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Molecular & Cellular Proteomics 6:1392-1405, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5 ^*,S

Mark E. Graham^,, Dean M. Kilby^,¶, Sue M. Firth^¶, Phillip J. Robinson and Robert C. Baxter^¶,||

From the Cell Signalling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia and the ^¶ Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia

Mass spectrometry is often used to determine post-translational modifications by analysis of tryptic digests of proteins. Here we demonstrate that the analysis of tryptic peptides together with analysis of the full-length protein provided optimal characterization of insulin-like growth factor-binding protein-5 (IGFBP-5) phosphorylation and glycosylation. IGFBP-5 binds insulin-like growth factors with high affinity and has important roles in cell survival, differentiation, and apoptosis. Until now, the primary structure of IGFBP-5 has been incompletely defined. We analyzed human IGFBP-5 from T47D cells by mass spectrometry to determine all of the in vivo post-translational modifications. In full-length IGFBP-5, 31% of the protein was unmodified, 37% was monophosphorylated, and 4% was diphosphorylated with no other modification. The remaining 27% was glycosylated, more than half of which was also monophosphorylated. The major phosphorylation site was Ser⁹⁶ in the central domain, and a minor phosphorylation site was Ser²⁴⁸ near the C terminus. Neither site was phosphorylated in vitro by casein kinase 2, ruling it out as the in vivo kinase. An in vivo phosphorylation site was also found in IGFBP-2 at an analogous position, Ser¹⁰⁶. IGFBP-5 was heterogeneously O-glycosylated mainly by sialylated core 1 type glycans. The most abundant structure contained N-acetylhexosamine, hexose, and two N-acetylneuraminic acid carbohydrates. A small amount of sialylated core 2 type glycan was also present. Phosphorylation and O-glycosylation both affected IGFBP-5 binding to heparin but not insulin-like growth factor binding or ternary complex formation with the acid-labile subunit. The results reveal the first description of the in vivo phosphorylation of IGFBP-5 and its glycan composition.

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