Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex

  1. Michael T Overgaard
  1. Department of Biotechnology, Chemistry, and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark
    1Division of Endocrinology, Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota, USA
  1. Correspondence should be addressed to M T Overgaard; Email: mto{at}bio.aau.dk
  1. Figure 1

    Overview of protein complexes investigated. The first column shows the nomenclature used throughout the text. The second and third columns illustrate the specific mutations introduced in IGF2 or IGFBP2 respectively. The numbering refers to the sequences of mature human IGF2 and murine IGFBP2 respectively (Fig. 2). The fourth column shows cartoons of the individual complexes, which will be used to aid nomenclature. IGFBP2 (N-terminal domain, blue; linker region, white; and C-terminal domain, green), IGF2 (red), and mutations (black circles).

  2. Figure 2

    Amino acid sequence alignments for homology modeling with annotations added. (Upper and middle panels) Sequences for the N-terminal domain (Glu1–Arg84) and C-terminal domain (Arg170–Gln271 plus adjacent linker region residues Pro161–Ala169) from mature murine IGFBP2 aligned with the corresponding parts of mature human IGFBP4. (Lower panel) Mature human IGF2 is aligned with human IGF1. IGFBP2 linker (Lys162–Arg165) and C-terminal domain (Lys209–His210, Lys216, and His253) HBD residues are colored in gray along with the proposed IGF2 HBD residues (Arg37–Arg40). The charged residues mutated to Ala in this study are boxed. IGF2-binding residues of IGFBP2 are highlighted in orange. IGF2 residues involved in IGF1R (turquoise) and IGF2R (olive green) binding are also highlighted (Butler et al. 1998, Clemmons 2001, Kuang et al. 2006, Williams et al. 2007, Brown et al. 2008). Disulfide bridges identified in this study are shown with black lines. Identified disulfide clusters are shown with gray lines. Numbering corresponds to mature murine IGFBP2 and human IGFBP4, IGF1, and IGF2 as indicated.

  3. Figure 3

    Analysis of protein-folding integrity. (A) SDS–PAGE analysis of the purified protein complexes. (B) Far-u.v. CD spectra of protein complexes in acetate buffer at pH 5.5 and 25 °C. θ*MRW is the corrected mean residue weight ellipticity.

  4. Figure 4

    Heparin affinities of IGF/IGFBP complexes. Columns indicate mean and bars the s.d. of triplicates at pH 5.5. Lettering indicates statistically unique values as determined from two-way ANOVA with pairwise comparisons (P<0.01). IGF2/IGFBP4 did not bind to the column at the applied loading conditions (κr∼8 mS/cm). Cartoons are added to aid the nomenclature with IGFBP3 N- and C-terminal domains in brown and orange respectively. IGFBP4 N- and C-terminal domains are in sea green and turquoise color, respectively, also IGF1 is in gray color. See Fig. 1 for additional color legend. Lines indicate κr for loading conditions (dotted) and PBS (solid).

  5. Figure 5

    Heparin affinities of free IGFBPs. Information in graph is the same as described for Fig. 4. Lettering indicates statistically unique values as determined from two-way ANOVA with pairwise comparisons (P<0.01).

  6. Figure 6

    Heparin affinities of IGFBP2, IGF2/IGFBP2, and IGF2 HBD mutated complex. Information in the graph is the same as described for Fig. 4. Lettering indicates statistically unique values as determined from two-way ANOVA with pairwise comparisons (P<0.001).

  7. Figure 7

    Heparin affinities of IGF2/IGFBP2 variants as a function of pH. Note that the IGF2/IGFBP_C-term-HBD lacked His209 and demonstrated reduced pH sensitivity. Notations are as given in Fig. 1. Connecting lines drawn to guide the eye. Error bars indicate s.d. (in most cases smaller than the symbol sizes). All values were statistically significant and different from each other as determined from two-way ANOVA with pairwise comparisons (P<0.001).

  8. Figure 8

    Three-dimensional, structural model of the IGF2/IGFBP2 complex based on homology modeling to the human IGF1/IGFBP4 complex. (A and B) Surface rendered model ((A) IGF2 shown as cartoons), highlighting the different features of the complex. IGFBP2 N- and C-terminal (including linker domain residues Pro161–Ala169) domains (blue and green respectively), IGF2-binding residues (orange), IGF2 (red), HBD residues (gray), IGF1R-binding residues (turquoise) and IGF2R-binding residues (olive green), attachment points for the linker region (purple), and control mutations (black). (C) Surface rendering with charge plot. The modeled IGFBP2 N-terminal domain is in good agreement with the one presented by Galea et al. (2012).

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