Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: Evidence for processing of cell-bound IGFBP-3

Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coliderived recombinant human IGFBP-3 (hIGFBP-3^E.coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3^CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [¹²⁵I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3^E.coli or hIGFBP-3^CHO produced a dose-dependent inhibition of IGF-I- but not insulin-stimulated [³H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGFBP-3^E.coli or hIGFBP-3^CHO potentiated subsequent IGF-I-stimulated [³H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3^E.coli for 24 h, [¹²⁵I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [³H] AIB uptake. The increase in [¹²⁵I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3^E.coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [¹²⁵I]IGF-I to cell-associated 29,000 M_r hIGFBP-S^E.coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3^E.coli forms of 12,000-27,000 M^r. Cell-associated IGFBP-3^E.coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3^E.coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.