The microtubule-associated protein tau is extensively modified with O-linked N-acetylglucosamine.

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimer's disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk β(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimer's disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk ␤(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.
Tau is a group of closely related microtubule-associated pro-teins that constitute an important component of the neuronal cytoskeleton (1). Tau proteins are generated by differential mRNA splicing of a single gene resulting in multiple isoforms in the adult mammalian brain (2)(3)(4). Tau is a phosphoprotein (5)(6)(7), and this post-translational modification clearly regulates both its function and metabolism (8 -10). Hyperphosphorylated tau is the primary component of the paired helical filaments (PHFs) 1 observed in Alzheimer's disease brains (11,12). Analysis of PHF-tau by ion spray mass spectrometry and peptide sequencing has revealed that 19 sites are at least partially phosphorylated (7,13). Other post-translational modifications of PHF-tau have been reported. For example, PHF-tau is both nonenzymatically glycated (14) and ubiquitinated (15)(16)(17). Recently, the presence of N-linked and (mucin-type) O-linked oligosaccharides on PHF-tau has also been suggested (18). However, the presence of these types of oligosaccharides on cytosolic proteins is highly controversial and has yet to be definitively documented for any protein (19,20).
Cytoskeletal proteins such as neurofilaments (21), synapsins (22), and cytokeratins (23) are modified by O-linked N-acetylglucosamine (O-GlcNAc). Recently, the high molecular weight microtubule associated proteins MAP1, MAP2, and MAP4 have been shown to contain O-GlcNAc (24). This type of glycosylation is dynamic, consisting of a single monosaccharide, N-acetylglucosamine, glycosidically linked to side chain hydroxyls of serine and threonine residues on nuclear and cytoplasmic proteins of eukaryotes (25) (for reviews see Refs. 26 -28). Although the exact function of this modification is not well understood, many lines of evidence suggest it to be an essential and abundant regulatory modification present in virtually all eukaryotes. For example, unlike other forms of protein glycosylation, O-GlcNAc turns over much more rapidly than the peptide backbone (29). O-GlcNAc is postulated to have modulatory functions similar to phosphorylation (30,31). This hypothesis is supported by the finding that O-GlcNAc bearing sites on certain proteins have been demonstrated to coincide with known sites of phosphorylation (32,33). Furthermore, O-GlcNAcylation appears to regulate DNA binding by the p53 tumor suppressor protein (34) and to regulate protein synthesis by controlling the activity of EIF2-kinase (35,36). Given phosphorylation's likely role in the formation of PHF-tau and the discovery that many cytoskeletal phosphoproteins are also O-GlcNAcylated (20, 37), we decided to investigate the possible glycosylation of normal tau. This study demonstrates that tau isolated from bovine brain is extensively modified by O-GlcNAc. antibody Tau2 (6) was a generous gift from Dr. L. Binder. All other reagents were of the highest commercial grade available.

Materials
Purification of Bovine Tau-Tau was purified to homogeneity from bovine brains (RJO Biologicals) as described previously (38,39).
Galactosyltransferase Labeling of Tau Proteins and Separation of 3 H-Labeled Tau Glycopeptides-Tau polypeptides were probed for terminal GlcNAc using Gal␤(1-4)galactosyltransferase (galactosyltransferase) and UDP-[ 3 H]galactose as described by Roquemore et al. (40). Bovine tau was separated into individual isoforms by electrophoresis on SDS-polyacrylamide gels. Individual polypeptide bands were excised from the gel with a blade and extracted with 20 mM Tris, pH 7.5, 0.1% SDS. Radiolabeled tau glycopeptides were isolated from SDS-polyacrylamide gels after digesting with trypsin as described by Rosenfeld et al. (41). Glycopeptides were separated by RP-HPLC on a Rainin HPLC system equipped with a Vydac C18 column (4.6 ϫ 250 mm) through a linear 2-h gradient of acetonitrile (0 -40%) in buffer containing 0.1 M sodium perchlorate and 1% phosphoric acid, pH 2.1, at a flow rate of 1 ml/min (42). Absorbance of the eluent was monitored at 214 nm, and tritium was detected with an on-line Flo1␤ scintillation detector.
␤-Elimination and Product Analysis-Purified bovine tau polypeptides were labeled with [ 3 H]galactose using galactosyltransferase followed by ␤-elimination and saccharide analysis, which was performed as described previously (21,43).
Stoichiometry Estimation of O-GlcNAc on Tau-Stoichiometry analysis was performed in triplicate as described by Roquemore et al. (44) with some modifications. Briefly, purified bovine tau polypeptides in 20 mM MES, 1 mM EGTA, 1 mM MgSO 4 were desalted by gel filtration, precipitated with six volumes of acetone, and resuspended in deionized water, and protein levels were determined by Micro bicinchoninic acid (45) or Amido Schwarz (46). A relative molecular mass of 46.2 kDa, which represented an average of all bovine isoforms (3), was used in all calculations. Prior to hydrolysis, [ 3 H]glucosamine (100,000 cpm) was added to each sample to determine percent recovery. Amino sugars were isolated on 0.5 ml of Dowex AG 50W-X8 ([H ϩ ] form) columns with 2 M HCl after washing with 20% methanol. The amount of glucosamine released from acid hydrolysis was quantified by peak areas of glucosamine standards on a Dionex CarboPac PA1 column. Identity of the glucosamine peak was confirmed by comigration with GlcNAc after re-N-acetylation (47).
Protein Determination-Protein concentrations were determined by Lowry et al. (49), micro bicinchoninic acid assay (Pierce), or Amido Schwarz dye binding assay using bovine serum albumin as a standard.

Galactosyltransferase Labels Terminal GlcNAc Moieties on Tau-The presence of terminal GlcNAc residues on bovine tau
is demonstrated by labeling tau with [ 3 H]galactose using galactosyltransferase. Tau purified from bovine brain migrates on SDS-polyacrylamide gels as five polypeptides with relative molecular masses between 45 and 60 kDa (3,38), and all are reactive with the tau monoclonal antibody Tau2 (Fig. 1A). The identification of terminal GlcNAc residues with galactosyltransferase is highly specific (26), resulting in the formation of [ 3 H]Gal␤(1-4)GlcNAc moieties on a glycoprotein. After incubating tau with galactosyltransferase in the presence of UDP-[ 3 H]galactose, labeling of all tau isoforms is observed (Fig. 1B). The radiolabel observed in lane 2 is due to incorporation into bovine tau because there is no detectable labeling of the enzyme alone (lane 1).
The lectin sWGA also documents the presence of multiple terminal GlcNAc residues on bovine tau. sWGA binds specifically to GlcNAc (50). Lectin blots shown in Fig. 2 demonstrate that sWGA binds to purified bovine tau. The specificity of the sWGA lectin for GlcNAc is illustrated by effectively competing the lectin with 0.5 M GlcNAc, whereas 0.5 M galactose has little effect (Fig. 2). Together, these data indicate the existence of multiple terminal GlcNAc containing moieties on purified bovine tau.
Tau Is Glycosylated with O-linked GlcNAc-To determine the nature of the [ 3 H]galactose-labeled residues on tau, labeled protein was first treated with peptide:N-glycosidase F (PNGase F), an enzyme that specifically cleaves N-linked carbohydrates from the peptide backbone (51). Treatment of tau with PNGase F did not remove the [ 3 H]galactose from tau, indicating that the carbohydrates on tau are not N-linked. In contrast, [ 3 H]galactosylated ovalbumin, a protein known to contain Nlinked carbohydrates (52), is PNGase F-sensitive (data not shown). The [ 3 H]galactose-labeled carbohydrates on tau are, however, susceptible to alkaline ␤-elimination (Fig. 3A), indicating that they are linked to the polypeptide via O-glycosidic linkages. TSK-Gel filtration of the [ 3 H]galactose-labeled compounds released by alkaline ␤-elimination demonstrate their migration as reduced disaccharides (Fig. 3B). Further analysis of the [ 3 H]galactose-labeled material by Dionex high pH anion exchange chromatography-pulsed amperometric detection shows that the labeled saccharides are derived from O-GlcNAc. Fig. 3C shows the co-migration of the [ 3 H]galactose-labeled compounds with Gal␤(1-4)GlcNAcitol, the expected product of a galactose-labeled O-GlcNAc released by reductive, alkaline ␤-elimination. Thus, purified bovine tau is modified with O-GlcNAc.
All Bovine Tau Isoforms Are Extensively Glycosylated-To determine if any differences exist in the glycosylation of the different bovine tau isoforms, tryptic maps of each [ 3 H]galactose-labeled tau isoform were generated by RP-HPLC. The purified galactosyltransferase-treated polypeptide mixture was separated into its major species by SDS-polyacrylamide gel electrophoresis (Fig. 4A). In-gel trypsin digestion was performed on each isoform, and the peptides released from the gel were separated by RP-HPLC (Fig. 4B). The resulting profiles clearly suggest that the tau isoforms contain numerous sites of O-GlcNAcylation. The similar retention times for many of the peaks indicate that all of the isoforms appear to share similar sites of glycosylation. The minor differences between the different isoforms could represent some stoichiometry differences at individual sites of glycosylation or differential trypsin cleavage due to amino acid sequence diversity.
The number of glycosylation sites observed on tau appears to be relatively high as compared with certain other known O-GlcNAc-modified proteins (21,29,44). The stoichiometry of tau glycosylation was determined and found to be 4.2 Ϯ 0.9 mol O-GlcNAc/mol tau (n ϭ 3 separate determinations). Initial site mapping data indicate that one major attachment site for O-GlcNAc on tau is localized to a domain involved in microtubule binding (2,3). These data are further supported by the finding that a synthetic peptide based upon these mapping studies, and containing the tau sequence VKSKIGSTENLKHQ is a substrate in vitro (data not shown) for the O-GlcNAc transferase (53). Further analysis of the in vivo tau glycosylation sites is being facilitated by the development of more sensitive methods (54,55).
The high degree of glycosylation of bovine tau indicates a possible structural role for these glycoconjugates on tau. The O-GlcNAcylation of other microtubule-associated proteins, MAP1, MAP2, and MAP4, also suggests a role for O-GlcNAc in mediating the interactions of the MAPs with tubulin. Recently, tau has been shown to be associated with the neural plasma membrane (56) and also localized to the nucleus (57). The O-GlcNAcylation of tau may play a role in its subcellular localization. The demonstration of O-GlcNAc attachment to known phosphorylation sites of nuclear proteins, c-Myc (33) and RNA polymerase II (32), and the exclusive reciprocity of O-GlcNAc and phosphate on some O-GlcNAc modified proteins (33) suggest that tau glycosylation and phosphorylation may also be reciprocally related. Phosphorylation modulates tau's interactions with tubulin and the proteolysis of tau (58). Therefore, tau O-GlcNAcylation could potentially regulate tau degradation. The fact that O-GlcNAc has not been detected on PHFs (14), which are extensively phosphorylated at numerous sites (7), supports this hypothesis concerning the reciprocal nature of these two modifications. Thus, these data suggest the intriguing possibility that in Alzheimer's disease, PHF-tau formation may result from the defective O-GlcNAcylation of tau, thus allowing or even promoting abnormal tau hyperphosphorylation. O-GlcNAc's putative involvement in the etiology of Alzheimer's disease is made more intriguing by the recent finding of O-GlcNAc on the cytoplasmic domain of the ␤-amyloid precursor protein (59) and by the direct observation of altered O-GlcNAcylation in the brains of Alzheimer's disease patients (60). Future investigations will directly evaluate the role(s) of O-GlcNAcylation in microtubule assembly and organization. One obvious implication predicted from the present study is that inhibitors of enzymes specific for the removal of O-GlcNAc (61) could prevent PHF-tau formation by not allowing tau hyperphosphorylation to occur.