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

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glyco protein which specifically inhibits insulin-like growth factor (IGF)-II act ions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminogl ycans and proteolysis, both of which reduce the IGF binding affinity of oth er IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefo ld greater than that of glycosylated (g) IGFBP-6. When bound to glycosamino glycans, IGFBP-6 had approximate to 10-fold reduced binding affinity for IG F-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 i nhibited by increasing salt concentrations, which is typical of glycosamino glycan interactions. O-glycosylation also protected human IGFBP-6 from prot eolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affi nity of IGFBP-6 for IGF-II, even with a relatively small reduction in appar ent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGF BP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-termi nal peptide was removed and peptide bonds involved in the putative high aff inity 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-glycosyla tion inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes a nd inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity , soluble form and so contributing to its inhibition of IGF-II actions.