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Identification of the amniotic fluid insulin‐like growth factor binding protein‐1 phosphorylation sites and propensity to proteolysis of the isoforms
Author(s) -
Dolcini Lorenzo,
Sala Alberto,
Campagnoli Monica,
Labò Sara,
Valli Maurizia,
Visai Livia,
Minchiotti Lorenzo,
Monaco Hugo L.,
Galliano Monica
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07318.x
Subject(s) - proteolysis , phosphorylation , biochemistry , serine , threonine , trypsin , protease , chemistry , amniotic fluid , serine protease , biology , peptide sequence , binding protein , growth factor , enzyme , fetus , receptor , pregnancy , gene , genetics
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 central, weakly conserved, region, at positions 95, 98, 101 and 119, whereas one, Ser169, is in the C‐terminal domain. The post‐translational modification predominantly involves the hydrophilic stretch of amino acids representing a potential PEST sequence (proline, glutamic acid, serine, threonine) and our results show that the phosphorylation state influences the propensity of IGFBP‐1 to proteolysis.