Please use this identifier to cite or link to this item: http://ahro.austin.org.au/austinjspui/handle/1/9243
Title: 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.
Authors: Marinaro, J A;Neumann, G M;Russo, V C;Leeding, K S;Bach, Leon A
Affiliation: University of Melbourne, Department of Medicine, Austin & Repatriation Medical Centre, Heidelberg, Victoria, Australia.
Issue Date: 1-Sep-2000
Citation: European Journal of Biochemistry / Febs; 267(17): 5378-86
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 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.
Internal ID Number: 10951195
URI: http://ahro.austin.org.au/austinjspui/handle/1/9243
URL: http://www.ncbi.nlm.nih.gov/pubmed/10951195
Type: Journal Article
Subjects: Amino Acid Sequence
Animals
Glycosaminoglycans.metabolism
Glycosylation
Humans
Hydrolysis
Insulin-Like Growth Factor Binding Protein 6.chemistry.metabolism
Insulin-Like Growth Factor II.metabolism
Mass Spectrometry
Molecular Sequence Data
PC12 Cells
Protein Binding
Rats
Recombinant Proteins.metabolism
Trypsin.metabolism
Appears in Collections:Journal articles

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