Human fibroblasts secrete a serine protease that cleaves insulin-like growth factor-binding protein-5.
We have previously reported the presence of proteolytic activity in conditioned medium from human fibroblast cultures that cleaves insulin-like growth factor-binding protein-5 (IGFBP-5) into non-IGF-I-binding fragments. Coincubation of IGF-I or IGF-II and IGFBP-5 with fibroblast cultures decreased proteolysis. The protease was purified by heparin-Sepharose affinity chromatography. The purified protease cleaved IGFBP-5 into 22-, 20-, and 17-kilodalton non-IGF-I-binding fragments. Protease inhibitor profiles obtained using partially purified enzyme showed that it was a calcium-dependent serine protease. After chelation with EDTA, the activity could only be partially restored with zinc, indicating that it was probably not a metalloprotease. The protease was specific for IGFBP-5 and did not cleave pure IGFBP-1, -2, -3, or -4. IGF-I and IGF-II caused minimal inhibition of proteolysis in vitro. This suggests that the IGF-I-induced increase in IGFBP-5 in fibroblast medium is only partially due to direct protease inhibition. Heparin, antithrombin-III (AT-III), and heparin cofactor-II had inhibitory activity, and heparin potentiated the activity of AT-III. Synthetic peptides, that contained the active sites of AT-III and alpha 1-antichymotrypsin, were also inhibitory. Peptides containing sequences found in two basic regions of IGFBP-5 were tested, and one had inhibitory activity. In summary, fibroblasts secrete a serine protease that cleaves IGFBP-5 and is specific for this form of IGFBP. The protease has properties that are similar to kallikreins, a family of serine proteases that is known to cleave epidermal and nerve growth factor-binding proteins.
Nam
T J
TJ
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599.
Busby
W H
WH
Clemmons
D R
DR
D000990:Antithrombin III / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism* / Q000502:physiology*
D002352:Carrier Proteins; D018975:Insulin-Like Growth Factor Binding Protein 5; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D000990:Antithrombin III; D006493:Heparin; D004492:Edetic Acid; D012697:Serine Endopeptidases