-I actions, understanding the chemical identity and reg ulation of this protease is important for understanding how IGF-I stimulate s anabolic functions. The protease was purified from human fibroblast-condi tioned medium by hydrophobic interaction, lectin affinity, and heparin Seph arose affinity chromatography followed by SDS polyacrylamide gel electropho resis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Se quencing of the high pressure liquid chromatography-purified peptides yield ed 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 electr ophoresis with silver staining showed two bands, and IGFBP-5 zymography sho wed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa b and contained only Clr and Cls. Clr in the fibroblast medium underwent auto activation, and the activated form cleaved Cls. Cls purified from the condi tioned medium cleaved C-4, a naturally occurring substrate. The purified pr otease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 i nhibitor, a protein known to inhibit activated Cls, was shown to inhibit th e 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 f indings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.