Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I in response to heparin

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 (ATg²⁰¹-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 (K_a) for IGF-I were similar to native IGFBP-5. When 10 μg/ml of heparin was added, the K_a of native IGFBP-5 decreased 17-fold, and the K_a of the K134A/ R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the ATg²⁰¹-ATg²¹⁸ region decrease the affinity shift to 1.1-3.2-fold. Lys²¹¹ 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/R136A 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 K211N 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, Lys²⁰⁶, 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.