Year-end Sale % OFF 
Abstract
Heparin binding to insulin-like growth factor (IGF)-binding protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for IGF-I, and a region that contains several basic amino acids (Arg201-Arg218) may be involved in this affinity shift. In the present study, mutagenesis was used to analyze the effect of substitutions for basic amino acids in the Arg201-Arg218 region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared. Their association constants (Ka) for IGF-I were similar to native IGFBP-5. When 10 microg/ml of heparin was added, the Ka of native IGFBP-5 decreased 17-fold, and the Ka of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg2O1-Arg218 region decrease the affinity shift to 1.1-3.2-fold. Lys 211 was especially important. When a mutant containing that single substitution was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity. Affinity cross-linking studies showed that heparin was equipotent in inhibiting the formation of 125I-IGF-I-K134A/Rl36A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between 125I-IGF-I and the other mutants. The heparin-binding activity of each mutant was determined. Four mutants, R201A/K202N, K202A/K206A/R207A, R201A/K202N/K206N/K208N, and K211N/R214A/K217A/R218A, had reduced heparin binding compared to native IGFBP-5. The other five mutants, including the K21IN mutant, showed no change in heparin binding. The four mutants with reduced heparin binding could be dissociated from heparin-Sepharose with much lower NaCl concentrations, indicating that they had reduced affinity. These findings suggest that Arg201 Lys202, LysS206, and Arg214 are important for heparin binding. In contrast, LyS211 is not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift.
Highlights
The abbreviations used are: Insulin-like growth factors (IGFs)-I, insulin-like growth factor-I, insulin-like growth factor-binding proteins (IGFBPs)-5, insulin-like growth factor binding protein-5; EMEM, Eagle’s minimum essential medium; ECM, extracellular matrix, AT-III, antithrombin III; PAI, plasminogen activator inhibitor
This result is consistent with our previous observations which showed that a peptide containing the Arg201–Arg218 sequence of IGFBP-5 did not alter 125I-IGF-I binding to native IGFBP-5 [7]
In this study we extended our previous observations [7] to report that site-directed mutagenesis of specific basic residues in IGFBP-5 results in a reduction of the capacity of this protein to associate with heparin
Summary
The abbreviations used are: IGF-I, insulin-like growth factor-I, IGFBP-5, insulin-like growth factor binding protein-5; EMEM, Eagle’s minimum essential medium; ECM, extracellular matrix, AT-III, antithrombin III; PAI, plasminogen activator inhibitor. Since heparin and heparan sulfate are composed of repeating disaccharides and are highly sulfated [8], they are strongly anionic These groups are believed to align with the basic residues in heparin-binding proteins. The heparin-induced affinity shift of IGFBP-5 for IGF-I has been proposed to be a two-step process, heparin-binding followed by a conformational change of IGFBP-5 that results in a decrease in its affinity. The purpose of this study was to determine the effect of substitutions for basic amino acids on the binding of IGFBP-5 to heparin and on the heparin-induced reduction in affinity for IGF-I. We prepared nine mutants of IGFBP-5 in which basic amino acids were substituted by neutral ones Their heparin-binding activities and affinity shifts in response to heparin were compared
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
