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(Received for publication, September 5, 1996, and in revised form, September 27, 1996)
From the ABSTRACT 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 INTRODUCTION Tau is a group of closely related microtubule-associated proteins
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, 9, 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, 27, 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.
EXPERIMENTAL PROCEDURES UDP-[6-3H]galactose (38 Ci/mmol)
was obtained from Amersham Corp. [3H]Glucosamine (40 Ci/mmol) was from DuPont NEN. Bovine milk galactosyltransferase was
from Sigma. Tosylphenylalanyl chloromethyl
ketone-treated trypsin was from Worthington, and saccharide standards
were from Dionex. Horseradish peroxidase-conjugated succinyl wheat germ agglutinin, Triticum vulgaris (sWGA-horseradish peroxidase),
was from E-Y Laboratories. The tau monoclonal antibody Tau2 (6) was a
generous gift from Dr. L. Binder. All other reagents were of the
highest commercial grade available.
Tau was purified to homogeneity
from bovine brains (RJO Biologicals) as described previously (38,
39).
Tau polypeptides were
probed for terminal GlcNAc using Gal Purified bovine tau
polypeptides were labeled with [3H]galactose using
galactosyltransferase followed by 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 MgSO4 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, [3H]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+] 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).
Tau proteins were resolved on 8%
SDS-polyacrylamide gels (48), stained with Coomassie Brilliant Blue
R-250 (Bio-Rad), treated with 2-Hydroxybenzoic acid, dried, and exposed
to X-OMAT film (Kodak).
Proteins were
transferred to polyvinylidine difluoride (PVDF) or nitrocellulose
membranes equilibrated in 25 mM Tris, pH 8.5, 190 mM glycine, and 20% methanol. After blocking the membranes with 5% bovine serum albumin in 125 mM NaCl, 20 mM Tris, pH 8.0, 0.1% Tween 20 (TBST), proteins were
probed for GlcNAc with sWGA-horseradish peroxidase in TBST (1:5,000)
and developed with 3,3 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.
RESULTS AND DISCUSSION The presence of terminal GlcNAc residues on bovine tau is
demonstrated by labeling tau with [3H]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
[3H]Gal
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.
To determine the
nature of the [3H]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
[3H]galactose from tau, indicating that the carbohydrates
on tau are not N-linked. In contrast,
[3H]galactosylated ovalbumin, a protein known to contain
N-linked carbohydrates (52), is PNGase
F-sensitive (data not shown). The [3H]galactose-labeled
carbohydrates on tau are, however, susceptible to alkaline
To
determine if any differences exist in the glycosylation of the
different bovine tau isoforms, tryptic maps of each
[3H]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 ± 0.9 mol
O-GlcNAc/mol tau (n = 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 FOOTNOTES Acknowledgments We thank K. Morrison for peptide sequencing,
M. Zinnerman for technical assistance, Dr. L. Binder for generously
providing the monoclonal antibody Tau2, and the members of the Hart lab for helpful discussion.
REFERENCES
Volume 271, Number 46,
Issue of November 15, 1996
pp. 28741-28744
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
§,
, and§§
Department of Biochemistry and Molecular
Genetics and the ¶ Department of Psychiatry and Behavioral
Neurobiology, Schools of Medicine and Dentistry, The University of
Alabama at Birmingham, Birmingham, Alabama 35294
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
(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.
(1-4)galactosyltransferase
(galactosyltransferase) and UDP-[3H]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 × 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
-elimination and saccharide
analysis, which was performed as described previously (21, 43).
-diaminobenzidine in the presence of hydrogen
peroxide or by enhanced chemiluminescence. Blots were probed for tau
with the monoclonal antibody Tau2 (1:10,000), followed by horseradish
peroxidase-conjugated goat anti-mouse-IgG in TBST (1:2000) prior to
developing.
(1-4)GlcNAc moieties on a glycoprotein. After
incubating tau with galactosyltransferase in the presence of
UDP-[3H]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).
Fig. 1.
Galactosyltransferase labeling of purified
bovine tau. A, purified bovine tau polypeptides were
resolved on 8% SDS-polyacrylamide gels and stained with Coomassie Blue
(lane 1) or transferred to PVDF and probed with the
monoclonal antibody Tau2 (lane 2). B, bovine tau
polypeptides were labeled with [3H]galactose as described
under "Experimental Procedures." Lane 1 contains
galactosyltransferase only, and lanes 2 and 3 are
labeled bovine tau and ovalbumin, a positive control, respectively.
Positions of molecular mass standards (kDa) are indicated in the
margins.
[View Larger Version of this Image (48K GIF file)]
Fig. 2.
sWGA binding of
N-acetylglucosamine on tau. Equal amounts of purified
bovine tau polypeptides were resolved on a 8% SDS-polyacrylamide gel,
transferred to nitrocellulose, and probed with the monoclonal antibody
Tau2 (lane 1) or with the lectin sWGA-horseradish peroxidase
(lanes 2-5). Specificity of sWGA-horseradish peroxidase for
GlcNAc is demonstrated by probing the membrane in the presence of 0.1 M GlcNAc (lane 3). Lane 4 was
incubated with 0.5 M GlcNAc, and lane 5 was
incubated with 0.5 M galactose. Positions of molecular mass
standards (kDa) are indicated on the left.
[View Larger Version of this Image (94K GIF file)]
-elimination (Fig. 3A), indicating that
they are linked to the polypeptide via O-glycosidic
linkages. TSK-Gel filtration of the [3H]galactose-labeled
compounds released by alkaline -elimination demonstrate their
migration as reduced disaccharides (Fig. 3B). Further
analysis of the [3H]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
[3H]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.
Fig. 3.
Characterization of carbohydrate on tau
proteins. Purified bovine tau was labeled with
[3H]galactose, and the -elimination products were
analyzed by: A, G-50 gel filtration chromatography.
Vo, void volume; Vi,
included volume. B, TSK-Fractigel chromatography.
Arrows indicate the elution positions of
[3H]galactose-labeled GlcNAc polymers. Numbers correspond
to GlcNAc residues. C, high pH anion exchange
chromatography-pulsed amperometric detection analysis of
-elimination products on a Dionex CarboPAc-MA1 column. Arrows
1 and 2 denote elution positions of
Gal1-3GalNAcitol and Gal1-3GlcNAcitol standards, respectively.
Arrow 3 represents the elution time of Gal1-4GlcNAcitol,
the expected product of a galactose-labeled O-GlcNAc.
[View Larger Version of this Image (19K GIF file)]
Fig. 4.
Separation of
[3H]galactose-labeled bovine tau isoforms and RP-HPLC of
trypsin digested polypeptides. A, purified bovine tau
(lane 0) and each of the purified individual bovine tau
isoforms (lanes 1-5) were separated by SDS-polyacrylamide
gel electrophoresis, transferred to PVDF membranes, and probed with the
monoclonal antibody Tau2. Positions of molecular mass standards (kDa)
are indicated at the left. B, RP-HPLC of tau
[3H]glycopeptides generated from trypsin digests. Numbers
at the left of each profile correspond to respective
proteins separated in A.
[View Larger Version of this Image (19K GIF file)]
-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.
§
Supported by predoctoral fellowship 5-T32-GM08111-10 from the
National Institutes of Health.
Supported by National Institutes of Health National Research
Service Award NS09415.
**
Present address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold
Spring Harbor, NY 11724.
Present address: Dept. of Pathology, Johns Hopkins Medical
Institutions, Baltimore, MD 21205.
§§
To whom correspondence should be addressed. Tel.: 205-934-4786;
Fax: 205-975-6685; E-mail: GWHART@BMG.BHS.UAB.EDU.
1
The abbreviations used are: PHF, paired helical
filament; PHF-tau, paired helical filament tau; O-GlcNAc,
O-linked N-acetylglucosamine; HPLC, high
performance chromatography; RP, reverse phase; galactosyltransferase, Gal(1-4)galactosyltransferase; sWGA, succinyl wheat germ
agglutinin; MES, (N-morpholino)ethanesulfonic acid; PVDF,
polyvinyl difluoride; PNGase F,
peptide:N-glycosidase F.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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