The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.
In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of >150 kDa. To investigate molecular interactions between proteins involved in IGF⅐IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of >440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs. The insulin-like growth factors (IGF-I and IGF-II)1 are important regulators of growth, differentiation, and survival.Both their mitogenic and metabolic effects are for the most part mediated through binding and activation of the IGF-I receptor (1). The interaction of IGFs with the IGF-I receptor is controlled by a family of six high affinity IGF-binding proteins (IGFBP-1 to IGFBP-6) exhibiting strong sequence homology. The cysteine-rich N-and C-terminal domains are conserved across all IGFBPs, and both domains appear to be involved in IGF binding (2, 3). In the circulation, the majority of the IGFs are sequestered into ternary 150-kDa complexes with either IGFBP-3 or IGFBP-5 and the 85-kDa acid-labile subunit, prolonging the half-life of IGFs, controlling their transcapillary transport to the target tissues, and functioning as a circulating reservoir of IGFs (4 -6). The IGFBPs can also form binary complexes with IGFs. Limited proteolysis of IGFBPs appears to be the major mechanism for the release of IGFs from binary and ternary IGFBP complexes, resulting in the generation of IGFBP fragments with reduced affinities for IGFs (7,8). Several cation-and serine-dependent serum proteases, such as a disintegrin and metalloprotease (ADAM) 12S, matrix metalloprotease-3, ␣-kallikrein, plasmin, thrombin, and the...
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