From the Department of Biological Chemistry, The
Johns Hopkins University School of Medicine, Baltimore, Maryland
21205-2185 and the § Department of Internal Medicine,
Division of Endocrinology, Metabolism and Diabetes, The University of
Utah, Salt Lake City, Utah 84132
Received for publication, November 16, 2000, and in revised form, January 4, 2001
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ABSTRACT |
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Dynamic modification of cytoplasmic and nuclear
proteins by O-linked N-acetylglucosamine
(O-GlcNAc) on Ser/Thr residues is ubiquitous in higher
eukaryotes and is analogous to protein phosphorylation. The enzyme for
the addition of this modification, O-GlcNAc transferase, has been cloned from several species. Here, we have cloned a human brain O-GlcNAcase that cleaves O-GlcNAc off
proteins. The cloned cDNA encodes a polypeptide of 916 amino acids
with a predicted molecular mass of 103 kDa and a pI value of
4.63, but the protein migrates as a 130-kDa band on SDS-polyacrylamide
gel electrophoresis. The cloned O-GlcNAcase has a pH
optimum of 5.5-7.0 and is inhibited by GlcNAc but not by GalNAc.
p-Nitrophenyl (pNP)- Since the description of O-linked
N-acetylglucosamine
(O-GlcNAc)1 as an
abundant modification in murine lymphocytes (1), a myriad of
cytoplasmic and nuclear proteins in all metazoans have been found to
carry this modification. Such proteins cover a broad range, including
many transcription factors, RNA polymerase II, oncogenes, nuclear pore
proteins, viral proteins, and tumor repressors (for details, see Refs.
2 and 3 and citations within). Unlike classic O- or
N-linked protein glycosylations, the O-GlcNAc
modification involves only a single GlcNAc moiety linked to the
hydroxyl group of Ser/Thr residues, generally is not elongated, and is
found exclusively in the cytoplasm and nucleoplasm.
Protein O-GlcNAcylation is highly dynamic, and the cycle of
addition/removal of the sugar moiety is rapid, analogous to protein phosphorylation/dephosphorylation catalyzed by kinases and phosphatases (2). Indeed, existing evidence suggests that this modification has a
"yin-yang" relationship with protein phosphorylation in some cases
(4). Many O-GlcNAcylation sites have been mapped to
phosphorylation sites or adjacent sites (5-7). Such spatial localization indicates that O-GlcNAc may regulate the target
protein by competing with protein kinases (4). Recent studies using phosphatase and kinase inhibitors have provided direct evidence for a
general reciprocal relationship between O-GlcNAcylation and
phosphorylation on some proteins (8, 9).
O-GlcNAcylation appears to be involved in gene
transcription. Most transcription factors examined so far, including
Sp1, AP1, AP2, AP4 (10), serum response factor (11), the estrogen
receptor (7, 12), the insulin promoter
factor-1,2 and peroxisome
proliferator-activated receptor- O-GlcNAc transferase (OGT), which transfers GlcNAc from the
donor substrate UDP-GlcNAc to target proteins, has been purified and
cloned from several species including human, rat, and
Caenorhabditis elegans (17-19). It does not share any
significant homology with any other known proteins, including
glycosyltransferases, and is highly conserved from C. elegans to human. Disruption of the ogt gene is lethal
in mouse embryonic stem cells, further underscoring the importance of
O-GlcNAc modification in cellular functions (20).
O-GlcNAcase, the enzyme that removes O-GlcNAc
from such proteins, was purified several years ago from rat spleen
(21). It is a neutral cytosolic Purification of O-GlcNAcase from Bovine Brain--
All
chromatographic materials were purchased from Amersham Pharmacia
Biotech (Piscataway, NJ). Purification procedure is substantially modified from that of Dong and Hart (21). All steps were conducted at
4 °C or on ice.
Step 1. Tissue Homogenization--
Three bovine brains (~1 kg)
were frozen in liquid N2 and shipped on dry ice from Pel
Freez Biologicals (Rogers, AR) and stored at Step 2. Ammonium Sulfate Precipitation--
The cytosolic
supernatant was subjected to 30-50% ammonium sulfate precipitation.
The pellet was resuspended in 500 ml of buffer A (20 mM
sodium phosphate, pH 7.5, 5 mM 2-mercaptoethanol) and
centrifuged to clarify the solution. The solution was then thoroughly
dialyzed against buffer A and centrifuged again to eliminate any
insoluble materials that had resulted from dialysis.
Step 3. DE52 Cellulose Ion Exchange Chromatography--
The
dialyzed sample was loaded onto a DE52 column (900-ml bed vol) at a
flow rate of 2 ml/min using a peristaltic pump. After washing the
column with 3 liters of buffer A, bound proteins were eluted with a
linear gradient of 0-1 M NaCl in 4 liters of buffer A at a
flow rate of 4 ml/min. The protein profile was monitored by absorbance
at 280 nm. Fractions (16 ml) enriched in O-GlcNAcase activity were pooled.
Step 4. Concanavalin A-Sepharose 4B
Chromatography--
MgCl2 was added to the pooled
fractions at a final concentration of 1 mM. The preparation
was then applied to a ConA column (60 ml) equilibrated in ConA buffer
(20 mM sodium phosphate, pH 7.5, 5 mM
2-mercapoethanol, 150 mM NaCl, 1 mM
MgCl2). The column was washed with 200 ml of ConA buffer.
The flow-through and the wash were combined.
Step 5. Affinity Blue A Chromatography--
The enzyme solution
from Step 4 was concentrated by 60% ammonium sulfate precipitation,
dialyzed, and applied three times to a Blue A-Sepharose column (25 ml)
equilibrated in buffer A. Again the activity was present in the
flow-through fraction. The protein was pooled and clarified by centrifugation.
Step 6. Re-chromatography on DE52 Column--
The sample from
Step 5 was injected to the DE52 cellulose column (same size as above),
and protein was eluted with a linear gradient of 50-350 mM
NaCl in 4 liters of buffer A. Activity was recovered as in Step 3 and
precipitated with 60% ammonium sulfate. The pellet was resuspended in
20 ml of Mono-Q buffer (20 mM Tris, pH 7.5, 5 mM 2-mercaptoethanol, 10% glycerol, 1 mM EDTA
plus protease inhibitor mixture (22) and 1 mM PMSF),
dialyzed, and clarified by centrifugation.
Step 7. Native Polyacrylamide Gel Electrophoresis--
Native
PAGE was performed using a preparative Prepcell apparatus (Bio-Rad,
Hercules, CA). The sample from Step 6 was divided into three equal
volumes (45 mg of protein each) and loaded batch-wise onto a 6% native
polyacrylamide gel (5-cm-long separating gel). The gel was run for
24 h at 12 watts of constant power. Protein was eluted in
Mono-Q buffer at a flow rate of 0.75 ml/min. 5-min fractions
were collected and assayed for protein content and enzyme activity.
Step 8. Mono-Q Chromatography--
The
O-GlcNAcase-containing fractions from each native PAGE run
was resolved on a Mono-Q column (HR10/10) with a linear
gradient of 0-500 mM NaCl in 450 ml of Mono-Q
buffer at a flow rate of 3 ml/min. Fractions (4.5 ml) rich in
O-GlcNAcase activity were pooled and then separated for a
second time on the Mono-Q column. The final preparation was
concentrated using Millipore concentrators to a final volume of 0.4 ml.
Glycerol (40% final) and 1 mM PMSF and protease inhibitor
mixture were added to the preparation. The enzyme was stored at
Identification of Proteins by Mass Spectrometry--
The final
preparation from Step 8 was separated by 10% SDS-PAGE and stained with
Coomassie Blue G-250 or with silver. The desired protein bands were
excised individually, reduced, alkylated, and digested in-gel with
modified trypsin (Worthington, Freehold, NJ) as described previously
(23). The tryptic peptides from each protein were analyzed by capillary
reversed phase high pressure liquid chromatography with in-line tandem
MS/MS on a Finnigan LCQ ion-trap mass spectrometer. Proteins
were identified by the SEQUEST algorithm with sequencing of at least
seven tryptic peptides for each protein (24).
Cloning of O-GlcNAcase--
A putative O-GlcNAcase
with a theoretical length of 916 amino acids in human was identified by
the above proteomic approach. A cDNA fragment, KIAA0679
(GenBankTM accession number AB014579), which contains the
coding sequence for 767 amino acids of the C terminus of the human
O-GlcNAcase and a 2.0-kb 3'-untranslated region, was
obtained from the Kazusa DNA Research Institute, Japan, in the vector
pBluescript. The coding sequence of this fragment was subsequently
transferred to pcDNA3.1His A using XhoI and
XbaI, which were located within the polycloning cloning site
of pBluescript and in the 3'-untranslated region of the cDNA,
respectively. The missing 5'-end fragment of the full-length coding
cDNA (447 bp) was amplified by PCR from a human brain Marathon
cDNA library (CLONTECH, Palo Alto, CA) using
the forward primer GGATGGTGCAGAAGGAGAGTCAAGCGAC and the reverse primer
TAGAAACCTCTTCGATGGACTCTACTGG. The forward primer sequence was based on
published data (25), and the reverse primer was located in the KIAA0679
clone. PCR conditions were 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 3 min for 30-35 cycles. A second round of PCR
using the first PCR product as template and a forward primer
incorporating a NotI site
(CCGGGCGGCCGCGGATGGTGCAGAAGGAGAG) and the same reverse
primer was performed. The product was digested with NotI and
HindIII (unique site in the PCR product) and ligated in-frame into the pcDNA3.1His A-KIAA0679 construct. This gave rise
to a full-length cDNA in the vector pcDNA3.1His A. The final construct was sequenced.
O-GlcNAcase Assays--
Unless stated otherwise,
O-GlcNAcase activity was assayed as described in 50 mM sodium cacodylate, pH 6.5, 0.3% bovine serum albumin, 2 mM pNP- Overexpression and Purification of O-GlcNAcase from Cos-7
Cells--
A plasmid of pcDNA3.1His A containing the full-length
O-GlcNAcase cDNA was prepared using a Qiagen kit
(Qiagen, Valencia, CA). Transfection was mediated by LipofectAMINE Plus
(Life Technologies Inc., Gaithersburg, MD) using 50-90% confluent
Cos-7 cells. Cells were harvested 2 days post-transfection and
sonicated for 2 × 12 s in 20 mM Tris (pH 7.5),
10% glycerol, 150 mM NaCl, 1 mM
dithiothreitol, 0.1 mM EDTA, 1 mM PMSF and
protease inhibitor mixture, and clarified by centrifugation. For
characterizations, the recombinant protein was purified over a nickel
affinity column.
Cell Fractionation and Western Blot--
After transfection with
O- GlcNAcase, Cos-7 cells were separated into cytoplasmic
and nuclear fractions as described (26) with one modification: The 25- to 50-µl nuclear pellet was carefully washed in 500 µl of hypotonic
buffer A to minimize cross-contamination. Immunoblot analysis was
performed using antibodies recognizing the nuclear protein
retinoblastoma (Rb) (Santa Cruz Biotechnology, Santa Cruz, CA),
cytoplasmic protein Northern Blot--
Northern blot analyses were performed on a
human multiple tissue Northern blot (CLONTECH)
using the manufacturer's protocol. To prepare an
O-GlcNAcase-specific probe, the full-length coding sequence
(2.75 kb) was amplified by PCR and labeled by random primer using
[ Native Polyacrylamide Gel Electrophoresis Aides in O- GlcNAcase
Purification from Bovine Brain--
Historically,
O- GlcNAcase, or neutral hexosaminidase C, has been
difficult to purify. For example, an early report described a
purification of only 25- to 40-fold from bovine brain despite the
extensive use of chromatographic steps (27). However, in rat brain, the
enzyme has been purified over 2000-fold to a major band (28). More
recently, renewed effort has gone into its purification from rat spleen
and bovine brain in the pursuit to cloning the cDNA (21, 29).
We have taken an approach, partly based on published literature (21)
but yet incorporating some novel steps in the purification of
O-GlcNAcase from bovine brain. Notably, we have discovered that the enzymatic activity survives the harsh conditions of native PAGE (high pH and high ionic strength) and migrates more slowly (RF = 0.28 in 6% native gel) than most other
proteins in the gel (data not shown). This property allows the
effective separation of O-GlcNAcase from other proteins with
higher RF values on a preparative scale native gel
(Fig. 1b). This step, in
conjunction with other chromatographic steps outlined in the protocol,
purified the protein ~1500-fold, with a specific activity of 1840 nmol/min/mg of protein.
The final preparation still shows seven well defined bands on SDS-PAGE
following silver staining, even after extensive purification (Fig.
2). We do not understand the basis for
this difficulty, but we have observed that the peaks for
O-GlcNAcase activity are very broad throughout the
purification procedure (Fig. 1). One example of this is illustrated in
the Mono-Q step, where the general protein peaks are sharp
but yet the activity peak spreads over 50 ml (Fig. 1c). We
have also tried ion exchange on Superose Q and hydroxylapatite columns
or hydrophobic interaction chromatography on a phenyl-Sepharose column.
They, too, give poor separations or the enzyme binds very tightly to
phenyl-Sepharose resulting in >50% activity loss (data not
shown).
Mass Spectrometry Identifies O-GlcNAcase on SDS-PAGE Gel--
The
seven bands on the silver-stained SDS-PAGE gel were excised
individually, digested with trypsin, and sequenced by electrospray MS/MS. The fragmentation data were used to search protein and DNA data
bases. This approach identified six proteins with known functions in
six of the bands (Fig. 2). Another protein, which runs as a 130-kDa
band on SDS-PAGE, is a hypothetical protein without any clearly
defined functions (GenBank, KIAA0679). BLAST searches indicated
that the hypothetical protein shared significant homology with a
protein from C. elegans called "similar to
hyaluronoglucosaminidase" (GenBank, AAA68333.1). Because
hyaluro- noglucosaminidase degrades hyaluronic acid, which is
a GlcNAc O-GlcNAcase Is Unique and Conserved during Evolution--
BLAST
searches of data bases reveal that O-GlcNAcase is conserved
in higher eukaryotic species, and the homologue is absent in yeast or
prokaryotes. The sequences and alignment of O-GlcNAcase from
human, C. elegans, and Drosophila are shown in
Fig. 3. In a pairwise alignment, the
human sequence shares 55 and 43% homology with that of
Drosophila and C. elegans, respectively, whereas Drosophila and C. elegans are 43% similar. Close
inspection of the sequences indicates that the N-terminal
~400 and the C-terminal ~350 amino acids in the human sequence are
conserved to a higher degree. These two domains are separated by a
highly variable region of ~150 amino acids. Another feature is that
most of the aromatic residues are conserved among the species. For
example, out of the 13 Trp residues found in the human sequence, 9 are
invariant in Drosophila and C. elegans, two are
conservative (substituted by Tyr or Phe), and only two are
variable.
The O-GlcNAcase sequence is conserved at a strikingly higher
level in mammals. Four overlapping EST sequences from cow, which cover
46% of the human protein, show that these two species are 100%
identical in these regions (BE481597, BE588694, BF043559, and
AW463869). Five EST entries for mouse, most of which are overlapping,
show that human and mouse are 97.8% identical (AW907793, AW324047,
AI530529, AW762257, and AA240394). In the case of zebrafish, two
overlapping EST sequences covering 33.8% of the human protein indicate
that zebrafish and human are 85% identical and 92% similar (AI882982
and AI722710).
Apart from the above-described homologues, O-GlcNAcase
does not show significant homology with any other proteins, including known glycosidases. Short stretches of ~200 amino acids of the polypeptide do show loose homology to a number of proteins such as
hyaluronidase (AAA23259.1), a putative acetyltransferase (AL158057), eukaryotic translation elongation factor-1 Overexpression of O-GlcNAcase in Cos-7 Cells--
To ascertain
that the cloned cDNA indeed encoded O-GlcNAcase, we
subcloned the entire coding region in-frame into the mammalian expression vector pcDNA3.1His and overexpressed for activity in Cos-7 cells. Transient transfection resulted in a 6-fold increase in
O-GlcNAcase activity over endogenous activity in the cells (Fig. 4a). After nickel
affinity purification, the activity from the
O-GlcNAcase-transfected cells was 230 nmol/min/mg of protein but was not detectable from control transfected cells (Fig.
4b). These data show that the activity is due to
overexpression from the plasmid. Fig. 4c shows that a
distinct band of the correct molecular mass (135 kDa) was isolated
after nickel purification from transfected cells. This band was
immunoreactive with the Xpress antibody, which was specific for a
peptide sequence in the overexpressed protein.
Recombinant O-GlcNAcase Has Distinct Properties from Lysosomal
The two enzymes also responded differently to inhibitors. GalNAc, a
widely used inhibitor of acidic Recombinant O-GlcNAcase Shows Strict Substrate Specificity for
O-GlcNAcase Cleaves O-GlcNAc from Glycopeptides--
A true
O-GlcNAcase should cleave O-GlcNAc attached to
proteins or peptides. We synthesized two glycopeptides containing
one repeat of the (CTD) C- terminal
domain of RNA polymerase II linked to Overexpressed O-GlcNAcase Is Localized to the Cytoplasm--
As
stated earlier, sequence analyses suggest that O-GlcNAcase
is localized in the cytoplasm and nucleus. This is consistent with the
localization of the O-GlcNAc modification. To obtain direct
evidence on its localization, we performed cellular fractionation and
assayed O-GlcNAcase activity in the cytoplasm and the
nucleus. The data show that, in nontransfected cells,
O-GlcNAcase activity was distributed in both the cytoplasm
and the nucleus. However, when O-GlcNAcase was
overexpressed, it was predominantly found in the cytoplasm (Fig.
8a). We also probed for its
localization by Western blots (Fig. 8b). Retinoblastoma
protein (Rb) and O-GlcNAcase Has One Transcript in Human Tissues--
To estimate
the number of transcripts of O-GlcNAcase and their levels of
expression, we performed Northern analyses using a human multiple
tissue blot consisting of RNA from eight different tissues. Labeled PCR
product of the entire coding region of the O-GlcNAcase
cDNA was used as a probe. The Northern blot analysis showed only
one transcript of ~5.5 kb for O-GlcNAcase (Fig.
9). Exposing the film for extended time
(32 h) did not reveal any additional bands (data not shown). The gene
was expressed in every tissue on the blot but was the highest in the
brain, followed by placenta, and pancreas. Lung and liver had the
lowest expression (Fig. 9). This pattern of expression largely agrees
with that of the ogt gene. ogt is expressed the
highest in the pancreas, followed by heart, brain, and placenta but the
lowest in lung, liver, and kidney (18). Unlike ogt gene,
which is on the X chromosome (20), the O-GlcNAcase gene is
localized on chromosome 10 (Ref. 25, and available on the Web from
Kazusa DNA Research Institute, Japan).
Two categories of The discovery of O-GlcNAc provides clues to the likely
physiological function of neutral glucosaminidase (hexosaminidase C) (2). This modification consists of the addition of a single GlcNAc
moiety to the hydroxyl group of Ser/Thr residues (2). The enzyme
responsible for GlcNAc addition, OGT, was identified and cloned several
years ago (17-19). On the other hand, the identity of the enzyme for
the removal of O-GlcNAc from proteins,
O-GlcNAcase, has remained obscure. Judged from the known
properties of hexosaminidase C, we speculate that it might in fact be
the O-GlcNAcase. We thus have extensively purified
hexosaminidase C from bovine brains. Proteomic analysis allowed us
to identify its DNA sequence. The sequence was originally isolated
during screening of a meningioma expression library (25). Based on its
initial characterization and sequence homology with an unidentified
protein called "similar to hyaluronoglucosaminidase" from C. elegans, the authors suggested that it was a new type of
hyaluronidase. We have here demonstrated that the cloned enzyme has all
the expected properties of O-GlcNAcase. Most importantly, it
specifically cleaves O-GlcNAc but not O-GalNAc from glycopeptides. We conclude that we have cloned hexosaminidase C,
which we have called O-GlcNAcase herein.
The recombinant protein differs from acidic lysosomal hexosaminidase in
a number of ways. It has a neutral, instead of acidic, pH optimum. This
pH optimum is expected due to its physiological functions in the
cytoplasm and nucleus. The protein displays strict substrate
requirement in the linkage of the sugar and is also sensitive to the C4
orientation of the sugar. As such, it hydrolyzes only O-GlcNAcase is a unique protein with no other obvious family
members. It is conserved in evolution down to the nematode C. elegans, and shows striking homology in mammals. It does not, however, possess any known signal peptides, domains, or motifs. Consistent with the literature that antibodies that recognize lysosomal
hexosaminidases A or B do not cross-react with hexosaminidase C (35),
O-GlcNAcase does not share any significant homology with
these enzymes, or with any other known proteins.
The predicted localization of O-GlcNAcase by sequence
analysis is consistent with the literature that O-GlcNAc
modification and OGT are found both in the cytoplasm and nucleus (19,
36). It is therefore somewhat unexpected to find that overexpressed O-GlcNAcase in Cos-7 cells is almost exclusively localized
to the cytoplasm (Fig. 8). Nevertheless, it is not uncommon that overexpressed proteins often form aggregates in the cytoplasm and
cannot be correctly translocated. We cannot exclude the possibility of
an unidentified isoform of O-GlcNAcase that is specifically localized to the nucleus. We are currently generating an
O-GlcNAcase-specific polyclonal antibody based on its
protein sequence. Immunofluorescence microscopic studies will determine
the relative distribution of the endogenous protein.
The cloning of O-GlcNAcase provides a valuable tool to
further study the biological function of O-GlcNAc
modification and may help to understand the mechanisms of a number of
prevalent diseases such as the Alzheimer's and diabetes. Recent data
suggest that many proteins in the brain, including Synapsin I and
neurofilaments (6, 37), are O-glycosylated. Some of the
glycosylated proteins may be involved in the development of the
Alzheimer's diseases. For example, Tau, whose hyperphosphorylation
leads to the formation of neurofibrillary tangles in the neurons of
Alzheimer's brains, is extensively O-glycosylated (38). The
-GlcNAc, but not
pNP-
-GalNAc or pNP-
-GlcNAc, is a
substrate. The cloned enzyme cleaves GlcNAc, but not GalNAc, from
glycopeptides. Cell fractionation suggests that the overexpressed
protein is mostly localized in the cytoplasm. It therefore has all the
expected characteristics of O-GlcNAcase and is
distinct from lysosomal hexosaminidases. Northern blots show
that the transcript is expressed in every human tissue examined but
is the highest in the brain, placenta, and pancreas. An understanding
of O-GlcNAc dynamics and O-GlcNAcase may be key
to elucidating the relationships between O-phosphate and O-GlcNAc and to the understanding of the
molecular mechanisms of diseases such as diabetes, cancer, and neurodegeneration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, as well as RNA polymerase II (13)
and chromatin (14) are O-GlcNAcylated. O-GlcNAcylation of Sp1 appears to enhance its activity in
transcription, and, conversely, blocking the GlcNAc residues with
lectin wheat germ agglutinin suppresses the transcriptional activity
(10). O-GlcNAcylation of Sp1 also controls its degradation
by the proteasome (15). Hyperglycemia-induced superoxide production
increases Sp1 glycosylation resulting in the activation of genes that
contribute to the pathogenesis of diabetes (16).
-glucosaminidase or hexosaminidase C
(EC 3.2.1.52). To further study the function of this modification, we
have now extensively purified O-GlcNAcase from bovine brain, sequenced the protein by mass spectrometry, and cloned the cDNA. The O-GlcNAcase is evolutionarily conserved, distinct from
lysosomal acidic hexosaminidases A and B. The recombinant protein has
all the expected characteristics of O-GlcNAcase, including
the ability to cleave O-GlcNAc from glycopeptides.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until use. The
brains were smashed into smaller pieces and homogenized in 5 volumes
(v/w) of homogenization buffer (20 mM sodium phosphate, pH
7.5, 15 mM 2-mecaptoethanol, 10 mM
MgCl2, 1 mM PMSF, 1 mM EDTA) in a
Hamilton Beach blender with 5 × 20-s bursts. The homogenate was
centrifuged at 18,000 × g for 30 min. The pellet was
discarded, and the cytosolic supernatant was pooled in a 5-liter beaker.
20 °C.
-GlcNAc, 50 mM GalNAc
(21). Purified bovine kidney lysosomal
-hexosaminidase (Roche
Molecular Biochemicals, Indianapolis, IN) activity was assayed in
citrate phosphate buffer, pH 4.5, 0.3% bovine serum albumin, 2 mM pNP-
-GlcNAc. To test the ability of
recombinant O-GlcNAcase to cleave O-GlcNAc from glycopeptides, two glycopeptides, CTD-GlcNAc
(N-YSPTS(GlcNAc)PSK-C) or CTD-GalNAc
(N-YSPTS(GalNAc)PSK-C), were synthesized by
standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry.
The peptides were purified on a C18 column under reversed phase high
pressure liquid chromatography conditions and used as a substrate for
cloned O-GlcNAcase. The reaction products were analyzed by
matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF).
-tubulin (Sigma Chemical Co., St. Louis, MO), or
with anti-Xpress antibody, which is specific for the sequence DLYDDDDK
located at the N terminus of the overexpressed O-GlcNAcase
fusion protein (Invitrogen, Carlsbad, CA).
-32P]dCTP (Stratagene, La Jolla, CA). After stripping
in 0.5% SDS at 100 °C for 10 min, the blot was reprobed for
-actin.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification steps of
O-GlcNAcase from bovine brain. The cytosolic
fraction of bovine brain proteins were sequentially purified by
precipitation with 30-50% ammonium sulfate, DE52 ion exchange, ConA,
and Blue A affinity columns, then re-separated on DE52 cellulose ion
exchange chromatography (a), native polyacrylamide gel
electrophoresis (b), and Mono-Q ion exchange
chromatography (c). The protein profiles in a and
c were continuously monitored by absorbance at 280 nm with a
UV detector during purification on a fast protein liquid chromatography
system, and are presented by solid lines without symbols. In
b, the protein contents were manually determined by Bio-Rad
assay (A595) for each fraction after the protein
was eluted from the native gel. Therefore, it is presented with
symbols. See "Experimental Procedures" for details. ,
protein profile;
, O-GlcNAcase activity.
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Fig. 2.
Silver staining and protein identification by
mass spectrometry. The final purification was separated on 10%
SDS-PAGE and silver stained. Each protein band was individually excised
and digested with modified trypsin, and the resultant peptides were
separated and sequenced by liquid chromatography-MS/MS. Seven to 22 peptides were identified for each protein.
1-4GlcUA polymer, it was possible that a
hyaluronoglucosaminidase may share some homology with
O-GlcNAcase. Furthermore, careful comparisons of O-GlcNAcase activity and protein patterns on the SDS gels of
different pools during the purification procedure indicated that this
protein was one of only two bands that corresponded with activity (the other band was Protein 1 in Fig. 2, data not shown). We
therefore hypothesized that this may be the O-GlcNAcase, and
cloned the cDNA. Further characterization of the expression product
of the cDNA confirmed that band 2 on Fig. 2 was, indeed,
O-GlcNAcase (see below).
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Fig. 3.
The predicted amino acid sequence of
O-GlcNAcase and its alignment with hypothetical
proteins in C. elegans ("similar to
hyaluronoglucosaminidase"; AAA68333.1) and
Drosophila (AAF55867.1). The alignment was done
with ClustalW in MacVector. Identical and conservative residues are
indicated by asterisks and dots, respectively.
The underlined sequences are the typtic peptides that are
identified by liquid chromatography-MS/MS. Boldface and
underlined letters indicate conserved aromatic residues in
the three species.
(Z11531, S26649), and the
11-1 polypeptide (X07453, S00485). Sequence analyses by a computer
program PSORT II (available on the Web) show that
O-GlcNAcase does not possess any known signal peptides,
domains, or motifs. The analyses do, however, suggest that the
endogenous protein is localized in the cytoplasm (p = 0.522) and the nucleus (p = 0.391).
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Fig. 4.
Overexpression of
O-GlcNAcase in Cos-7 cells. Cos-7 cells were
transiently transfected with the vector pcDNA3.1His alone or with
pcDNA3.1His containing O-GlcNAcase cDNA using
LipofectAMINE Plus. 48 h post-transfection, soluble proteins were
isolated from the cells. a, O-GlcNAcase activity
in total cell extract. The activity in control transfected cells is
arbitrarily set as one-fold. b, O-GlcNAcase
activity in control and transfected cells after nickel purification.
c, Western blot and Coomassie Blue G-250 staining of nickel
affinity-purified O-GlcNAcase from transfected cells. The
primary antibody was anti-Xpress, which recognized a peptide sequence
(DLYDDDDK) present in the vector pcDNA3.1His. The arrow
indicates the band of O-GlcNAcase.
-Hexosaminidase--
We further characterized the properties of the
cloned O-GlcNAcase and compared them with those of lysosomal
-hexosaminidase purified from bovine kidney. As expected,
the lysosomal
-hexosaminidase had an acidic pH optimum
(pH 3.5-5.5) with little activity at pH 7.0 or above (Fig.
5a). On the other hand, the
cloned O-GlcNAcase had a pH optimum of 5.7-7.0 and retained
significant activity (~30%) at pH 7-8. This pH profile is
consistent with the expected localization of O-GlcNAcase in
the cytoplasm and the nucleus.
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Fig. 5.
Some key properties of the cloned
O-GlcNAcase. A lysosomal -hexosaminidase
purified from bovine kidney was used as a control enzyme. a,
pH optima of the two enzymes. Inhibition studies by GalNAc
(b), GlcNAc (c), and PUGNAc (d). In
b, c, and d, the activity was assayed
at pH 4.5 for the lysosomal hexosaminidase and pH 6.5 for the
O-GlcNAcase. The highest activity at optimal pH
(a) or in the absence of any inhibitor (b,
c, d) is arbitrarily set as 100%.
,
O-GlcNAcase;
, lysosomal
-hexosaminidase.
-hexosaminidase,
inhibited the lysosomal enzyme 50% at 5.0 mM and 88% at
50 mM. The cloned O-GlcNAcase was not inhibited
at all by GalNAc up to 50 mM (Fig. 5b). GlcNAc
and its synthetic analogue PUGNAc inhibited both enzymes but were more
potent with the O-GlcNAcase (Fig. 5, c and
d).
-Linked GlcNAc--
O-GlcNAcase also differed from
lysosomal
-hexosaminidase in substrate requirements. In the in
vitro assays, purified recombinant O-GlcNAcase cleaved
only pNP-
-GlcNAc, but not pNP-
-GalNAc or pNP-
-GlcNAc (Fig. 6). The
activity using the latter two compounds as substrates was not
detectable. This substrate specificity was in contrast to the lysosomal
-hexosaminidase, which also cleaved pNP-
-GalNAc,
albeit with slightly lower efficiency compared with pNP-
-GlcNAc.
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Fig. 6.
Substrate specificity of cloned
O-GlcNAcase. pNP- - GlcNAc,
pNP-
-GalNAc, and pNP-
-GlcNAc (all 2 mM) were tested as substrates for purified
O-GlcNAcase from transfected Cos-7 cells. The lysosomal
hexosaminidase was used as a control. The assays were done at pH 4.5 for the lysosomal enzyme and at pH 6.5 for O-GlcNAcase. The
activity using pNP-
-GlcNAc as substrate is set as
100%.
-GlcNAc or
-GalNAc through the hydroxyl group of a serine residue (CTD-GlcNAc
or CTD-GalNAc). The design of these glycopeptides is based on earlier
information that this serine residue is glycosylated in vivo
(13). These peptides were tested as substrates for the purified
recombinant O-GlcNAcase, and the product peptides were analyzed by
matrix-assisted laser desorption ionization-time of flight (MALDI-TOF).
Cleavage of GlcNAc or GalNAc from the peptides should result in a
downshift of 203 in the molecular weight (the weight of GlcNAc or
GalNAc minus 18). O-GlcNAcase did not cleave GalNAc from the
peptide (Fig. 7, a and
b) but did successfully cleave GlcNAc from the peptide, as
judged by the expected shift in molecular weight (1069.8 to 866.6, 1091.8 to 888.6) (Fig. 7, c and d).
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Fig. 7.
Recombinant O-GlcNAcase
cleaves O-GlcNAc, but not O-GalNAc,
from glycopeptides. Sythetic peptides, CTD-GlcNAc
(N-YSPTS(GlcNAc)PSK-C) or CTD-GalNAc
(N-YSPTS(GalNAc)PSK-C) were tested as substrates
for purified recombinant O-GlcNAcase. The reactions
containing 0.1 mM peptide were incubated at pH 6.5, 37 °C overnight. The mock reactions were done using nickel
purification from nontransfected Cos-7 cells. The peptides were then
cleaned up by zip tips and analyzed by MALDI-TOF. The values 1091.7 (or
1091.8) and 1069.7 (or 1069.8) represented molecular weights of the
Na+ and H+ form of the peptides, respectively.
After the GlcNAc was cleaved in CTD-GlcNAc peptide by
O-GlcNAcase, the mass expectedly shifted down to 888.6 and
866.6, respectively. The numbers on the right-hand
side of each spectrum (2744, 2930, 5158, and 2100) are the total
ion counts recorded by the detector in MALDI-TOF analysis.
-tubulin, which were exclusively localized in the
nucleus and cytoplasm, respectively, were used as markers. In agreement
with activity assays, overexpressed O-GlcNAcase protein was
only detected in the cytoplasm of overexpressed cells.
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Fig. 8.
Overexpressed O-GlcNAcase is
distributed in the cytoplasm. Cos-7 cells were transfected with
O-GlcNAcase. Two days post-transfection, the cells were
fractionated into cytoplasmic or nuclear fractions. a,
O-GlcNAcase activity (n = 3). Assays were
done with 100 mM GalNAc as inhibitor. b, Western
blots with anti-Xpress (for recombinant O-GlcNAcase
detection), anti- -tubulin (cytoplasmic marker), and
anti-retinoblastoma (Rb) (nuclear marker).
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Fig. 9.
O-GlcNAcase transcript is
expressed in every human tissue examined but is the highest in the
brain, placenta, and pancreas. a, a human multiple
tissue Northern blot was probed with
[ -32P]dCTP-labeled full-length O-GlcNAcase
coding sequence and exposed to film for 18 h. b, after
stripping, the blot was reprobed for
-actin and exposed to film for
7 h.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hexosaminidases are known to exist in
eukaryotic cells. One category, which comprises the A, B, I, S
isozymes, is exclusively localized in the lysosomes and are responsible for the degradation of complex glycans. This group of hexosaminidases, particularly the A and B isozymes, have been extensively studied because their deficiency leads to Tay-Sachs and Sandhoff diseases (30,
31). These enzymes are characterized by their acidic pH optima,
inhibition by both GlcNAc and GalNAc, the ability to use both
artificial glucosaminide and galactosaminide as substrates, and their
thermostability (32). The second category, however, consists of two
neutral hexosaminidases, the GlcNAc-specific glucosaminidase (hexosaminidase C) and the GalNAc-specific galactosaminidase
(hexosaminidase D) (28, 33, 34). In contrast to lysosomal
hexosaminidases, they reside in the cytosol, have neutral pH optima,
and are heat labile. Despite the wide occurrence of these neutral
isoforms in tissues, their natural substrates have not been previously identified (28).
-linked GlcNAc
but is totally ineffective toward
-linked GlcNAc or
-linked
GalNAc. This substrate specificity is in contrast with that of
lysosomal hexosaminidases, which hydrolyze both GlcNAc and GalNAc
substrates. It also determines that GlcNAc, but not GalNAc, is a
competitive inhibitor of O-GlcNAcase. Unlike lysosomal hetero-oligmeric hexosaminidases A
(
a
b) or B
(2x
a
b) (32), the cloned human
O-GlcNAcase has only one polypeptide of 916 amino acids with
a predicted molecular mass of 103 kDa. The apparent size on SDS-PAGE is
130 kDa, which is identical to the glucosaminidase purified from rat
brain (28). The oligomeric status of the hexosaminidase C is still not
known, because gel filtration, SDS-PAGE, and sucrose density gradient
centrifugation give inconsistent estimations of its native molecular
size (28).
-amyloid precursor protein, which gives rise to the neurotoxic
-amyloid peptide in Alzheimer's brains, is also
O-GlcNAcylated (39). Furthermore, Alzheimer's disease has
been correlated to the glycosylation of at least one protein, the
chathrin assembly protein-3 (40). Continuing efforts in several
laboratories are underway to understand the role of O-GlcNAc
in the development of such neurodegenerative diseases. In addition,
strong evidence suggests that O-GlcNAc is involved in the
development of insulin resistance in diabetes mellitus (4, 41).
Infusion of glucosamine or the overexpression of
glutamine:fructose-6-phosphate amidotransferase in animal models, both
of which increase cellular UDP-GlcNAc levels through the hexosamine
synthetic pathway (42, 43), leads to insulin resistance (44, 45). The
cloning of O-GlcNAcase will not only allow the selective
disruption of the gene, or tissue-specific overexpression of the
protein in animal models, but will also make it possible to directly
regulate and monitor O-GlcNAc-modified proteins and thus
facilitate the understanding of the role of this modification in the
development of these diseases.
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ACKNOWLEDGEMENTS |
---|
We thank the Kazusa DNA Research Institute, Japan for providing the KIAA0679 cDNA and Dr. N. Zachara for reading the manuscript.
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FOOTNOTES |
---|
* Supported by National Institutes of Health Grant HD13563.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-614-5993; Fax: 410-614-8804; E-mail: gwhart@jhmi.edu.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M010420200
2 Y. Gao and G. W. Hart, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
O-GlcNAc, O-linked N-acetylglucosamine;
GalNAc, N-acetylgalactosamine;
GlcNAc, N-acetylglucosamine;
OGT, O-GlcNAc transferase;
O-GlcNAcase, N-acetyl--D-glucosaminidase;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
pNP, p-nitrophenyl;
PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate;
bp, base pair(s);
CTD, C-terminal domain;
kb, kilobase(s);
EST, expressed sequence tag;
MALDI-TOF, matrix-assisted laser desorption
ionization-time of flight;
MS, mass spectrometry;
ConA, concanavalin A;
PMSF, phenylmethylsulfonyl fluroride.
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