title: Differential proteolysis of insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy.
abstract: The insulin-like growth factors and their binding proteins are important for placental and foetal growth. In this study, we have investigated the presence of proteolytic activity directed against insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy. In addition, the effect of protease activity on IGFBP-1 immunoreactivity and IGF binding was characterised. 125I-IGFBP-1 was incubated with maternal and foetal serum, amniotic fluid and placental extracts. Breakdown of 125I-IGFBP-1 was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The size distribution of endogenous IGFBP-1 was determined by Western immunoblotting. Protease inhibitor studies characterised the proteolytic activity, and Western ligand blotting with 125I-IGF-I was used to determine IGF binding capacity of proteolysed IGFBP-1. Amniotic fluid samples collected after labour onset contained proteolytic activity that generated 12- and 19-kDa IGFBP-1 fragments that did not bind to 125I-IGF-I. This activity was not detected in amniotic fluid collected prior to labour onset or in other tissues. Activity was blocked by aprotinin, leupeptin, phenyl methyl sulphonyl fluoride, and Kunitz soybean trypsin inhibitor but not by ethylene diamine tetraacetic acid or pepstatin. Incubation of IGFBP-1 with trypsin generated fragments of a similar size to the amniotic fluid protease. In conclusion, we have demonstrated the presence in vivo of a trypsin-like proteolytic activity that alters the IGF-binding function of IGFBP-1 in pregnancy.
authors.affiliation: Department of Natural Sciences Middlesex University, The Burroughs London NW4 4BT, UK. f.hills@mdx.ac.uk
authors.forename: Frank A
authors.initials: FA
authors.lastname: Hills
authors.forename: Ray K
authors.initials: RK
authors.lastname: Iles
authors.forename: Mark H
authors.initials: MH
authors.lastname: Sullivan
mesh_terms: D000595:Amino Acid Sequence; D000653:Amniotic Fluid / Q000378:metabolism; D005260:Female; D005312:Fetal Blood / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D007745:Labor Onset / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy / Q000097:blood / Q000378:metabolism*; D059748:Proteolysis; D014357:Trypsin / Q000378:metabolism
chemical_list: C032231:IGFBP1 protein, human; D018970:Insulin-Like Growth Factor Binding Protein 1; D010446:Peptide Fragments; D014357:Trypsin