Synthesis and Characterization of Biotinylated Forms of Insulin-like Growth Factor-1: Topographical Evaluation of the IGF-1/IGFBP-2 and IGFBP-3 Interface,
Аннотация:Activation of the insulin-like growth factor-1 (IGF)-1 receptor signaling pathways by IGF-1 and IGF-2 results in mitogenic and anabolic effects. The bioavailability of the IGFs is regulated by six soluble binding proteins, the insulin-like growth factor binding proteins (IGFBPs), which bind with ∼0.1 nM affinity to the IGFs and often serve as endogenous antagonists of IGF action. To identify key domains of IGF-1 involved in the interaction with IGFBP-2 and IGFBP-3, we employed IGF-1 selectively biotinylated on residues Gly 1, Lys 27, Lys 65, and Lys 68. All monobiotinylated species of IGF-1 exhibited high affinity (∼0.1−0.2 nM) for IGFBP-2 and IGFBP-3 in solid-phase-binding assays. However, different labeling intensities were observed in ligand blot analysis of IGFBP-2 and IGFBP-3. The NεLys65/68(biotin)-IGF-1 (NεLys65/68b-IGF-1) probe exhibited the highest signal intensity, while NαGly1b-IGF-1 and NεLys27b-IGF-1 demonstrated significantly lower signals. When taken together, these results suggest that, once bound to IGFBP-2 or IGFBP-3, the biotin moieties of NαGly1b-IGF-1 and NεLys27b-IGF-1 are inaccessible to NeutrAvidin-peroxidase, the secondary binding component. Ligand blots using IGF-1 derivatized with a long chain form of the N-hydroxysuccinimide biotin (NHS-biotin) to yield NαGly1(LC-biotin)-IGF-1 and NεLys27(LC-biotin)-IGF-1 demonstrated increased signal intensity compared with their NHS-biotin counterparts. In BIAcore analysis, IGFBP-2 and IGFBP-3 bound only to the NεLys65/68b-IGF-1-coated flowcell of a biosensor chip, confirming the inaccessibility of Gly 1 and Lys 27 when IGF-1 is bound to IGFBP-2 and IGFBP-3. These data confirm the involvement of the IGFBP-binding domain on IGF-1 in binding to IGFBP-2 and IGFBP-3 and support involvement of the IGF-1R-binding domain in IGFBP binding.
