Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles

O-linked glycosylation on Ser/Thr with single N-acetylglucosamine ( O-GlcNAcylation) is a reversible modification of many cytosolic/nuclear proteins, regulated in part by UDP-GlcNAc levels. Transgenic (T) mice that overexpress GLUT1 in muscle show increased basal muscle glucose transport that is resistant to insulin stimulation. Muscle UDP-GlcNAc levels are increased. To assess whether GLUT4 is a substrate for O-GlcNAcylation, we translated GLUT4 mRNA (mutated at the N-glycosylation site) in rabbit reticulocyte lysates supplemented with [35S]methionine. O-GlcNAcylated proteins were galactosylated and separated by lectin affinity chromatography; >20% of the translated GLUT4 appeared to be O-GlcNAcylated. To assess whether GLUT4 or GLUT4-associated proteins were O-GlcNAcylated in muscles, muscle membranes were prepared from T and control (C) mice labeled with UDP-[3H]galactose and immunoprecipitated with anti-GLUT4 IgG (or nonimmune serum), and N-glycosyl side chains were removed enzymatically. Upon SDS-PAGE, several bands showed consistently two- to threefold increased labeling in T vs. C. Separating galactosylated products by lectin chromatography similarly revealed approximately threefold more O-GlcNAc-modified proteins in T vs. C muscle membranes. RL-2 immunoblots confirmed these results. In conclusion, chronically increased glucose flux, which raises UDP-GlcNAc in muscle, results in enhanced O-GlcNAcylation of membrane proteins in vivo. These may include GLUT4 and/or GLUT4-associated proteins and may contribute to insulin resistance in this model.

Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.