Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1

Abstract

Signal transduction from the insulin receptor to downstream effectors is attenuated by phosphorylation at a number of Ser/Thr residues of insulin receptor substrate-1 (IRS-1) resulting in resistance to insulin action, the hallmark of type II diabetes. Ser/Thr residues can also be reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) monosaccharide, a dynamic posttranslational modification that offers an alternative means of protein regulation to phosphorylation. To identify sites of O-GlcNAc modification in IRS-1, recombinant rat IRS-1 isolated from HEK293 cells was analyzed by two complementary mass spectrometric methods. Using data-dependent neutral loss MS3 mass spectrometry, MS/MS data were scanned for peptides that exhibited a neutral loss corresponding to the mass of N-acetylglucosamine upon dissociation in an ion trap. This methodology provided sequence coverage of 84% of the protein, permitted identification of a novel site of phosphorylation at Thr-1045, and facilitated the detection of an O-GlcNAc-modified peptide of IRS-1 at residues 1027-1073. The level of O-GlcNAc modification of this peptide increased when cells were grown under conditions of high glucose with or without chronic insulin stimulation or in the presence of an inhibitor of the O-GlcNAcase enzyme. To map the exact site of O-GlcNAc modification, IRS-1 peptides were chemically derivatized with dithiothreitol following beta-elimination and Michael addition prior to LC-MS/MS. This approach revealed Ser-1036 as the site of O-GlcNAc modification. Site-directed mutagenesis and Western blotting with an anti-O-GlcNAc antibody suggested that Ser-1036 is the major site of O-GlcNAc modification of IRS-1. Identification of this site will facilitate exploring the biological significance of the O-GlcNAc modification.

Fig. 1. LC-MS/MS of trypsin-digested IRS-1 peptide 1027−1073 with and without O-GlcNAc modification

A, extracted ion chromatograms showing the elution profiles of residues 1027−1073 with (m/z 1510.2) and without (m/z 1442.6) O-GlcNAc modification. B, mass spectrum acquired at 33−33.2 min. C, MS/MS of precursor at m/z 1442.5 confirming the identity of the peptide 1027−1073 (TTGAAPPPSSTASASASVTPQGAAEQAAHSSLLGGPQGPGGMSAFTR). D, MS/MS of O-GlcNAcylated precursor at m/z 1510.3 confirmed the sequence of 1027−1073 and shows loss of the monosaccharide upon CID. Generation of the neutral loss ion at 1442.6 triggered MS³ and facilitated detection of this peptide. The expected mass shift due to O-GlcNAc modification is 203.2 Da. Fragment ions in both MS/MS spectra are labeled according the predicted b and y ions of the unmodified peptide.

Fig. 2. LC-MS/MS of phosphopeptide 1039−1055 (SASASVpTPQGAAEQAAH where pT is phosphothreonine)

MS/MS of the doubly charged precursor ion at m/z 833.1 revealed Thr-1045 as the site of phosphorylation. The asterisks indicate ions that exhibited neutral loss of phosphoric acid (98 Da).

Fig. 3. MALDI-TOF MS of IRS-1 tryptic peptides before and after β-elimination/derivatization with DTT and after thiol chromatography

The insets highlight mass range 4200−5000. A, trypsin digest of oxidized, alkaline phosphatase-treated IRS-1. The average calculated [M + H]⁺ of the oxidized peptide 1027−1073 with and without O-GlcNAc modification is 4559.8 and 4356.7, respectively. B, IRS-1 peptides following β-elimination and Michael addition with DTT. The average calculated [M + H]⁺ of oxidized, DTT-derivatized 1027−1073 is 4492.0. C, sulfhydryl-reactive peptides purified by thiol chromatography. Masses that correspond to predicted DTT-derivatized peptides of IRS-1 are indicated by asterisks and are listed in Table II. The expected increases in mass due to modification by N-acetylglucosamine or derivatization with DTT are 203.2 and 136.2 Da, respectively.

Fig. 4. MS/MS of the DTT-derivatized peptide 1027−1073 precursor ion at m/z 1498.0 (3⁺)

The fragmentation pattern is consistent with DTT modification at Ser-1036 (TTGAAPPPSSTASASASVTPQGAAEQAAHSSLLGGPQGPGGMSAFTR).

Fig. 5. O-GlcNAc modification of recombinant wild type and mutant IRS-1 (IRS-1 AAA)

Wild type (wt) or mutant (AAA) IRS-1 proteins were expressed in HEK293 cells treated for 18 h with 50 μm PUGNAc to increase the level of protein O-GlcNAc modification. Nickel affinity-purified protein was separated by SDS-PAGE and transferred to nitrocellulose. The presence of O-GlcNAc modification was assessed by probing with an anti-O-GlcNAc antibody. Total IRS-1 protein loaded was assessed by probing the stripped blot with the S-protein-HRP conjugate. WB, Western blot.