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 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.
Dolcini
Lorenzo
L
Department of Biochemistry 'A. Castellani', University of Pavia, Italy.
Sala
Alberto
A
Campagnoli
Monica
M
Labò
Sara
S
Valli
Maurizia
M
Visai
Livia
L
Minchiotti
Lorenzo
L
Monaco
Hugo L
HL
Galliano
Monica
M
D000653:Amniotic Fluid / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011247:Pregnancy; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D018970:Insulin-Like Growth Factor Binding Protein 1; D020033:Protein Isoforms