A catalytically inactive b1,4- N -acetylglucosaminyltransferase IIIGnT-III behaves as a dominant negative GnT-III inhibitor Hideyuki Ihara, Yoshitaka Ikeda, Souichi Koyota, Takeshi Endo,
Trang 1A catalytically inactive b1,4- N -acetylglucosaminyltransferase III
(GnT-III) behaves as a dominant negative GnT-III inhibitor
Hideyuki Ihara, Yoshitaka Ikeda, Souichi Koyota, Takeshi Endo, Koichi Honke and Naoyuki Taniguchi Department of Biochemistry, Osaka University Medical School, Suita, Osaka, Japan
b1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a
regulatory role in the biosynthesis of N-glycans, and it has
been suggested that its product, a bisecting GlcNAc, is
involved in a variety of biological events as well as in
regu-lating the biosynthesis of the oligosaccharides In this study,
it was found, on the basis of sequence homology, that
GnT-III contains a small region that is signi®cantly homologous
to both snail b1,4GlcNAc transferase and b1,4Gal
trans-ferase-1 Subsequent mutational analysis demonstrated an
absolute requirement for two conserved Asp residues
(Asp321 and Asp323), which are located in the most
homologous region of rat GnT-III, for enzymatic activity
The overexpression of Asp323-substituted, catalytically
inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no signi®cant decrease
in its expression, and led to a speci®c inhibition of the for-mation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells These ®ndings indicate that the mutant serves a dominant negative eect on
a speci®c step in N-glycan biosynthesis This type of Ôdomi-nant negative glycosyltransferaseÕ, identi®ed has potential value as a powerful tool for de®ning the precise biological roles of the bisecting GlcNAc structure
Keywords: GnT-III; glycosyltransferase; bisecting GlcNAc; N-glycan synthesis; dominant negative eect
b1,4-N-Acetylglucosaminyltransferase III (GnT-III)
cata-lyzes the transfer of GlcNAc from UDP-GlcNAc, a glycosyl
donor, to a core b-mannose residue in N-linked
oligosac-charides via a b1 ® 4 linkage, resulting in the formation of
a bisected sugar chain [1] The resulting GlcNAc residue is
referred to as a bisecting GlcNAc, and is known to play a
role in regulating the biosynthesis of N-glycans, as the
addition of this unique structure inhibits the action of other
N-acetylglucosaminyltransferases, such as GnT-IV and
GnT-V, both of which are involved in the formation of
multiantennary sugar chains [2] Thus, GnT-III can be
regarded as a key glycosyltransferase in N-glycan
biosyn-thetic pathways
It has been suggested that GnT-III and/or the bisecting
GlcNAc residue are involved in a variety of biological
processes, such as the intracellular sorting of
glycopro-teins [3], secretion of apo B100 from the liver [4], cell
adhesion [5] and cancer metastasis [6,7], as evidenced by
gene transfection experiments using GnT-III cDNA
Although studies using GnT-III-de®cient mice have
revealed that a defect in GnT-III suppresses
diethylni-trosamine-induced hepatocarcinogenesis [8,9], they do not
provide any explanations for the changes caused by an overexpression of GnT-III, as mentioned above The mechanisms that underlie such a variety of biological events, which are caused by ectopic expression, overex-pression and defects in GnT-III, remain obscure In addition, the issue of whether phenotypes associated with altered expression of GnT-III are the actual consequences
of N-glycan modi®cation by GnT-III remains unresolved
To address these issues, a catalytically inactive GnT-III and/or dominant negative mutant GnT-III would be valuable because it may potentially de®ne whether bisected N-glycan structures are actually important Alternatively, the issue of whether GnT-III protein is directly involved regardless of formation of bisected sugar chains could also be examined
The identi®cation of catalytic residues, the absence of which leads to the complete loss of activity, is required to produce a catalytically inactive GnT-III The enzymatic properties of this enzyme have been extensively studied in terms of substrate speci®city towards acceptors and donors, and these collective data provide an enzymatic basis for the role of the bisecting GlcNAc in the regulation
of N-glycan biosynthesis [1,10,11] Nevertheless, because the catalytic mechanism of GnT-III has not yet been analyzed in suf®cient detail, the chemical basis of the enzymatic reaction is not known The reaction catalyzed
by GnT-III is accompanied by inversion at the anomeric center of the transferred monosaccharide and the forma-tion of a b1 ® 4 linkage, while chemically similar reactions are also catalyzed by other glycosyltransferases such as b1,4GlcNAc transferase (b1,4GlcNAcT) from snail, Lymnaea stagnalis [12], and mammalian b1,4Gal transferases, (b1,4GalTs), both of which are mutually homologous [13] It seems more likely that the common properties of these three enzymes are associated with the catalytic mechanism, rather than substrate binding, as the
Correspondence to N Taniguchi, Department of Biochemistry, Osaka
University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871,
Japan Fax: + 81 6 6879 3429, Tel.: + 81 6 6879 3420,
E-mail: proftani@biochem.med.osaka-u.ac.jp
Abbreviations: GnT-III, b1,4-N-acetylglucosaminyltransferase III;
PNGase, peptide-N-glycosidase; HRP, horseradish peroxidase;
DMEM, Dulbecco's modi®ed Eagle's medium.
Enzyme: b1,4-N-acetylglucosaminyltransferase III (EC 2.4.1.144).
Note: a web site is available at http://www.med.osaka-u.ac.jp/pub/
biochem/index.html
(Received 17 April 2001, revised 15 October 2001, accepted 29 October
2001)
Trang 2donor and acceptor substrates are divergent Therefore, as
the residues that are conserved among these enzymes
would be expected to be involved in the presumed
common catalytic mechanism, such residues must be
essential for enzyme activity
In this study, amino-acid residues of GnT-III, which are
conserved in mammalian b1,4GalT-1 and snail
b1,4GlcN-AcT were identi®ed by a comparison of sequences using a
dot matrix analysis, and were then examined for their
requirement with respect to GnT-III activity In addition,
the effect of the inactive mutant GnT-III, in which a residue
identi®ed as being essential was replaced, was examined in
the biosynthesis of bisected sugar chains in cells These
experiments were performed, in order to determine if the
mutant serves as a Ôdominant negative glycosyltransferaseÕ
toward a speci®c step in the oligosaccharide biosynthesis
E X P E R I M E N T A L P R O C E D U R E S
Materials
Restriction endonucleases and DNA-modifying enzymes
were purchased from Takara (Kyoto, Japan), Toyobo
(Shiga, Japan) and New England Biolabs (UK)
UDP-GlcNAc and UDP-GlcNAc were obtained from Sigma (MO,
USA) Oligonucleotide primers were synthesized by Greiner
Japan (Tokyo, Japan) Antibodies were obtained from
following sources: monoclonal anti-(GnT-III) Ig from
Fujirebio Inc (Tokyo, Japan); horseradish peroxidase
(HRP)-conjugated anti-(mouse IgG) Ig from Promega
(WI, USA) Peptide-N-glycosidase F (PNGase F) was
obtained from Roche Diagnostics (IN, USA) Standards of
pyridylaminated sugar chains were purchased from Takara
and Seikagaku Corp (Tokyo, Japan) Other common
chemicals were obtained from Wako pure chemicals
(Osaka, Japan), Nacalai Tesque (Kyoto, Japan) and Sigma
Construction of expression plasmids
For transient expression in COS-1 cells, a cDNA encoding
rat GnT-III [14] was subcloned into the EcoRI sites of an
SV40-based expression vector, pSVK3 (Amersham
Phar-macia Biotech, Buckinghamshire, UK) When the enzyme
was stably expressed in Huh6 cells, the cDNA was
subcloned into the EcoRI sites of another expression vector,
pCXNII, which contains a neorgene [15] In this vector, the
GnT-III was expressed under the control of the b-actin
promoter and the CMV enhancer
Site-directed mutagenesis
Site-directed mutagenesis experiments were carried out
according to Kunkel [16], as described previously [17] A
0.6-kb fragment obtained by digestion of rat GnT-III cDNA
with EagI and HindIII was subcloned into pBluescript
KS+, and the resulting plasmid was used for transformation
of CJ236 (dut±, ung±) The uracil-substituted ssDNA was
prepared by infection of the transformed CJ236 with a
helper phage M13K07 This template was then used with
oligonucleotide primers to replace the conserved aspartic
acid residues with alanine The primers used in this study
were
5¢-TTTATCATCGACGCCGCGGACGAGATCC-3¢ for replacement of Asp321 (designated D321A),
5¢-ATCATCGACGACGCCGCGGAGATCCCTGCGT-3¢ for Asp323 (D323A), and 5¢-ATCCCTGCGCGTGCC GGCGTGCTGTTCCTGAAG-3¢ for Asp329 (D329A) The resulting mutations were veri®ed by dideoxy sequencing using a DNA sequencer (model 373 A, Applied Biosystems,
CA, USA), and the entire sequences that had been subjected
to mutagenesis were also veri®ed The corresponding region
of the wild-type cDNA was replaced by each mutant sequence The plasmids for the expression of these mutants were constructed, as were those for the wild-type enzyme, and used for transfection
Cell culture Huh6 cells, a human hepatoblastoma cell line, and COS-1 cells were maintained in Dulbecco's modi®ed Eagle's medium (DMEM) containing 10% fetal bovine serum,
100 UámL)1 penicillin, 100 lgámL)1 streptomycin and
5 gáL)1glucose under a humidi®ed atmosphere of 95% air and 5% CO2
Protein determination Protein concentration was determined with BCA Kit (Pierce, IL, USA) using BSA as a standard
Electrophoresis and immunoblot analysis SDS/PAGE was carried out on 8% gels, according to Laemmli [18] The separated proteins were transferred onto
a nitrocellulose membrane (PROTORAN, Schleicher & Schuell Inc., NH, USA) The resulting membrane was blocked with 5% skimmed milk and 0.5% BSA in NaCl/Pi
containing 0.05% Tween-20, and was then incubated with
an anti-(GnT-III) Ig After washing with NaCl/Pi that contained 0.05% Tween-20, the membrane was reacted with
a HRP-conjugated goat anti-(mouse IgG) Ig The immuno-reactive protein bands were visualized by chemiluminescence using an ECL system (Amersham Pharmacia) Ponceau staining of the transferred membrane was performed before blocking with skimmed milk and BSA to verify equal amounts of proteins loaded For digestion by PNGase F, the samples were denatured by boiling for 3 min in 20 mM
phosphate buffer (pH 7.0) containing 0.2% SDS, 1% 2-mercaptoethanol and 0.5% Triton X-100 Deglycosyla-tion by PNGase F was performed according to the manu-facturer's instructions
DNA transfection Expression plasmids were transfected into cells by electro-poration [19] using a Gene Pulser (Bio-Rad, CA, USA), as described previously [14] In a typical experiment, the cells were washed with Hepes-buffered saline and resuspended in the same solution Plasmids (30 lg), puri®ed by CsCl gradient ultracentrifugation, were added to the cell suspen-sion, followed by electri®cation For transient expression in COS-1 cells, the transfected cells were harvested after an appropriate growth period When stable transfectants of Huh6 cells were established, the transfected cells were subjected to selection by geneticin resistance The expression
of GnT-III was veri®ed by immunoblot analysis and enzyme activity assay for GnT-III
Trang 3Enzyme activity assays for glycosyltransferases
GnT-III and GnT-V activities were assayed using a
pyridylaminated biantennary sugar chain as an acceptor
substrate, as described previously [20,21] A large-scale
preparation of pyridylaminated biantennary sugar chain
was performed as reported previously [22±24] Standard
assays were performed in a ®nal volume of 15 ll of 125 mM
Mes/NaOH buffer (pH 6.25) containing 0.5% Triton
X-100, 200 mMGlcNAc The assay mixture also contained
10 mM MnCl2 for GnT-III assay or 10 mM EDTA for
GnT-V The concentration of pyridylaminated biantennary
sugar chain was 10 lM for both enzymes The
concentra-tions of the donor, UDP-GlcNAc, for GnT-III and GnT-V
were 20 and 40 mM, respectively After incubation for 2±4 h,
the reactions were terminated by rapidly heating to 100 °C
The reaction mixtures were then centrifuged at 10 000 g, for
10 min and the resulting supernatants were applied to an
HPLC equipped with a TSK-gel, ODS-80TM column
(4.6 ´ 150 mm) (Tosoh, Tokyo, Japan) in order to separate
and quantitate the products Elution was performed
isocratically at 55 °C using a 20-mM acetate buffer
(pH 4.0) containing 0.3% and 0.15% butanol for GnT-III
and GnT-V assays, respectively The column eluate was
monitored for ¯uorescence using a detector (model
RF-10AXL, Shimadzu, Kyoto, Japan) operating at
excita-tion and emission wavelengths of 320 and 400 nm,
respec-tively The amounts of products were estimated from the
¯uorescence intensity b1,4GalT activity was also assayed
using a pyridylaminated biantennary sugar chain as an
acceptor substrate [25] The assay mixture for b1,4GalT
consisted of 50 mMMops buffer (pH 7.4) containing 20 mM
MnCl2, 0.5% Triton X-100, 5 mM UDP-Gal and 10 lM
pyridylaminated biantennary sugar chain After incubation
for 2 h, the reaction was stopped, and the reaction mixture
was analyzed by normal phase HPLC In this assay, a
TSK-gel Amide-80 column (4.6 ´ 250 mm) (Tosoh) was used at
40 °C The elution buffer was 1.14% acetic
acid/triethyl-amine (pH 7.3) that also contained 62% acetonitrile
Structural analysis of sugar chains
Huh6 cells and the transfectant cells, which were harvested
from 10 10-cm dishes of con¯uent cultures were sonicated
and lyophilized Each preparation of the whole cells was
then hydrazinolyzed to liberate Asn-linked oligosaccharides,
as described previously [23,24,26] The free oligosaccharides
were then re-N-acetylated in 10 mL saturated ammonium
bicarbonate with 554 ll acetic anhydride, followed by
desalting on a column of AG 50 W-X12 resin (Bio-Rad)
Fluorescence labeling of the sugar chains was carried out by
reductive amination involving the use of a ¯uorescent
reagent, 2-aminopyridine and sodium cyanoborohydride
[24] After pyridylamination, the excess reagents were
removed by gel ®ltration with HW-40F (Toyopearl, Tosoh)
The pyridylaminated sugar chains were then desialylated,
degalactosylated and defucosylated by sialidase
(Arthro-bacter ureafaciens, Nacarai tesque), b-galactosidase (jack
bean, Seikagaku Corp.) and a-fucosidase (bovine kidney,
Sigma), respectively The digested samples were then
analyzed by HPLC using a TSK-gel ODS-80TM column
(4.6 ´ 150 mm), and elution was performed at 55 °C by a
linear gradient of butanol from 0.1% to 0.25% in 20 mM
ammonium/acetate buffer (pH 4.0) The eluted sugar chain peaks were identi®ed by comparison with standard pyr-idylaminated sugar chains (Takara and Seikagaku Corp.) RT-PCR
Total RNA from parental Huh6 cells and various transfec-tants was prepared using TRIZOL (Gibco-BRL, MD, USA), and the cDNAs were synthesized by reverse transcriptase with an oligo dT-adaptor primer from RNA
LA PCR Kit (Takara) In order to speci®cally detect the expression of endogenous human GnT-III, PCR was performed with the selective primers for human GnT-III
in a PCR Thermal Cycler 480 (Takara) The primers used in this study were designed to detect mRNA for human GnT-III but not rat GnT-GnT-III: 5¢-AAGACCCTGTCCTAT-3¢ (nucleotide position 85±99 in the ORF) for sense, and 5¢-GTTGGCCCCCTCAGG-3¢ (position 415±429) for antisense This differential detection was con®rmed by PCR using plasmid DNA containing human and rat GnT-III cDNAs as templates The primers to detect b-actin mRNA
as a control were 5¢-CAAGAGATGGCCACGGCTGCT-3¢ (nucleotide position 673±693 in the ORF of human b-actin)and5¢-TCCTTCTGCATCCTGTCGGCA-3¢(posi-tion 927±947), for sense and antisense, respectively The sizes of the products that were yielded by the PCR using these primers were expected to be 345 bp and 275 bp for human GnT-III and b-actin, respectively The absence of ampli®cation of the genomic DNA was veri®ed by subject-ing total RNA directly to the PCR without reverse transcription, because the open reading frame of GnT-III
is encoded by a single exon
R E S U L T S A N D D I S C U S S I O N
Comparison of the amino-acid sequence of GnT-III with snail b1,4GlcNAcT and mammalian b1,4GalT-1
In order to identify the essential residues that are important for GnT-III activity, the candidate residues for examination were selected on the basis of amino-acid sequence homology amongratGnT-III,mammalianb1,4-galactosyltransferase-1 (b1,4GalT-1) and snail b1,4-N-acetylglucosaminyltrans-ferase (b1,4GlcNAcT) Dot matrix analyses comparing the GnT-III sequence with that of either of the other enzymes showed signi®cant similarities in the small region, in which three aspartic acid residues and several other residues are perfectly conserved (Fig 1A,B) Only this homologous region was detected in all three enzymes These Asp residues correspond to Asp321, Asp323 and Asp329 in the rat GnT-III sequence, as shown by sequence alignment (Fig 1C) The sequence of Asp321-Val322-Asp323 appears to corre-spond to the D-X-D motif, as have been suggested for the catalytic importance in many other glycosyltransferases [27±41], and, thus, the sequence comparison suggests that these Asp residues represent likely candidates for investiga-tion as having a role in GnT-III activity
Site-directed mutagenesis of the conserved aspartic acid residues in GnT-III
To explore the requirement of the candidate amino acids, Asp321, Asp323 and Asp329, for enzyme activity, mutant
Trang 4GnT-IIIs in which these Asp residues were replaced by Ala
were prepared using site-directed mutagenesis, and the
respective mutants were designated as D321A, D323A and
D329A When these mutants were transiently expressed in
COS-1 cells, their protein expression levels were found to be
similar to that of wild-type, as indicated by immunoblot
analysis (Fig 2A,B) However, in the standard activity
assay, the D321A and D323A mutants had no detectable
catalytic activity, whereas the D329A mutant was fully
active, as shown by an activity similar to that of the
wild-type enzyme (Table 1) No activity was detected in the
D321A and D323A mutants even under the conditions of
longer incubation time and UDP-GlcNAc concentration as
high as 100 mM, indicating that the replacement of Asp321
and Asp323 lead to a complete loss of activity These data
suggest that Asp321 and Asp323 play an essential role in the
activity of enzyme, while Asp329 does not, even though this
Asp residue is conserved Our previous study showed that
three N-linked glycosylation sites in rat GnT-III are fully
glycosylated when expressed in COS cells [42], and all
mutants used in this study were also found to be fully
glycosylated, as indicated by the digestion with PNGase F
(Fig 2C) Immuno¯uorescence microscopic analysis
showed that the intracellular localization of the mutants are identical to that of the wild-type enzyme, indicating that the replacements of Asp321 and Asp323 have no effect on intracellular localization (data not shown) Thus, it seems unlikely that these mutations lead to gross conformational alterations or misfolding of the protein, but it is more likely that the loss of the activity is the result of the deletion of the active site residues
The possible role of Asp321 and Asp323
in the catalysis of GnT-III The absolute requirement for Asp321 and Asp323 and their conservation in b1,4GalT-1 and snail b1,4GlcNAcT suggest that the short sequence of Asp321-Val322-Asp323 in GnT-III plays a role that is analogous to the function of the D-X-D motif, found in b1,4GalT-1 [13] Although an essential role for the D-X-D motif has been demonstrated in many glycosyltransferases [27±35], the function of this motif seemed divergent While a large clostridial glucosyltransfer-ase requires the D-X-D motif for UDP or UDP-sugar binding [27], this motif appears not to be critical for nucleotide binding in GM2 synthase [34] and Fringe [31] in
Fig 1 Dotplot analyses for rat GnT-III vs snail b1,4GlcNAcT or bovine b1,4GalT-1 Dotplots for (A) rat GnT-III vs L stagnalis b1,4GlcNAcT and (B) rat GnT-III vs bovine b1,4GalT-1 are shown The amino-acid sequences were compared under the conditions of a window size of 10 residues and 50% of identity using the computer software program, ALIGN The numbers beside the axis indicate the residue number of each enzyme Diagonal plots show the homologous regions, which satisfy the above conditions The sequences of the most homologous region are given outside the matrices (C) Multiple alignment of the homologous regions of GnT-III, b1,4GalT-1 and L stagnalis b1,4GlcNAcT were carried out by
CLUSTALV The amino-acid residues which are conserved in all enzymes are highlighted by shaded boxes, and two homologous residues are also indicated by grey-shaded boxes The conserved aspartic acid residues that were examined by mutational analysis are indicated by arrowheads GenBank accession numbers for the glycosyltransferases are: GnT-III (human), D13789; GnT-III (rat), NM_019239; GnT-III (mouse), NM_010795; b1,4GlcNAcT (L stagnalis), X80228; b1,4 GalT-1 (human), X14085; b1,4 GalT-1 (bovine), X14558; b1,4 GalT-1 (mouse), J03880; b1,4 GalT-2 (mouse), AB019541.
Trang 5spite of its requirement for their enzyme activities On the other hand, crystallographic analyses of b1,4GalT-1 [36] as well as other glycosyltransferases [37±41] have suggested that this motif serves the coordination of a divalent cation such as Mn2+along with a phosphoryl group of the donor The motif would thereby allow the enzyme to interact with a nucleotide portion of the donor nucleotide sugar and also may facilitate the reaction via electrostatic catalysis involv-ing the divalent cation Although the function of the D-X-D motif is not known for GnT-III, it is possible that the D-X-D motif in GnT-III plays a similar role to that in b1,4GalT-1 because of the signi®cant sequence homology in the region containing this motif In a large clostridial glucosyltransferase mutant similar to the GnT-III D321A and D323A mutants, the activity could be recovered in the presence of extremely high concentrations of Mn2+, supporting the suggestion that the equivalent aspartic acid residues in the motif are involved in the coordination with the divalent cation [27] Nevertheless, in the case of GnT-III, activity was not detected even at concentrations of Mn2+as high as 100 mM, suggesting an absolute requirement of Asp321 and Asp323 for the coordination of Mn2+during the reaction of GnT-III (data not shown)
Expression of a catalytically inactive GnT-III
in Huh6 cells, a hepatoblastoma cell line
In order to determine whether a catalytically inactive GnT-III mutant serves a dominant negative function by prevent-ing the action of the wild-type endogenous enzyme, Huh6 cells, a human hepatoblastoma cell line, were transfected with the rat GnT-III mutants because a structural pro®le of the N-glycans in these cells had been previously character-ized through a structural analysis of N-linked sugar chains
of a-fetoprotein produced by the cells [43] Huh6 cells express relatively high levels of GnT-III and produce bisected sugar chains, the products of this glycosyltransfer-ase [43] Following the selection of the transfected cells by geneticin resistance, clones were grown separately, and the expression of the rat mutant enzyme was veri®ed by immunoblot analysis As a result, we obtained three clones, which overexpress the D323A GnT-III mutant (Fig 2D) In Huh6 cells, as was in COS-1 cells, the D323A mutant was expressed at a similar level to the wild-type, and appeared to
be fully glycosylated (Fig 2D,F) In the case of the other mutant, D321A, however, we were not successful in establishing such clones
The speci®c suppression of endogenous GnT-III activity by expression of the catalytically inactive D323A mutant in Huh6 cells
When assays for GnT-III activity were performed in the transfected cells, it was found that the activity in the transfected cells were as low as less than 5% of the activity in the parental or mock-transfected Huh6 cells, which were used as controls On the other hand, the activities of GnT-V, another GlcNAc transferase which is involved in the formation of b1,6-branches, and b1,4GalT were not essen-tially affected in the transfected cells, indicating that the overexpression of the D323A mutant has no effect on these glycosyltransferase activities (Fig 3) Therefore, it appears that the overexpression of the mutant does not impair
Fig 2 Expression of the wild-type and mutant GnT-III proteins The
wild-type and mutant enzymes were transiently expressed in COS-1
cells (A±C), and stably expressed in Huh6 cells (D±F) (A,D) The cell
homogenates were separated on 8% SDS-gels and were analyzed by
immunoblot using anti-(GnT-III) Ig (B,E) The amounts of protein
loaded were veri®ed by Ponceau staining, prior to the immunoblot
analysis (C,F) The samples were treated with PNGase F, followed by
SDS/PAGE and immunoblotting COS-1 and pSVK3 indicate
non-transfected and vector-non-transfected (mock) COS-1 cells, respectively.
Huh6, Huh6/D323A and Huh6/WT represent parental
nontrans-fected, D323A-transfected and wild-type-transfected Huh6 cells,
respectively Details of the conditions are described in Experimental
procedures.
Table 1 Activities of the wild-type and mutant GnT-IIIs transiently
expressed in COS-1 cells GnT-III activity was determined as described
in Experimental procedures The mock plasmid was transfected with a
vector, pSVK3 ND, not detectable.
Plasmid GnT-III activity(nmoláh )1 ámg protein )1 )
Not transfected ND
Wild-type 0.98
Trang 6glycosyltransferase activities in a nonspeci®c manner, for example, via the downregulation of their expression or damage to the Golgi apparatus Quite similar results were obtained in all three of the obtained clones These results suggest that the overexpression of the D323A mutant speci®cally suppresses the activity of endogenous GnT-III
To examine whether the mutant GnT-III inhibits the intrinsic GnT-III in vitro, the extracts from the parental cells were mixed with the extract from the D323A-transfected cells, and were then analyzed by the activity assay As shown in Fig 4, no inhibitory effect of the mutant was observed, and, thus, it seemed unlikely that the in vivo inhibition by the mutant is due to direct competition for substrate Furthermore, RT-PCR using a primer set that is speci®c to the human GnT-III sequence showed that the mRNAs for endogenous human GnT-III were not signif-icantly decreased (Fig 5), suggesting that the decreased GnT-III activity is not due to the downregulation of expression of the intrinsic human GnT-III gene Therefore,
it is more likely that the activity of the intrinsic human enzyme is decreased as the result of control at the translational or post-translational level including protein± protein interactions
Blockage of a speci®c step, namely the formation
of a bisecting GlcNAc residue, in N-glycan biosynthesis via the expression of the D323A mutant
In order to further examine the issue of whether the biosynthesis of bisected sugar chains are inhibited by overexpression of the D323A mutant, total N-linked sugar chains were prepared from the cells and labeled with 2-aminopyridine, a ¯uorescent reagent The resulting free oligosaccharides were digested with sialidase, b-galactosi-dase and a-fucosib-galactosi-dase, and then subjected to reversed phase HPLC, in order to analyze the core structures, i.e the addition of a bisecting GlcNAc and the extent of branching
As shown in Fig 6, when parental cells, which express various sugar chains with bi-, tri- and tetra-antennae were examined, substantial fractions (20±30%) of these sugar chains were found to contain the bisecting GlcNAc Almost
Fig 4 Eect of the D323A mutant on the endogenous GnT-III activity
in vitro After indicated amounts of the extracts from parental Huh6 cells and the D323A-transfected cells were mixed, GnT-III activity was assessed The data are expressed as the relative value to the activity determined in the absence of the mutant extract.
Fig 3 GnT-III, b1,4GalT and GnT-V activities in the
D323A-trans-fectant Activities of GnT-III (A), b1,4GalT (B) and GnT-V (C) were
assayed using a pyridylaminated oligosaccharide acceptor, as described
in Experimental procedures Data for the transfectants are expressed as
mean values from three dierent clones for D323A and 2 clones for the
wild-type GnT-IIIs and mock-transfected cells In the
D323A-trans-fectants, standard deviations are also shown.
Trang 7the same pro®le was obtained in the case of the
mock-transfected cells (data not shown) On the other hand, the
D323A-transfected cells were found to express negligible
levels of bisected sugar chains These results indicate that the
overexpression of the D323A mutant blocks the
biosynthe-sis of bisected sugar chains as the result of a decrease in the activity of endogenous GnT-III The results demonstrate that the catalytically inactive mutant enzyme acts as a dominant negative glycosyltransferase toward the forma-tion of a bisecting GlcNAc in vivo
Fig 5 Detection of mRNA for endogenous human III in the D323A-transfectant by RT-PCR mRNA expression of endogenous human GnT-III in parental Huh6 cells and the transfectants for the D323A and wild-type GnT-GnT-IIIs were investigated by RT-PCR (upper panel) Reverse transcription was omitted to verify the absence of ampli®cation of genomic DNA in the lanes indicated by RT (±) Human b-actin mRNA expression was also examined as a control (lower panel) Speci®city to the endogenous human enzyme was con®rmed by PCR using plasmid DNAs Lanes: human GnT-III and rat GnT-III indicate PCR-ampli®cation of plasmids containing cDNAs for human and rat enzyme, respectively Details regarding the PCR procedures are described in Experimental procedures.
Fig 6 Structural analysis of Asn-linked sugar
chains from parental and transfected Huh6
cells Elution pro®les of pyridylaminated sugar
chains in reversed phase HPLC are shown for
parental Huh6 cells (a), wild-type
GnT-III-transfectant (b) and D323A-GnT-III-transfectant (c).
Numbers at the top indicate peaks for bisected
sugar chains: 1, asialo-agalacto-bisected
biantennary sugar chain; 2,
agalacto-bisected tetraantennary sugar chain; 3,
asialo-agalacto-bisected triantennary sugar chain
containing a b1,4-GlcNAc residue on the
Mana1,3 arm Arrowheads indicate
nonbi-sected sugar chains: left arrowhead,
overlap-ping peaks of asialo-agalacto biantennary and
asialo-agalacto tetraantennary sugar chains;
right, asialo, agalacto triantennary sugar chain
containing a b1,4-GlcNAc residue on the
Mana1,3 arm These structures are shown
above the pro®le.
Trang 8C O N C L U S I O N S
Our present study identi®ed Asp321 and Asp323 as being
essential residues in the enzyme activity of GnT-III, and
provides further support for the view that the D-X-D motif
in glycosyltransferases is important and signi®cant, even
though the exact roles of Asp321 and Asp323 in GnT-III
remains to be elucidated On the other hand, the utility of
the catalytically inactive GnT-III as a dominant negative
glycosyltransferase toward bisecting GlcNAc formation is
clearly demonstrated The ®ndings contribute to the
estab-lishment of a distinct strategy for in vivo oligosaccharide
manipulation, which permits the speci®c inhibition of a
particular glycosylation step in the biosynthetic pathway in
a manner that is independent of gene targeting Although a
similar attempt was made for a1,3GalT [44], the mechanism
of the inhibition is not known It is generally thought that
dominant negatively acting molecules involve competition
with the corresponding endogenous wild-type molecules
Such competition could also occur in the case of the
dominant negative GnT-III Possible steps of the
competi-tion could involve: (a) competicompeti-tion for localizacompeti-tion in the
Golgi, as observed in competition for cell surface b1,4GalT-1
[45]; (b) homophilic interaction involved in the formation of
the active enzyme; (c) association with a presently unknown
molecule that activates the enzyme; and (d) activation of the
enzyme by post-translational modi®cation Although the
mechanism of action of the dominant negative mutant is not
yet known, an understanding of this mechanism could lead
to the discovery of a novel regulatory mechanism for
glycosyltransferase activity
A C K N O W L E D G E M E N T S
We thank Dr Milton S Feather for correcting this manuscript This
research was supported, in part, by a Grant-in-Aid for Scienti®c
Research on Priority Area no 10178104 from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and
by the Sumitomo Foundation.
R E F E R E N C E S
1 Narasimhan, S (1982) Control of glycoprotein synthesis
UDP-GlcNAc: glycopeptide beta 4-N-acetylglucosaminyltransferase III,
an enzyme in hen oviduct which adds GlcNAc in beta 1 ® 4
linkage to the beta-linked mannose of the trimannosyl core of
N-glycosyl oligosaccharides J Biol Chem 257, 10235±10242.
2 Schachter, H (1986) Biosynthetic controls that determine the
branching and microheterogeneity of protein-bound
oligosacchar-ides Biochem Cell Biol 64, 163±181.
3 Sultan, A.S., Miyoshi, E., Ihara, Y., Nishikawa, A., Tsukada, Y.
& Taniguchi, N (1997) Bisecting GlcNAc structures act as
nega-tive sorting signals for cell surface glycoproteins in
forskolin-treated rat hepatoma cells J Biol Chem 272, 2866±2872.
4 Ihara, Y., Yoshimura, M., Miyoshi, E., Nishikawa, A., Sultan,
A.S., Toyosawa, S., Ohnishi, A., Suzuki, M., Yamamura, K.,
Ijuhin, N & Taniguchi, N (1998) Ectopic expression of
N-acety-lglucosaminyltransferase III in transgenic hepatocytes disrupts
apolipoprotein B secretion and induces aberrant cellular
mor-phology with lipid storage Proc Natl Acad Sci USA 95, 2526±
2530.
5 Sheng, Y., Yoshimura, M., Inoue, S., Oritani, K., Nishiura, T.,
Yoshida, H., Ogawa, M., Okajima, Y., Matsuzawa, Y &
Taniguchi, N (1997) Remodeling of glycoconjugates on CD44
enhances cell adhesion to hyaluronate, tumor growth and
meta-stasis in B16 melanoma cells expressing b1,4-N-acetylglucosami-nyltransferase III Int J Cancer 73, 850±858.
6 Yoshimura, M., Ihara, Y., Matsuzawa, Y & Taniguchi, N (1996) Aberrant glycosylation of E-cadherin enhances cell±cell binding to suppress metastasis J Biol Chem 271, 13811±13815.
7 Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S & Taniguchi, N (1995) Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfec-tion Proc Natl Acad Sci USA 92, 8754±8758.
8 Bhaumik, M., Harris, T., Sundaram, S., Johnson, L., Guttenplan, J., Rogler, C & Stanley, P (1998) Progression of hepatic neo-plasms is severely retarded in mice lacking the bisecting N-acetylglucosamine on N-glycans: evidence for a glycoprotein factor that facilitates hepatic tumor progression Cancer Res.
58, 2881±2887.
9 Yang, X., Bhaumik, M., Bhattacharyya, R., Gong, S., Rogler, C.E & Stanley, P (2000) New evidence for an extra-hepatic role of N-acetylglucosaminyltransferase III in the progression of diethyl-nitrosamine-induced liver tumors in mice Cancer Res 60, 3313± 3319.
10 Schachter, H., Narasimhan, S., Gleeson, P & Vella, G (1983) Control of branching during the biosynthesis of asparagine-linked oligosaccharides Can J Biochem Cell Biol 61, 1049±1066.
11 Ikeda, Y., Koyota, S., Ihara, H., Yamaguchi, Y., Korekane, H., Tsuda, T., Sasai, K & Taniguchi, N (2000) Kinetic basis for the donor nucleotide-sugar speci®city of b1,4-N-acetylglucosaminyl-transferase III J Biochem (Tokyo) 128, 609±619.
12 Bakker, H., Agterberg, M., Van Tetering, A., Koeleman, C.A., Van den Eijnden, D.H & Van Die, I (1994) A Lymnaea stagnalis gene, with sequence similarity to that of mammalian b1 ® 4-galactosyltransferases, encodes a novel UDP-GlcNAc: GlcNAc b-R b1 ® 4-N-acetylglucosaminyltransferase J Biol Chem 269, 30326±30333.
13 Breton, C., Bettler, E., Joziasse, D.H., Geremia, R & Imberty, A (1998) Sequence±function relationships of prokaryotic and eukar-yotic galactosyltransferases J Biochem (Tokyo) 123, 1000±1009.
14 Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K & Taniguchi, N (1992) Puri®cation, cDNA cloning, and expression
of UDP-N-acetylglucosaminyltransferase III from rat kidney.
J Biol Chem 267, 18199±18204.
15 Niwa, H., Yamamura, K & Miyazaki, J (1991) Ecient selection for high-expression transfectants with a novel eukaryotic vector Gene 108, 193±199.
16 Kunkel, T.A (1985) Rapid and ecient site-speci®c mutagenesis without phenotypic selection Proc Natl Acad Sci USA 82, 488± 492.
17 Ikeda, Y., Fujii, J., Taniguchi, N & Meister, A (1995) Human c-glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit J Biol Chem 270, 12471±12475.
18 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680±685.
19 Chu, G., Hayakawa, H & Berg, P (1987) Electroporation for the ecient transfection of mammalian cells with DNA, Nucleic Acids Res 15, 1311±1326.
20 Taniguchi, N., Nishikawa, A., Fujii, S & Gu, J.G (1989) Gly-cosyltransferase assays using pyridylaminated acceptor: N-acetyl-glucosaminyltransferase III, IV, and V Methods Enzymol 179, 397±408.
21 Nishikawa, A., Fujii, S., Sugiyama, T & Taniguchi, N (1988) A method for the determination of N-acetylglucosaminyltransferase III activity in rat tissues involving HPLC Anal Biochem 170, 349±354.
22 Seko, A., Koketsu, M., Nishizono, M., Enoki, Y., Ibrahim, H.R., Juneja, L.R., Kim, M & Yamamoto, T (1997) Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg yolk Biochem Biophys Acta 1335, 23±32.
Trang 923 Patel, T., Bruce, J., Merry, A., Bigge, C., Wormald, M., Jaques, A.
& Parekh, R (1993) Use of hydrazine to release in intact and
unreduced form both N- and O-linked oligosaccharides from
glycoproteins Biochemistry 32, 679±693.
24 Hase, S., Ibuki, T & Ikenaka, T (1984) Reexamination of the
pyridylamination used for ¯uorescence labeling of
oligosacchar-ides and its application to glycoproteins J Biochem (Tokyo) 95,
197±203.
25 Morita, N., Hase, S., Ikehara, K., Mikoshiba, K & Ikenaka, T.
(1988) Pyridylamino sugar chain as an acceptor for
galactosyl-transferase J Biochem (Tokyo) 103, 332±335.
26 Nishiura, T., Fujii, S., Kanayama, Y., Nishikawa, A., Tomiyama,
Y., Iida, M., Karasuno, T., Nakano, H., Yonezawa, T.,
Tanigu-chi, N & Tarui, S (1990) Carbohydrate analysis of
immuno-globulin G myeloma proteins by lectin and high performance
liquid chromatography: role of glycosyltransferase Cancer Res.
50, 5345±5350.
27 Busch, C., Hofmann, F., Selzer, J., Munro, S., Jeckel, D &
Aktories, K (1998) A common motif of eukaryotic
glyco-syltransferases is essential for the enzyme activity of large
clostri-dial cytotoxins J Biol Chem 273, 19566±19572.
28 Wiggins, C.A.R & Munro, S (1998) Activity of the yeast MNN1
a-1,3-mannosyltransferase requires a motif conserved in many
other families of glycosyltransferases Proc Natl Acad Sci USA
95, 7945±7950.
29 Shibayama, K., Ohsuka, S., Tanaka, T., Arakawa, Y & Ohta, M.
(1998) Conserved structural regions involved in the catalytic
mechanism of Escherichia coli K-12 WaaO (RfaI) J Bacteriol.
180, 5313±5318.
30 Hagen, F.K., Hazes, B., de Rao, R.Sa, D & Tabak, L.A (1999)
Structure-function analysis of the UDP-N-acetyl- D
-galactos-amine: polypeptide N-acetylgalactosaminyltransferase Essential
residues lie in a predicted active site cleft resembling a lactose
repressor fold J Biol Chem 274, 6797±6803.
31 Munro, S & Freeman, M (2000) The notch signalling regulator
fringe acts in the Golgi apparatus and requires the
glycosyltrans-ferase signature motif DXD Curr Biol 10, 813±820.
32 Keusch, J.J., Manzella, S.M., Nyame, K.A., Cummings, R.D &
Baenziger, J.U (2000) Expression cloning of a new member of the
ABO blood group glycosyltransferases, iGb3 synthase, that directs
the synthesis of isoglobo-glycosphingolipids J Biol Chem 275,
25308±25314.
33 Keusch, J.J., Manzella, S.M., Nyame, K.A., Cummings, R.D &
Baenziger, J.U (2000) Cloning of Gb3 synthase, the key enzyme in
globo-series glycosphingolipid synthesis, predicts a family of
a1,4-glycosyltransferases conserved inplants, insects, and
mam-mals J Biol Chem 275, 25315±25321.
34 Li, J., Rancour, D.M., Allende, M.L., Worth, C.A., Darling, D.S.,
Gilbert, J.B., Menon, A.K & Young, W.W Jr (2001) The DXD
motif is required for GM2 synthase activity but is not critical for nucleotide binding Glycobiology 11, 217±229.
35 Maeda, Y., Watanabe, R., Harris, C.L., Hong, Y., Ohishi, K., Kinoshita, K & Kinoshita, T (2001) PIG-M transfers the ®rst mannose to glycosylphosphatidylinositol on the lumenal side of the ER EMBO J 20, 250±261.
36 Gastinel, L.N., Cambillau, C & Bourne, Y (1999) Crystal structures of the bovine b4-galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose EMBO J 13, 3546±3557.
37 Charnock, S.J & Davies, G.J (1999) Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms Biochemistry 38, 6380±6385.
38 Pedersen, L.C., Tsuchida, K., Kitagawa, H., Sugahara, K., Darden, T.A & Negishi, M (2000) Heparan/chondroitin sulfate biosynthesis Structure and mechanism of human glucuronyl-transferase I J Biol Chem 275, 34580±34585.
39 Unligil, U.M., Zhou, S., Yuwaraj, S., Sarkar, M., Schachter, H & Rini, J.M (2000) X-ray crystal structure of rabbit N-acetylglu-cosaminyltransferase I: catalytic mechanism and a new protein superfamily EMBO J 19, 5269±5280.
40 Persson, K., Ly, H.D., Dieckelmann, M., Wakarchuk, W.W., Withers, S.G & Strynadka, N.C (2001) Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs Nat Struct Biol.
8, 166±175.
41 Gastinel, L.N., Bignon, C., Misra, A.K., Hindsgaul, O., Shaper, J.H & Joziasse, D.H (2001) Bovine a1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases EMBO J.
20, 638±649.
42 Nagai, K., Ihara, Y., Wada, Y & Taniguchi, N (1997) N-Gly-cosylation is requisite for the enzyme activity and Golgi reten-tion of N-acetylglucosaminyltransferase III Glycobiology 7, 769± 776.
43 Ohno, M., Nishikawa, A., Koketsu, M., Taga, H., Endo, Y., Hada, T., Higashino, K & Taniguchi, N (1992) Enzymatic basis
of sugar structures of a-fetoprotein in hepatoma and hepato-blastoma cell lines: correlation with activities of a1,6,fucosyl-transferase and N-acetylglucosaminyla1,6,fucosyl-transferases III and V Int J Cancer 8, 315±317.
44 Ogawa, H., Kobayashi, I., Nagasaka, T., Namii, Y., Hayashi, S., Kadomatsu, K., Muramatsu, T & Takagi, H (1999) Suppression
of porcine xenoantigen expression by dominant-negative eect of a-1,3-galactosyltransferase (a-1,3-GT) splicing variants Trans-plant Proc 32, 58.
45 Evans, S.C., Lopez, L.C & Shur, B.D (1993) Dominant negative mutation in cell surface b1,4-galactosyltransferase inhibits cell±cell and cell±matrix interactions J Cell Biol 120, 1045±1057.