O‐glycosylation of insulin‐like growth factor (IGF) binding protein‐6 maintains high IGF‐II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis

Abstract

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had < 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a highaffinity, soluble form and so contributing to its inhibition of IGF-II actions.

Promotion of Cancer Cell Migration

“…These results suggest several potential mechanisms underlying IGFBP modulation of cell migration/invasion. IGFBP-6 does not have an RGD sequence, but it contains a heparin binding domain and so can bind to glycosaminoglycans (36). Interestingly, our previous studies investigating the role of IGFBP-6 in migration using a wound assay showed that IGFBP-6 inhibited migration in an IGF-II-dependent manner (13), which conflicts with the results presented here.…”

Glycosaminoglycans increase levels of free and bioactive IGF-I in vitro

“…Interestingly, despite their homogeneity, the IGFBPs appear to possess genuine differences in their response to GAGs: IGFBPs-3, -5 and -6 all show a reduced affinity for IGFs following GAG exposure, whereas the ligand affinity of IGFBP-4 appears to be unaffected. Data on IGFBP-2 are contradictory, but at least in some settings, IGFBP-2 has been reported to bind GAGs only in the presence of IGFs, while IGFBP-1 does not bind GAGs at all (23)(24)(25). Finally, GAGs appear to exert a larger impact on the binding of ALS to preformed complexes of IGF-I and IGFBP-3 than on the binding of IGF-I to IGFBP-3 (14).…”

“…However, both options appear to exist. The proximity of the HBD and the binding sites for IGF and ALS may explain that several different GAGs are reported to affect IGF:IGFBP as well as IGFBP-3:ALS complex formation (13,14,(22)(23)(24)(25). Interestingly, despite their homogeneity, the IGFBPs appear to possess genuine differences in their response to GAGs: IGFBPs-3, -5 and -6 all show a reduced affinity for IGFs following GAG exposure, whereas the ligand affinity of IGFBP-4 appears to be unaffected.…”

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma

“…7,40 Presumably, these multifunctional roles for IGFBP-3 are influenced by posttranslational modifications and susceptibility to proteases. 8,13,23,33 In addition to IGF-I, several other molecules such as interleukin-1, tumor necrosis factor-a, transforming growth factor-b (TGFb), retinoic acid, as well as the PI3K/AKT and MAPK/ERK1/2 pathways have been implicated in the regulation of IGFBP-3 expression. 12,40,48 The overexpression of some IGFBP isoforms has been well documented in gliomas, in particular, glioblastoma (GBM).…”