The N-terminal disulfide linkages of human insulin-like growth factor-binding protein-6 (hIGFBP-6) and hIGFBP-1 are different as determined by mass spectrometry

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1-6), IGFBP-6 differs from o ther IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20-100-fold lower affinity. IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines, The complete dis ulfide linkages of IGFBP-6 were determined using electrospray ionization ma ss spectrometry of purified tryptic peptide complexes digested with combina tions of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGF BP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys(1)-Cys(2), Cys(3)-Cys(4), and Cys(5)-Cys(6). The next two linkages are Cys(7)-Cys(9) and Cys(8)-Cys(10), which are analogous to those previously determined for IGFBP-3 and IGFBP-5, The C-terminal linkages are Cys(11)-Cys(12), Cys(13)-Cys(14), and Cys(15)-Cys(16), analogous to those previously determined for IGFBP-2, Disulfide linkages of IGFBP-1 were parti ally determined and show that Cys(1) is not linked to Cys(2) and Cys(3) is not linked to Cys(4), Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys(9)- Cys(11) and Cys(10)-Cys(12) of IGFBP-1 are also disulfide-linked, The N-ter minal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctiv e IGF binding properties of IGFBP-6.