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

Mark E Graham,Dean M Kilby,Sue M Firth,Phillip J Robinson,Robert C Baxter

doi:10.1074/mcp.m700027-mcp200

Abstract

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 Ser(96) in the central domain, and a minor phosphorylation site was Ser(248) 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, Ser(106). 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.

Highlights

Mass spectrometry is often used to determine post-translational modifications by analysis of tryptic digests of proteins
In the case of IGFBP-3 and insulin-like growth factor-binding protein-5 (IGFBP-5), binary complexes with Insulin-like growth factor (IGF) can associate with a third protein, the acid-labile subunit (ALS), to form high molecular weight ternary complexes that act as relatively stable reservoirs of circulating IGF-I and IGF-II
IGFBP-5 Is Secreted as a Phosphoprotein—Metabolic labeling with 32Pi indicated that IGFBP-5 is secreted as a phosphoprotein by T47D human breast carcinoma cells

Summary

EXPERIMENTAL PROCEDURES

Metabolic 32P Labeling and Purification of IGFBP-5 from T47D Human Breast Carcinoma Cells—The method of in vivo radiolabeling of IGFBP-5 was similar to that used by Pattison et al [17]. Intact IGFBP-5 protein, either AdIGFBP-5 or protein purified from T47D cell medium, was reconstituted in formic acid and diluted to 0.1% formic acid It was concentrated and desalted using a Poros R2 microcolumn to a final concentration of 20 ␮M and sprayed into the QSTAR XL mass spectrometer using a nanoelectrospray capillary in 0.5% formic acid, 50% acetonitrile aqueous solution. Ternary complex formation assays were performed using 125IALS (10,000 cpm/100 ␮l) to which IGFBP-5 (0.01–5 ng) was allowed to bind in the presence of either IGF-I (10 ng) or IGF-II (10 ng) in a final volume of 300 ␮l of buffer containing 50 mM sodium phosphate, pH 6.5, 0.25% BSA for 2 h at 22 °C. In-house IGFBP-5 antiserum (SFK Y.1, 5 ␮l) was added and incubated overnight at 4 °C, and complexes were precipitated using sheep anti-chicken ␥-globulin (2 ␮l) followed by 1 ml of 60 g/liter polyethylene glycol 6000 in 0.15 M NaCl and centrifugation

RESULTS

HexNAc and Hex

DISCUSSION