title: Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex.
abstract: IGF1 and IGF2 are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF-binding proteins (IGFBP1-IGFBP6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulfated glycosaminoglycans (GAGs), e.g. heparin and heparan sulfate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 complex to the bone matrix. In order to obtain a more detailed understanding of the interactions between the IGF2/IGFBP2 complex and GAGs, we investigated heparin-binding properties of IGFBP2 and the IGF2/IGFBP2 complex in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin-binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared with free intact IGFBP2.
authors.affiliation: Department of BiotechnologyChemistry, and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, DenmarkDivision of EndocrinologyEndocrine Research Unit, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Lund
authors.affiliation: Department of BiotechnologyChemistry, and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, DenmarkDivision of EndocrinologyEndocrine Research Unit, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Mads T
authors.initials: MT
authors.lastname: Søndergaard
authors.affiliation: Department of BiotechnologyChemistry, and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, DenmarkDivision of EndocrinologyEndocrine Research Unit, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Conover
authors.affiliation: Department of BiotechnologyChemistry, and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, DenmarkDivision of EndocrinologyEndocrine Research Unit, Mayo Clinic, Rochester, Minnesota, USA mto@bio.aau.dk.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics*; D002846:Chromatography, Affinity; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts / Q000378:metabolism; D010012:Osteogenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment
chemical_list: C035170:IGF2 protein, human; D018971:Insulin-Like Growth Factor Binding Protein 2; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II; D006493:Heparin
keywords: IGF2/IGFBP2 complex; glycosaminoglycans; heparin affinity; ternary binding model