Adenoviral-mediated expression of human insulin-like growth factor-binding protein-3.

Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGFBP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGFBP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3(Ad)) was purified from the culture medium of virus-infected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3(Ad) was similar in size (43- to 45-kDa glycoform doublet) to IGFBP-3(Pl) derived from plasma. In addition, IGFBP-3(Ad) was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3(Ad) had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3(Pl). IGFBP-3(Ad) showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3(Pl), but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3(Ad) also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.

Structural determinants of ligand and cell surface binding of insulin-like growth factor-binding protein-3.

Among the well defined insulin-like growth factor (IGF)-binding proteins (IGFBPs), IGFBP-3 is characterized by its interaction with an acid-labile glycoprotein (ALS) in the presence of IGFs. To identify the structural determinants on IGFBP-3 required for ligand binding and cell association, five recombinant human IGFBP-3 variants were expressed in Chinese hamster ovary cells: deletions of amino acids 89-264, 89-184, and 185-264, and site-specific mutations 228KGRKR --> MDGEA and 253KED --> RGD. The basic carboxyl-terminal region of IGFBP-3 was required for binding to heparin. The deletion variants had greatly decreased IGF binding ability as assessed by ligand blotting and solution binding assays; affinity cross-linking indicated at least a 20-fold decrease in IGF affinity. The RGD mutant had a 4-6-fold reduced affinity for both IGFs, but the MDGEA mutant bound IGF-I with near normal affinity and IGF-II with a 3-fold reduction in affinity. The three deletion variants were incapable of binding ALS; but of the site-specific variants, the MDGEA mutant bound ALS with 90% lower affinity (Ka = 2.5 +/- 0.9 liters/nmol) than seen for rhIGFBP-3 (Ka = 24.3 +/- 5.2 liters/nmol), whereas the RGD mutation had no effect on ALS affinity (Ka = 21.7 +/- 4.5 liters/nmol). The ability of IGFBP-3 to associate with the cell surface was lost in variants lacking residues 185-264 and in the 228KGRKR --> MDGEA mutant. We conclude that residues 228-232 of IGFBP-3 are essential for cell association and are required for normal ALS binding affinity.