Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain.

Abstract

Within the IGF axis, the insulin-like growth factor-binding proteins (IGFBPs) are known to play a pivotal role in cell proliferation and differentiation. Defined proteolysis of the IGFBPs is proposed to be an essential mechanism for regulating IGF bioavailability. The generated IGFBP fragments in part exhibit different IGF-dependent and -independent biological activities. Characterizing naturally occurring forms of IGFBPs in human plasma, we identified both a N- and a C-terminal fragment of IGFBP-4 by means of immunoreactivity screening. As a source for peptide isolation, we used large amounts of human hemofiltrate obtained from patients with chronic renal failure. Purification of the IGFBP-4 peptides from hemofiltrate was performed by consecutive cation-exchange and reverse-phase chromatographic steps. Mass spectrometric and sequence analysis revealed an M(r) of 13 233 for the purified N-terminal fragment spanning residues Asp(1)-Phe(122) of IGFBP-4 and an M(r) of 11 344 for the C-terminal fragment extending from Lys(136) to Glu(237). Proteolytic digestion and subsequent biochemical analysis showed that the six cysteines of the C-terminal IGFBP-4 fragment are linked between residues 153-183, 194-205, and 207-228 (disulfide bonding pattern, 1-2, 3-4, and 5-6). Plasmon resonance spectroscopy, ligand blot analysis, and saturation and displacement studies demonstrated a very low affinity of the C-terminal IGFBP-4 fragment for the IGFs (IGF-II, K(d) = 690 nM; IGF-I, K(d) > 60 nM), whereas the N-terminal fragment retained significant IGF binding properties (IGF-II, K(d) = 17 nM; IGF-I, K(d) = 5 nM). This study provides the first molecular characterization of circulating human IGFBP-4 fragments formed in vivo exhibiting an at least 5-fold decrease in the affinity of the N-terminal IGFBP-4 fragment for the IGFs and a very low IGF binding capacity of the C-terminal fragment.