(Received for publication, February 5, 1996)
From the
We have examined the post-translational modification of high
molecular weight microtubule-associated proteins (MAPs) and have shown
that MAP1, MAP2, and MAP4 are glycosylated. The presence of
carbohydrate residues on these proteins was indicated by labeling with
biotin hydrazide following periodate oxidation, a specific and well
established method for detecting saccharide moieties on proteins. Both
MAP2 and MAP4 were also labeled in vitro by
UDP-[H]galactose in the presence of
galactosyltransferase. Labeling by galactosyltransferase indicated that
MAP2 and MAP4 contained terminal nonreducing GlcNAc residues, and they
appeared to be O-linked to the proteins as shown by their
sensitivity to
-elimination. Chromatographic analysis showed that
the GlcNAc residues were directly linked to the proteins as
monosaccharides. Thus, we have added MAP2 and MAP4 to the list of
intracellular O-GlcNAc-modified proteins, which includes other
cytoskeletal proteins such as cytokeratins 8, 13, and 18 and
neurofilament proteins NF-L and NF-M. We further characterized the O-GlcNAc modification of MAP2, and stoichiometric analysis
indicated that nearly 10% of the MAP2 isolated from rat brain is
modified by O-GlcNAc. However, this estimate is thought to
reflect the minimal level of O-GlcNAc modification present on
MAP2. We have also shown that both the O-GlcNAc and biotin
hydrazide-reactive carbohydrate moieties are located on the projection
domain of MAP2. Three O-GlcNAc-containing peaks were observed
following fast protein liquid chromatography of a tryptic digest of
MAP2, suggesting that multiple modification sites exist. The specific
modification sites and functional significance of the O-GlcNAc
glycosylation on the high M
MAPs remain to be
determined.
Microtubules (MTs) ()are one of the major components
of the cytoskeleton and play an important role in the organization of
the cytoplasm. As an example, disruption of MTs by various
MT-destabilizing agents results in major changes in cytoplasmic
organization, including collapse of intermediate filaments and
redistribution of the Golgi apparatus and the endoplasmic reticulum (1) . MTs are also involved in such diverse cellular functions
as maintenance of cell shape, movement of eukaryotic cilia and
flagella, formation of the mitotic spindle, and regulation of organelle
distribution and vesicle movements(2) . Regulation of the
dynamic properties of MTs is thought to play a role in many of these
processes (3) .
A group of proteins that bind to MTs in vivo and copurify with MTs, collectively defined as microtubule-associated proteins (MAPs), modulate MT dynamics and function. MAPs can be categorized into two major classes according to their primary function, i.e. motor proteins, which include the kinesin superfamily and the dynein family, and non-motor proteins, which are traditionally further divided into the high molecular weight MAPs (including MAP1A, MAP1B, MAP2, and MAP4) and the lower molecular weight tau proteins. Among the identified in vivo functions of MAPs in neuronal cells, kinesin and cytoplasmic dynein are responsible for anterograde and retrograde axonal transport, respectively, while tau protein and MAP2 appear to be required for initial neurite growth as shown using antisense mRNA (4, 5) . It is well known that the functions of MAPs can be regulated through phosphorylation. For example, phosphorylation of MAPs can alter their affinity for MTs in vitro and affect their ability to stabilize MTs(6) . Furthermore, the phosphorylation of MAP4 has been coupled to the onset of mitosis in vivo, and this phosphorylation may play a functional role in regulating MT dynamics during the interphase to mitosis transition(7) . Post-translational modification of MAPs other than phosphorylation may also play a role in regulating MAP function; however, little is known about other potential forms of MAP modification.
During the past decade, a unique glycosylation of cytoplasmic and nuclear proteins has been characterized and identified as an O-GlcNAc modification. This modification is composed of a single N-acetylglucosamine O-linked to either a serine or threonine residue(8, 9) . Virtually all of the O-GlcNAc-modified proteins identified to date are also phosphorylated, and they form reversible multimeric complexes in a regulated manner. Analogous to reversible phosphorylation reactions catalyzed by specific kinases and phosphatases, the corresponding O-GlcNAc-transferases and glycosidases have also been identified(10, 11) . It has been proposed that O-GlcNAc modifications may play a regulatory role similar to that of phosphorylation. A recent report has shown that O-GlcNAc residues on the associated 67-kDa protein (p67) of eukaryotic initiation factor 2 (eIF-2) are indispensable for its ability to protect eIF-2 from inactivation following phosphorylation by eIF-2 kinase(12) . Interestingly, this rapidly growing list of O-GlcNAc-modified proteins includes several cytoskeletal components, namely, cytokeratin 13(13) , cytokeratins 8 and 18(14) , and neurofilaments(15) . This prompted us to ask if glycosylation, O-GlcNAc modification in particular, could be another general means of post-translational modification of MAPs.
We report here that the high M MAPs are
glycosylated as indicated by their labeling with biotin hydrazide
following periodate oxidation. The biotin hydrazide labeling method is
a common technique used to detect saccharide moieties on proteins (16) . More important, both MAP2 and MAP4 contain O-linked N-acetylglucosamine residues in the
monosaccharide form, adding these MAPs to the growing list of O-GlcNAc-modified proteins. We have further shown that the
biotin hydrazide-labeled carbohydrate and the O-GlcNAc
modification are both localized to the MAP2 projection domain. Analysis
of MAP2 tryptic digests indicates that there may be several O-GlcNAc modification sites on MAP2. Our findings have
revealed a previously unknown form of post-translational modification
on high M
MAPs, which may prove to be critical in
understanding their intracellular functions, especially their
interactions with MTs and other cellular components.
Rat MAP2 and tubulin were purified from
the rat MT pellet by adding NaCl to a final concentration of 0.35 M. After incubation in a boiling water bath for 8 min, the
sample was centrifuged at 15,800 g (Brinkmann
Instruments Model 5402 microcentrifuge) at 4 °C for 15 min.
Heat-stable MAP2 was recovered in the supernatant. Alternatively, after
addition of NaCl to 0.35 M, the Taxol-stabilized rat MTs were
incubated at 37 °C for 15 min, followed by centrifugation at 15,800
g at 37 °C for 15 min. The salt-extracted pellet
was resuspended in PEM buffer and used as Taxol-stabilized tubulin.
HeLa MAP4 was kindly provided by Yunhi Choi of this laboratory and was
purified essentially as described by Vallee and Collins(17) .
To determine
whether glycosylation might be another common post-translational
modification of high M MAPs, we employed a well
established glycoprotein detection method(16) . In essence,
this approach is based upon the oxidation of the vicinal hydroxyls on
carbohydrate moieties by periodate and the subsequent formation of
aldehyde groups. The newly generated aldehyde groups are available to
react with various hydrazide probes, the binding of which can be
detected following completion of the reaction. Using a biotin
hydrazide/streptavidin detection system, we observed that MAP1A, MAP1B,
and MAP2 present in rat brain MT samples were all labeled following
periodate oxidation (Fig. 1, MT). The specificity of
this reaction was confirmed by the negative results obtained in the
experiments omitting either periodate oxidation (Fig. 1, MT, -Per) or biotin hydrazide conjugation (Fig. 1, MT, -Hyd). Isolation of
heat-stable MAP2 following boiling of the rat brain MT preparation
confirmed the reaction of this MAP with the biotin hydrazide-labeled
carbohydrate detection system (Fig. 1, MAP2). These
results indicate that brain high M
MAPs contain
some form of carbohydrate modification. Furthermore, MAP4 isolated from
HeLa MT preparations was also strongly labeled by biotin hydrazide (Fig. 1, MAP4). Thus, the presence of carbohydrate
residues on high M
MAPs was not restricted to
brain samples, but also included the major MAP found in non-neuronal
cells.
Figure 1: Glycoprotein detection using biotin hydrazide/streptavidin following metaperiodate oxidation. Rat brain MTs (MT) were separated on a 4% urea-SDS gel and transferred to nitrocellulose. Nitrocellulose transfers were then analyzed using the glycoprotein detection kit as described under ``Experimental Procedures.'' Control experiments were done by omitting either the sodium metaperiodate oxidation step (-Per) or the biotin hydrazide conjugation step (-Hyd). The positions of MAP1A, MAP1B, and MAP2 as determined by Coomassie staining and immunoblot analysis (data not shown) are indicated as 1A, 1B, and 2, respectively. Boiled MAP2 and boiled MAP4 were separated by 4% urea-SDS-PAGE and 7.5% SDS-PAGE, respectively. They were either stained with Coomassie Brilliant Blue (C) or analyzed by the glycoprotein detection kit (G). This highly specific reaction indicated that MAP1, MAP2, and MAP4 contained carbohydrate moieties.
The isolated MAPs were labeled using a
UDP-[H]galactose/galactosyltransferase system
that specifically transfers [
H]galactose to a
terminal nonreducing N-acetylglucosamine. Labeled samples were
then separated by SDS-PAGE and stained with Coomassie Brilliant Blue,
and the stained samples were subjected to fluorography. Ovalbumin
containing N-linked terminal nonreducing N-acetylglucosamine was used as a control (Fig. 2A, lanes 1 and 4). Our results
showed that both mitotic MAP4 and interphase MAP4 isolated from HeLa
cells were labeled (Fig. 2A, lanes 2 and 5 and lanes 3 and 6, respectively) and that rat
brain MAP2 was also heavily labeled (lanes 7 and 8)
by [
H]galactose. Rat brain MAP1 was only weakly
labeled by [
H]galactose (data not shown). In
addition to the MAP4 band, another heavily labeled band was present in
both of the MAP4 samples. These additional labeled bands were minor
Coomassie staining bands, suggesting that this band could be a
breakdown product of MAP4, or alternatively, it could be another
heat-stable HeLa MAP that contains a high amount of N-acetylglucosamine. Interestingly, both MAP4 and the lower
molecular weight protein showed a higher degree of labeling in the
mitotic sample, suggesting a potential cell cycle-dependent regulation
of this glycosylation.
Figure 2:
Galactosyltransferase labeling and
glycosidic linkage analysis of MAP2 and MAP4. Samples were
galactosyltransferase-labeled as described under ``Experimental
Procedures.'' A, labeled samples were separated by either
7-17% (lanes 1-6) or 7% (lanes 7 and 8) SDS-PAGE and stained by Coomassie Brilliant Blue (C), followed by fluorography analysis (Fl) of the
same gels. Lanes 1 and 4, ovalbumin; lanes 2 and 5, mitotic MAP4; lanes 3 and 6,
interphase MAP4; lanes 7 and 8, rat brain MAP2. B, labeled ovalbumin (1), MAP2 (2), mitotic
MAP4 (3), and interphase MAP4 (4) were incubated at
37 °C for 18 h with -galactosidase (&cjs2108;), with
-elimination buffer (&cjs2106;), or with distilled water (
)
as control. Radioactivity retained after various treatments was plotted
as a percentage of the total radioactivity in control samples. Note the
high sensitivity of MAP2 and MAP4 to
-elimination buffer as
compared with ovalbumin.
To prove that the label was covalently linked
to the protein via O-glycosidic bonds, equal amounts of the
labeled samples were incubated with either -galactosidase or
-elimination buffer overnight. The radioactivity retained by the
samples following this treatment was analyzed after binding of the
protein to polyvinylidene difluoride and was compared with that of
control samples that were incubated with distilled water only (Fig. 2B).
-Galactosidase, an enzyme that
specifically cleaves terminal nonreducing galactose from carbohydrate,
removed at least 70% of the incorporated radioactivity from MAP2,
mitotic MAP4, and interphase MAP4. Furthermore,
-elimination
buffer, which cleaves O-linked carbohydrate from protein but
leaves N-linked carbohydrate intact, removed 90% of the
radioactivity from MAP2 and 70% from mitotic MAP4 and interphase MAP4.
These data indicate that MAP2, mitotic MAP4, and interphase MAP4 all
contain O-linked terminal nonreducing N-acetylglucosamines. On the other hand, ovalbumin showed high
sensitivity to
-galactosidase, but resistance to
-elimination
buffer, as expected for a protein containing N-linked terminal N-acetylglucosamine (Fig. 2B).
Ovalbumin
was used as a control, and as expected, -elimination did not
significantly change the elution position of the
[
H]galactose-containing peak (Fig. 3A). As shown in Fig. 3B, the
radioactivity associated with the MAP2 control sample also eluted at
the V
position. However, the
[
H]galactose-labeled fraction obtained after
-elimination of the MAP2 sample migrated as a single peak between
GlcNAc
1-4GlcNAc and glucosamine. These results exclude the
possibility that the labeled terminal GlcNAc on MAP2 was associated
with a complex polysaccharide moiety. We thus conclude that the
original carbohydrate moiety on MAP2 is an O-linked N-acetylglucosamine monosaccharide. Similar results were
obtained with [
H]galactose-labeled MAP4 (data not
shown).
Figure 3:
Chromatographic analysis of
-elimination products from galactosyltransferase-labeled MAP2 and
ovalbumin. Galactosyltransferase-labeled ovalbumin (A) and
MAP2 (B) were analyzed by Bio-Gel P-4 chromatography following
incubation with (
) or without (
)
-elimination
buffer. The column V
and V
volumes were established by using cytochrome c and
galactose, respectively. Glucosamine (2) and
GlcNAc
1-4GlcNAc (1) were also used to calibrate the
column. The volume of each fraction is 0.63 ml. The
-elimination
product of galactosyltransferase-labeled MAP2 migrated as a single peak
between glucosamine and GlcNAc
1-4GlcNAc (B), as
expected for an O-GlcNAc-modified
protein.
To determine the extent of O-GlcNAc modification,
identical amounts of MAP2 were galactosyltransferase-labeled for
different time periods at 0 °C, and the total incorporated
radioactivity was measured (Fig. 4A). The reaction
proceeded rapidly, and by 180 min, all of the available GlcNAc residues
were labeled. Higher temperature (37 °C) or longer incubation time
(up to 5 h) did not generate a significant difference in the total
incorporated radioactivity (data not shown). The 180-min samples were
used to deduce the maximum amount of [H]galactose
incorporated. The average ratio obtained from eight separate
experiments was one O-GlcNAc in every 11 MAP2 molecules (Fig. 4B). While it is possible that this number
reflects the in vivo stoichiometry, some O-GlcNAc
residues may have been cleaved by glycosidase activity during the
purification process or may have been inaccessible to the
galactosyltransferase. Therefore, this result probably represents a
minimal estimate of the O-GlcNAc modification of rat brain
MAP2.
Figure 4:
Stoichiometry of O-GlcNAc-modified MAP2. An equal amount of rat brain MAP2 was
galactosyltransferase-labeled for 1, 3, 6, 15, 90, or 180 min, and the
total incorporated radioactivity was detected by polyvinylidene
difluoride adsorption assay. The 180-min samples were used to deduce
the mole ratio of O-GlcNAc to protein. A, a
representative graph of galactosyltransferase labeling reactions on
MAP2; B, the average mole ratio of incorporated
[H]galactose to MAP2, calculated from eight
experiments.
Figure 5:
Localization of carbohydrates on MAP2. Rat
MAP2 was incubated with either distilled water or thrombin at 37 °C
for 1 h. The samples were subsequently analyzed for their MT binding
ability (B) or tested for the presence of carbohydrates (C). The projection fragments (PF) and binding
fragments (BF) are indicated. A, shown is a schematic
drawing of MAP2. PSR, protease-sensitive region. B,
Taxol-stabilized tubulin was used to test the MT binding ability of
undigested MAP2 (lanes 3-5) or thrombin-digested MAP2 (lanes 6-8). Lane 1, molecular mass markers
(97, 66, 55, 42, 40, and 30 kDa); lane 2: Taxol-stabilized
tubulin. The whole mixture (T), pellet (P), and
supernatant (S) were separated by 4-12% SDS-PAGE and
stained with Coomassie Brilliant Blue. C, samples were
galactosyltransferase-labeled, separated by 4-12% SDS-PAGE, and
analyzed by fluorography (Fl) following Coomassie staining (C). Alternatively, samples were separated by SDS-PAGE,
transferred to nitrocellulose paper, and analyzed by the glycoprotein
detection kit (Gl). Lanes 1, 3, and 5, undigested MAP2; lanes 2, 4, and 6, thrombin-digested MAP2. The results indicate that the
projection domain of MAP2 contains the carbohydrate moieties. Also note
a slight difference in labeling patterns between the
[H]galactose-labeled fragments and the biotin
hydrazide-labeled fragments, suggesting that multiple carbohydrate
moieties may be present.
To
elucidate if glycosylation may affect MAP2 binding to MT in a similar
manner, we examined the location of the glycosylation sites on MAP2.
Briefly, rat MAP2 was digested with thrombin, and the various
proteolytic fragments were examined either for their MT binding ability
or the presence of carbohydrate. Comparison of the results allowed us
to determine which binding or projection fragments of MAP2 contained
carbohydrate. Galactosyltransferase and the biotin
hydrazide/streptavidin system were both used to track the
carbohydrate-containing fragments. As expected after limited
proteolysis, several high molecular weight projection fragments of MAP2
were generated that lost their MT binding ability and remained in the
supernatant, while several low molecular weight binding fragments
around 30 kDa cosedimented with MT after incubation with
Taxol-stabilized tubulin (Fig. 5B). Most of the large
projection fragments of MAP2 contained both the
[H]galactose-labeled O-GlcNAc residues
and the biotin hydrazide-reactive carbohydrate, while none of the small
MT-binding fragments were labeled with either reagent (Fig. 5,
compare B and C). The largest proteolytic fragment
retained MT binding ability and was also glycosylated, presumably
containing both the binding domain and a partial projection domain due
to incomplete proteolysis. Since carbohydrate was absent from the
binding domain, it is unlikely that the carbohydrate directly regulates
the MT binding ability of MAP2; instead, the carbohydrate modification
might play a role in maintaining MAP2 conformation or mediating its
interaction with other cellular components.
Figure 6:
Tryptic mapping of rat MAP2. Boiled rat
brain MAP2 was labeled by [H]galactose,
lyophilized, and digested with trypsin as specified under
``Experimental Procedures.'' The peptides were then applied
to a PepRPC HR 5/5 FPLC column and eluted at a flow rate of 0.5 ml/min.
The volume of each fraction is 0.5 ml. 30-µl solutions from each
fraction were analyzed by scintillation counting, and the counts/minute
of each fraction were plotted against the elution position and
superimposed with the concentration of acetonitrile. Three
H-containing peaks eluting at
25, 33, and 38%
acetonitrile were obtained.
The results presented here demonstrate that high M MAPs can be considered as glycoproteins as
indicated by both their reactivity to biotin hydrazide following
periodate oxidation and labeling with
[
H]galactose following galactosyltransferase
incubation. Using galactosyltransferase, we have shown that brain MAP2
and HeLa MAP4 incorporate [
H]galactose,
demonstrating the presence of terminal nonreducing GlcNAc residues.
Furthermore, we have shown that the GlcNAc residues on MAP2 are
directly O-linked to the protein as a monosaccharide. MAP4 is
also modified by single GlcNAc residues as the final
-elimination
product of MAP4 eluted as a single peak at the same position as the
-elimination product of MAP2 (data not shown). Our results also
suggested that there was an increase in the level of the O-GlcNAc modification in the mitotic MAP4 sample as compared
with the interphase sample. This observation is similar to a previous
report that showed that keratins 8 and 18 both have an elevated O-GlcNAc modification level in mitotic arrested human
epithelial cells (27) and may indicate a cell cycle-dependent
regulation of the O-GlcNAc modification on MAP4.
In an
effort to ascertain the location of the saccharide moiety associated
with the MAPs, we generated fragments of MAP2 following thrombin
digestion. The brief thrombin-induced digestion of MAP2 generated a
limited number of high molecular weight bands that did not bind to
Taxol-stabilized MTs. The complexity of the bands was probably due to
incomplete digestion of the MAP2 sample. All the high molecular weight
projection fragments could be labeled by both galactosyltransferase and
biotin hydrazide; however, the comparative labeling intensity of the
individual bands was obviously different. This may simply reflect the
inability of galactosyltransferase to access all of the available O-GlcNAc residues, or it could indicate that in addition to
the simple O-linked GlcNAc residues identified by the
galactosyltransferase, there are other carbohydrate modifications of
the MAP detected by the biotin hydrazide reaction. Tryptic mapping of
[H]galactose-labeled rat brain MAP2 using
reverse-phase FPLC showed that three distinct peaks were present.
Therefore, at least three different sites on the projection domain of
MAP2 may be O-GlcNAc-modified. However, confirmation of this
result will require identification of the O-GlcNAc-modified
peptides and sequence analysis.
Despite the presence of multiple modification sites, we obtained results suggesting a low overall stoichiometry of O-GlcNAc on MAP2. We believe that this labeling density may be an underestimate of the in vivo amount of O-GlcNAc on MAP2 for several reasons. First, our detection methods using galactosyltransferase might only label a portion of the total O-GlcNAc residues present on the MAP due to inaccessibility of the galactosyltransferase. Second, no precautions were taken to preserve the glycosylation state of the protein during purification, and some of the O-GlcNAc residues could have been lost due to the exposure of the protein to glycosidases in the brain extract. Third, there might be different subpopulations of glycosylated MAPs in vivo. However, whether such subpopulations of MAP2 exist remains to be determined. It is also possible that a single MAP2 molecule could contain multiple O-GlcNAc modification sites in vivo as well.
Evidence for glycosylation, both in the form of O-GlcNAc and more complex carbohydrate, has also been demonstrated in other proteins associated with the cytoskeleton. Cytokeratins 13 (13) and 8 and 18 (14) have all been shown to contain the O-GlcNAc modification. In addition, four keratin isoforms present in human keratinocytes have been demonstrated to be reactive to several monoclonal antibodies raised against keratan sulfate, and immunocytochemical analysis using these keratan sulfate antibodies confirms that keratin filaments are one source of cellular immunoreactivity in these keratinocytes(28) . Neurofilament subunits NF-L and NF-M have also been shown to contain O-GlcNAc, with stoichiometries similar to those reported here for MAP2 of 0.1 and 0.15 mol of GlcNAc/mol of protein for NF-L and NF-M, respectively(15) . It should be noted, however, that the level of modification determined for the neurofilament proteins was only obtained after analysis on Dionex CarboPAc PA1 of the GlcNAc residues released following acid hydrolysis. When measuring the ratio of incorporated galactose to proteins, as we have reported here for MAP2, levels of only 0.011 and 0.028 mol of Gal/mol of protein for NF-L and NF-M, respectively, were obtained(15) . Thus, the ratio of modification we obtained for MAP2 should also be considered as the minimal degree of MAP2 modification.
Various studies with O-GlcNAc-modified proteins have revealed that the O-GlcNAc residues provide different functions in different
proteins(9) . These functional properties could be dependent on
the particular glycosylation site, requiring site-specific transferases
analogous to phosphorylation consensus sequences. One of the putative
functions of O-GlcNAc is to regulate protein phosphorylation.
For example, O-GlcNAc modification may modulate
phosphorylation by occupying or blocking the phosphorylation site.
Mutually exclusive modifications of phosphorylation and glycosylation
have been shown in the C terminus of RNA polymerase II; this fragment
was either phosphorylated or O-GlcNAc-modified, but not
both(29) . This mutually exclusive model is supported by the
notion that the identified O-GlcNAc modification sites on many
proteins resemble a number of proline-directed kinase sites.
Interestingly, the high M MAPs contain several
consensus proline-directed kinase phosphorylation sites, and MAP4 has
been shown to be a substrate for p34
(7) .
Alternatively, the O-GlcNAc modification of one protein could
regulate phosphorylation on a second protein, as in the case of O-GlcNAc-modified p67. When deglycosylated, p67 loses its
ability to protect the eIF-2
-subunit from
phosphorylation(12) . O-GlcNAc modification does not
necessarily inhibit phosphorylation, however, as both phosphorylation
and O-GlcNAc modification levels on keratins 8 and 18 in the
mitotic arrested human colonic cell line HT29 were higher compared with
control cells(27) . In addition to phosphorylation, it has been
hypothesized that O-GlcNAc may be crucial for intracellular
protein-protein recognition in a manner similar to lectins or in the
regulation of a protein's susceptibility to protease(9) .
Some or even all of these putative roles for O-GlcNAc could
be potentially important for MAP2 and MAP4 functions. Both MAP2 and
MAP4 have been shown to interact with many cytoplasmic components in
addition to MT. With regard to MAP4, a recent report has shown that
cyclin B binds to MAP4(7) . Thus, bound cyclin B localizes
p34 kinase, a potential regulator of M phase MT
dynamics, to the MT. Furthermore, other kinase activities have been
found to associate with other MAPs(30) . How the binding of
these cellular components to MAPs is regulated remains largely
undefined. It is known that MAP2 and MAP4 are phosphorylated at
multiple sites and in a regulated manner. The MAP phosphorylation state
might in turn control the interaction between MAPs and other cellular
components. The phosphorylation state of MAPs could in part be
regulated by O-GlcNAc modification as observed for other O-GlcNAc-modified proteins. Alternatively, the O-GlcNAc on MAP2 and MAP4 may directly regulate the binding
and/or phosphorylation of MAP-associated proteins, such as kinases,
that may be crucial for the biological functions of these associated
proteins.
From the results that we have obtained, it would appear
that glycosylation of the high M MAPs is a general
post-translational modification of these proteins. Previous studies
have provided evidence suggesting that MAPs might be glycosylated in vivo. It has been reported that tau protein, a low
molecular weight MAP, is present in a glycated form in paired helical
filaments that are components of neurofibrillary tangles in brain
tissue of Alzheimer's disease patients(22, 23) .
It was also reported that glycation of tau decreased the binding
affinity of tau proteins for tubulin, which is another characteristic
of tau obtained from Alzheimer's tissue. Claustrin, a chicken
brain homologue of MAP1B, has been shown to be sensitive to keratanase
treatment, and the protein was reported to be reactive with monoclonal
antibodies raised against cartilage keratan sulfate(24) . In
addition, claustrin was shown to incorporate
[
H]glucosamine when added to the culture medium
of chick glial cultures, further indicating its glycoprotein nature.
Briones and Wiche (31) demonstrated that certain antibodies
specific for MAP1 and MAP2 identified a sulfoglycoprotein component of
the extracellular matrix secreted by 3T3 cells. While these results
were interpreted as cross-reaction of these MAP antibodies with a
distinct extracellular glycoprotein, it is also possible that the
sulfoglycoprotein antigen detected in the extracellular matrix was a
secreted form of MAP. This would correlate with the hypothesis that the
keratin sulfate-containing proteoglycan claustrin might represent a
secreted form of MAP1B(24) . It has also been suggested that
MAP1B may exist as a transmembrane cell-surface protein in rat cortical
cell cultures and may be involved in synaptogenesis(32) . All
these reports indicate the possibility that high M
MAPs may exist in different populations that may differ in their
amino acid composition, post-translational modification
(phosphorylation and glycosylation), and/or distribution.
We have
also found that high M MAPs, such as MAP2 and
MAP4, exhibit a high degree of sensitivity to keratanase digestion. (
)However, our observations indicate that keratanase
digestion did not generate a stable core protein for each of the high M
MAPs. We also observed that the addition of
keratan sulfate to the reaction mixture failed to inhibit the
keratanase digestion of MAPs. Furthermore, the chemical deglycosylation
of MAPs with trifluoromethanesulfonic acid did not induce an observable
molecular weight shift in the protein following SDS-PAGE analysis. We
were also unable to stain rat brain MAPs with the keratan
sulfate-specific antibody 5D4.
Thus, it appears unlikely
that MAP2 or MAP4 contains complex carbohydrate modifications, such as
has been reported for claustrin. Indeed, if other high M
MAPs contain complex carbohydrates like keratan sulfate, it would
be against prevailing models of glycosylation. Complex glycoproteins
generally exist either at the cell surface or within luminal
compartments. Nevertheless, several reports have indicated that some
glycoproteins are present in the nucleus and cytosol. For example, it
has been shown that while chondroitin sulfate proteoglycans are
exclusively extracellular in 7-day postnatal brains, they become
predominantly cytoplasmic in adult brains(33, 34) .
The question of how various complex carbohydrate structures have
reached the nucleus and/or cytoplasm has been answered in at least one
case. A unique type of nuclear heparan sulfate has been shown to be
originally synthesized by conventional glycosylation pathways onto
heparan sulfate proteoglycans, which are subsequently secreted. After
being specifically endocytosed, the free heparan sulfate is released
from the proteoglycans, further modified, and transported into the
nucleus(34) . Tentative complex carbohydrate moieties on high M
MAPs such as MAP1B could be added via similar
pathways. Alternatively, high M
MAPs could be
glycosylated by an unknown cascade of glycosyltransferases in the
cytosol. In fact, several glycosyltransferase activities have been
demonstrated in the cytosol and nucleus(8) .
The biological
functions of complex carbohydrates on cytoplasmic and nuclear proteins
are poorly understood; however, it has been suggested that they might
serve to stabilize proteins in an otherwise unfavorable conformation.
High M MAPs have long projection domains that are
thought to extend away from the surface of the MT. It is possible that
carbohydrate moieties associated with the projection domain help
maintain this extended conformation. Carbohydrate moieties located
within the projection domain could also prevent untimely proteolysis of
this extended region of the protein within the cytoplasm.
In
summary, this report has shown for the first time that glycosylation is
a common post-translational modification shared by high M MAPs. In addition, we have added MAP2 and MAP4
to the growing list of O-GlcNAc-modified proteins. We are
currently in the process of identifying the sites modified by O-linked GlcNAc on MAP2. Further work will be necessary to
investigate the keratanase-sensitive feature of the high M
MAPs and to determine the functional
consequences of MAP O-glycosylation.