Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Alberto Sala,Stefano Capaldi,Monica Campagnoli,Beniamino Faggion,Sara Labò,Massimiliano Perduca,Assunta Romano,Maria E Carrizo,Maurizia Valli,Livia Visai,Lorenzo Minchiotti,Monica Galliano,Hugo L Monaco

doi:10.1074/jbc.m504304200

Alberto Sala, Stefano Capaldi + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m504304200

Copy DOI

Abstract

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

Highlights

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thought to play a key role at the fetal-maternal interface
The first 15 residues of a homogeneous 11-kDa polypeptide chain were VTNIKKWKEPCRIEL, and a search in the SWISSPROT data bank revealed that this sequence was identical to an internal amino acid sequence of IGFBP-1 starting from residue 166
The anion exchange chromatography retention times of the isoforms correlate with their more acidic pI values, and both acquire the same net charge of the non-phosphorylated protein after treatment with alkaline phosphatase. These results show that the IGFBP-1 isoforms present in human amniotic fluid are differently phosphorylated forms of the protein

Summary

Introduction

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and the domain has a biological activity on its own. IGFBP-1 is normally expressed in a tissue-specific manner in the liver, kidney, decidualized endometrium, and luteinizing granulosa cells [12] It is the predominant IGFBP in amniotic fluid [13], a major IGF-binding protein in fetal plasma, and its concentration is increased in maternal circulation during pregnancy. The mature polypeptide chain of IGFBP-1 is composed of 234 amino acids (the unprocessed precursor is 259 amino acids long) and is phosphorylated at

Methods

Results

Conclusion