Site-Specific IGFBP-1 Hyper-Phosphorylation in Fetal Growth Restriction: Clinical and Functional Relevance Majida Abu Shehab,† Javad Khosravi,‡ Victor K. M. Han,†,§,| Brian H. Shilton,§ and Madhulika B. Gupta*,†,§,| Department of Pediatrics, University of Western Ontario, London, Ontario, Canada, Diagnostic Systems Laboratories Inc., Toronto, Ontario M5G 1L7, Canada, Department of Biochemistry, University of Western Ontario, London, Ontario N6C 2V5, Canada, and Children’s Health Research Institute, University of Western Ontario, London, Ontario N6C 2V5, Canada Received November 11, 2009
Phosphorylation enhances IGFBP-1 binding to IGF-I, thereby limiting the bioavailability of IGF-I that may be important in fetal growth. Our goal in this study was to determine whether changes in sitespecific IGFBP-1 phosphorylation were unique to fetal growth restriction. To establish a link, we compared IGFBP-1 phosphorylation (sites and degree) in amniotic fluid from FGR (N ) 10) and controls (N ) 12). The concentration of serine phosphorylated IGFBP-1 showed a negative correlation with birth weight in FGR (P ) 0.049). LC-MS/MS analysis revealed all four previously identified phosphorylation sites (Ser98, Ser101, Ser119, and Ser169) to be common to FGR and control groups. Relative phosphopeptide intensities (LC-MS) between FGR and controls demonstrated 4-fold higher intensity for Ser101 (P ) 0.026), 7-fold for Ser98/Ser101 (P ) 0.02), and 23-fold for Ser169 (P ) 0.002) in the FGR group. Preliminary BIAcore data revealed 4-fold higher association and 1.7-fold lower dissociation constants for IGFBP-1/IGF-I in FGR. A structural model of IGFBP-1 bound to IGF-I indicates that all the phosphorylation sites are on relatively mobile regions of the IGFBP-1 sequence. Residues Ser98, Ser101, and Ser169 are close to structured regions that are involved in IGF-I binding and, therefore, could potentially make direct contact with IGF-I. On the other hand, residue Ser119 is in the middle of the unstructured linker that connects the N- and C-terminal domains of IGFBP-1. The model is consistent with the assumption that residues Ser98, Ser101, and Ser169 could directly interact with IGF-I, and therefore phosphorylation at these sites could change IGF-I interactions. We suggest that site-specific increase in IGFBP-1 phosphorylation limits IGF-I bioavailability, which directly contributes to the development of FGR. This study delineates the potential role of higher phosphorylation of IGFBP-1 in FGR and provides the basis to substantiate these findings with larger sample size. Keywords: IGFBP-1 • phosphorylation • fetal growth restriction • mass spectrometry • IEMA, ELISA • BIAcore
Introduction
linked with FGR need to be elucidated for appropriate and timely interventions and management of the disease.
Fetal growth restriction (FGR) is the pathological reduction of the fetal weight below the 10th percentile for gestational age.1 FGR affects 3-10% of pregnancies2 and is a serious contributor to perinatal/neonatal morbidity and mortality.3,4 Studies by Barker demonstrate that FGR fetuses are at higher risk for health complications as adults.5-9 The current diagnostic and prognostic procedures are challenging, due to the complex etiology of FGR.2 Furthermore, the molecular mechanisms
During pregnancy, IGFs and IGF binding proteins (IGFBPs) are important regulators for the growth and development of the fetal and the maternal tissues.10,11 In animal models, overexpression of human IGFBP-1 in the fetal liver12 or placenta13 was accompanied by poor placental function and growth restriction. In human pregnancies, the circulating levels of IGFBP-1 in maternal and fetal blood were negatively correlated with birth weight.14-18
* To whom correspondence should be addressed at the Children’s Health Research Institute, VRL Room A5-136 (WC), 800 Commissioners Road E., London, ON N6C 2V5 Canada. Tel: (519) 685-8500Ext. 55099. Fax: (519) 6858186. E-mail:
[email protected]. † Department of Pediatrics, University of Western Ontario. ‡ Diagnostic Systems Laboratories Inc. § Department of Biochemistry, University of Western Ontario. | Children’s Health Research Institute, University of Western Ontario.
IGFBP-1 phosphorylation enhances the binding affinity to IGF-I and leads to reduced availability of free IGF-I to interact with the cell receptor.19 Although the significance of increase in IGFBP-1 phosphorylation in FGR has consistently been reiterated, numerous attempts to demonstrate the clinical relevance of IGFBP-1 phosphorylation by quantitative ELISAs have not been successful.20-24
10.1021/pr900987n
2010 American Chemical Society
Journal of Proteome Research 2010, 9, 1873–1881 1873 Published on Web 02/09/2010
research articles
Abu Shehab et al.
a
Table 1
(A) Summary of the Study Group Characteristics and the Clinical Outcomes of the Subjects study group
no. of infants (N)
birth wt, g
placental wt, g
wt percentile
mean maternal age, years
mean gestational age, weeks
control C1 C2 FGR P
8 4 10 NA
3427 ( 223.3 NA 1762 ( 663.6b 0.00009
679.5 ( 42.1 NA 407 ( 123.8b 0.0008
>25th (100%) NA E10%(100%) NA
31 ( 4.1 NA 27.9 ( 5.1 0.557
39 ( 1.12 NA 34.8 ( 3.55b 0.0004c
(B) Distribution of the Weight Percentile in the FGR Group Based on the Gestational Age FGR newborn percentile
28 weeks of gestation (n)
25th percentile) for standardization to establish the techniques; thus, the clinical data were not noted. The samples were collected during vaginal or C/S deliveries, and then they were filtered and centrifuged. The clear supernatant was collected, and protease inhibitor cocktail (Complete mini, Roche Diagnostics Gmbh, Mannheim, Germany) was added, after which the samples were stored in small aliquots (50 µL) at -80 °C until used. Total protein concentrations in the samples were determined by a BCA Protein Assay Kit (Pierce, Rockford, IL). IGFBP-1 Immunoenzymometric Assay (IEMA). The concentration of IGFBP-1 in amniotic fluid from both control groups (C1 and C2) and FGR samples was determined by IEMA assay (Oy Medix Biochemica, Kauniainen, Finland). The samples were diluted 400 times in the IEMA kit zero standard and analyzed in duplicate following the manufacturer’s protocol by the Multiskan EX microplate reader (Thermo Electron Corp., Helsinki, Finland) using a 414 nm filter. Phosphorylated IGFBP-1 ELISA. The level of IGFBP-1 phosphorylation was determined by a selective ELISA kit which detects the serine phosphorylated IGFBP-1 as described previously.30,36 Amniotic fluid samples from FGR (N ) 10) and control C2 (N ) 4) were diluted 10 times and analyzed in
Hyper-Phosphorylation of IGFBP-1 in Fetal Growth Restriction triplicate using the Labsystems Multiskan Multisoft microplate reader (Labsystems, Helsinki, Finland). Two-Dimensional Immunoblot Analyses. The phosphoisoforms of IGFBP-1 in the amniotic fluid samples, control (C1 and C2) (N ) 12) and FGR (N ) 10) (∼25 µg total protein each), were detected by 2-D immunoblot analyses as previously described.30 The samples were diluted in 125 µL of Rehydration (RH) buffer, and isoelectric focusing (IEF) was performed using a 7 cm immobilized pH gradient (IPG) strip with pH range 4-7 using Protean IEF Cell (BioRad). The phosphorylation of IGFBP-1 was confirmed by dephosphorylation using the control (N ) 12) and FGR (N ) 10) samples (25 µg protein each, ∼200 ng of IGFBP-1). Samples were incubated with 100 IU of calf intestinal alkaline phosphatase (ALP) (Sigma Aldrich, St. Louis, MO) at 37 °C and tested by 2-D immunoblotting. Analysis by Mass Spectrometry (MS). We had recently reported an enrichment strategy that required
