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 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 approximately 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 high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.
Marinaro
J A
JA
University of Melbourne, Department of Medicine, Austin & Repatriation Medical Centre, Heidelberg, Victoria, Australia.
Neumann
G M
GM
Russo
V C
VC
Leeding
K S
KS
Bach
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D006025:Glycosaminoglycans / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000737:chemistry / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006025:Glycosaminoglycans; D018976:Insulin-Like Growth Factor Binding Protein 6; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D014357:Trypsin