Abstract
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.
Highlights
Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size
As a prerequisite to application of this approach clinically, we evaluated representative amniotic fluid (AF) samples to determine whether or not differential patterns of insulin-like growth factor-binding protein-1 (IGFBP-1) phosphorylation exist in FGR and whether these changes could be attributed to augmentation of insulin-like growth factor-I (IGF-I) affinity in the disease
Phosphorylation of IGFBP-1 mediates acute metabolic regulation of insulin-like growth factor (IGF)-I bioavailability; it is significant in FGR
Summary
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. 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), and Ser(P)169 sites, a clear association was recorded with Ser(P)119. 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 be broadly applicable to other phosphoproteins with clinical and functional significance. Several studies have considered the clinical implications of IGFBP-1 phosphorylation, focusing on correlating variable ratios of high to low concentrations of IGFBP-1 phosphoisoforms with fetal outcome in FGR pregnancies [15,16,17,18,19].
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