tailieunhanh - Báo cáo khoa học: Identification of the amniotic fluid insulin-like growth factor binding protein-1 phosphorylation sites and propensity to proteolysis of the isoforms

Insulin-like growth factor binding protein-1 (IGFBP-1) is the major secreted protein of human decidual cells during gestation and, as a modula-tor of insulin-like growth factors or by independent mechanisms, regulates embryonic implantation and growth. | ỊFEBS Journal Identification of the amniotic fluid insulin-like growth factor binding protein-1 phosphorylation sites and propensity to proteolysis of the isoforms Lorenzo Dolcini1 Alberto Sala1 Monica Campagnoli1 Sara Labo1 Maurizia Valli1 Livia Visai1 2 Lorenzo Minchiotti1 Hugo L. Monaco3 and Monica Galliano1 1 Department of Biochemistry A. Castellani University of Pavia Italy 2 Center for Tissue Engineering C. I. T University of Pavia Italy 3 Biocrystallography Laboratory Department of Biotechnology University of Verona Italy Keywords IGFBP insulin-like growth factor binding protein-1 mass spectrometry phosphorylation proteolysis Correspondence M. Galliano Department of Biochemistry A. Castellani University of Pavia viale Taramelli 3b 27100 Pavia Italy Fax 39 0382 423108 Tel 39 0382 987724 E-mail galliano@ Received 16 March 2009 revised 27 July 2009 accepted 19 August 2009 doi Insulin-like growth factor binding protein-1 IGFBP-1 is the major secreted protein of human decidual cells during gestation and as a modulator of insulin-like growth factors or by independent mechanisms regulates embryonic implantation and growth. The protein is phosphorylated and this post-translational modification is regulated in pregnancy and represents an important determinant of its biological activity. We have isolated from human normal amniotic fluid collected in the weeks 16-18 the intact nonphosphorylated IGFBP-1 and five electrophoretically distinct phosphoisoforms and have determined their in vivo phosphorylation state. The unmodified protein was the most abundant component and mono- bi- tri-and tetraphosphorylated forms were present in decreasing amounts. The phosphorylation sites of IGFBP-1 were identified by liquid chromatography-tandem mass spectrometry analysis of the peptides generated with trypsin chymotrypsin and Staphylococcus aureus V8 protease. Five serines were found to be phosphorylated and of these four are localized in the