The Complement Component C1s Is the Protease That Accounts for Cleavage of Insulin-like Growth Factor-binding Protein-5 in Fibroblast Medium

Abstract

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 C1r and C1s. C1r in the fibroblast medium underwent autoactivation, and the activated form cleaved C1s. C1s 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 C1s, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete C1r and C1s 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.

Specifying the cellular responses to IGF signals: roles of IGF-binding proteins

“…Both effects require relatively high concentrations and are fairly modest. These seemingly contradictory observations are probably due to the presence of a specific IGFBP-5 protease in these cells (Duan et al 1996, Busby et al 2000. Most, if not all of the added IGFBP-5 is degraded after more than 48 h of incubation, while a substantial proportion of IGFBP-5 6).…”

C1q Protein Binds to the Apoptotic Nucleolus and Causes C1 Protease Degradation of Nucleolar Proteins

“…Besides C4, C2, and the C1 inhibitor, only three other molecules, all expressed extracellularly, have been reported to be cleaved by C1s: insulin-like growth factor binding protein 5, MHC class I, and the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (42)(43)(44). However, on the basis of a new C1s substrate formula established using a library of peptides centered around the known C1s cleavage sites in C4, C2, and the C1 inhibitor, many intracellular proteins have been predicted to contain C1s cleavage sites (33).…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…Complement activation also occurs at the cartilage surface; both intact and degraded fibronectin can activate the complement cascade, and cartilage degradation and fibronectin release may be an important mechanism in promoting joint inflammation (7,26). Serine proteinase C1s can also degrade insulinlike growth factor binding protein 5 (IGFBP-5) to release active IGF-1 (27), a growth factor integral to controlling cartilage damage. Conversely, C1s can also degrade type I collagen, type II collagen, and gelatin (28), although the typical three-quarter-and one- quarter-size fragments effected by collagenasemediated collagenolysis are not observed.…”

“…Enzymes that target IGFBPs include C1s (27), HtrA1 (172), plasmin (43,173), thrombin (173), CTG (174), and NE (174), while furin, plasmin, thrombin, NE, and tryptase can all process latent TGF␤ proteins (for review, see ref. 42).…”