Functional and complementary phosphorylation state attributes of human insulin-like growth factor-binding protein-1 (IGFBP-1) isoforms resolved by free flow electrophoresis.
Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size. High concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) have been associated with FGR. Phosphorylation of IGFBP-1 is a mechanism by which insulin-like growth factor-I (IGF-I) bioavailability can be modulated in FGR. In this study a novel strategy was designed to determine a link between IGF-I affinity and the concomitant phosphorylation state characteristics of IGFBP-1 phosphoisoforms. Using free flow electrophoresis (FFE), multiple IGFBP-1 phosphoisoforms in amniotic fluid were resolved within pH 4.43-5.09. The binding of IGFBP-1 for IGF-I in each FFE fraction was determined with BIAcore biosensor analysis. The IGF-I affinity (K(D)) for different IGFBP-1 isoforms ranged between 1.12e-08 and 4.59e-07. LC-MS/MS characterization revealed four phosphorylation sites, Ser(P)(98), Ser(P)(101), Ser(P)(119), and Ser(P)(169), of which Ser(P)(98) was new. Although the IGF-I binding affinity for IGFBP-1 phosphoisoforms across the FFE fractions did not correlate with phosphopeptide intensities for Ser(P)(101), Ser(P)(98), and Ser(P)(169) sites, a clear association was recorded with Ser(P)(119). Our data demonstrate that phosphorylation at Ser(119) plays a significant role in modulating affinity of IGFBP-1 for IGF-I. In addition, an altered profile of IGFBP-1 phosphoisoforms was revealed between FGR and healthy pregnancies that may result from potential site-specific phosphorylation. This study provides a strong basis for use of this novel approach in establishing the linkage between phosphorylation of IGFBP-1 and FGR. This overall strategy will also be broadly applicable to other phosphoproteins with clinical and functional significance.
Nissum
Mikkel
M
BD Diagnostics, Am Klopferspitz 19a, 82152 Planegg, Germany.
Abu Shehab
Majida
M
Sukop
Ute
U
Khosravi
Javad M
JM
Wildgruber
Robert
R
Eckerskorn
Christoph
C
Han
Victor K M
VK
Gupta
Madhulika B
MB
D015374:Biosensing Techniques; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism*; D007525:Isoelectric Focusing / Q000379:methods*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism*
D018970:Insulin-Like Growth Factor Binding Protein 1; D020033:Protein Isoforms