From the
Giannina Gaslini Institute, 16147 Genova,
Italy, the
Department of Biochemistry and Cell
Biology, Institute for Cell and Developmental Biology, State University of New
York at Stony Brook, New York 11794-5215,
¶Department of Experimental Medicine, University
of Genova and Center of Excellence for Biomedical Research, 16132 Genova,
Italy, and the ||Department of Pediatrics, Meyer
Children Hospital, Rambam Medical Center and B. Rappaport School of Medicine,
Technion, 31096 Haifa, Israel
Received for publication, April 17, 2003 , and in revised form, May 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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Fucose is present in both N-linked and mucin-type
O-linked glycoproteins, where it is linked to the oligosaccharide
chains as a terminal modification that is not further elongated
(7). Recently, a new class of
fucose-containing glycoproteins has been identified in which fucose is
directly O-linked to a serine/threonine residue
(89).
O-fucose modifications have been reported to occur in two distinct
sequence contexts: epidermal growth factor-like
(EGF)1 repeats
(8) and thrombospondin type 1
repeats (TSR) (10). Serum
proteins, involved in blood clotting or clot dissolution, such as human factor
VII, factor XII, uPA (urokinase-type plasminogen activator) and tPA (tissue
type plasminogen activator), are modified by O-fucose as a simple
monosaccharide at a specific consensus sequence within their EGF repeats
(1116).
Elongation of the O-fucose monosaccharide on EGF repeats to a
tetrasaccharide can occur in a protein-specific manner (e.g. factor
IX, Notch and Notch ligands, Refs.
1719).
Elongation of O-fucose is initiated by Fringe, an
O-fucose-specific 1,3-N-acetylglucosaminyltransferase.
Subsequent action of galactosyl- and sialyltransferases results in the
formation of the final tetrasaccharide
(Sia-
2,3/6-Gal-
1,4-GlcNAc-
1,3-Fuc-
1-O(S/T)
(2021).
Many reports have demonstrated an essential functional role for the O-fucose modification on proteins such as Notch, Cripto/Nodal, and uPA (Refs. 15 and 2223; for recent reviews, see Refs. 24 and 25). The Notch receptor plays an essential role in numerous stages of development (26), and recent work has demonstrated that O-fucose is essential for Notch signaling. Reduction or elimination of the enzyme responsible for addition of O-fucose to proteins, protein O-fucosyltransferase 1 (O-FucT-1), causes strong Notch-like phenotypes in both Drosophila (27) and mice (28). In addition, the presence of an O-fucose glycan is essential in the Notch receptor system for the Fringe-mediated modulation of Notch activation in vivo in both Drosophila and mammalian cells (2021). The fact that a defect in O-fucosylation of Notch results in embryonic lethality in mice raised the question of whether Notch is properly O-fucosylated in LAD II/CDGS IIc patients.
Recent work by Hofsteenge and co-workers
(10,
29) has demonstrated that
several extracellular matrix glycoproteins, including thrombospondin-1,
F-spondin and properdin, are modified with O-fucose at a consensus site within
TSRs. In contrast to EGF repeats, the O-fucose on TSRs exists as
either the monosaccharide Fuc-O-Ser/Thr or the disaccharide
Glc-Fuc-O-Ser/Thr. A similar modification (Glc-1,3Fuc) has also
been observed in CHO cells, where it represents a quite abundant form of
glycosylation (30). Although
the function of the O-fucose on TSRs is not yet known, it occurs in a
predicted cell interaction domain, suggesting that the modification may play a
role in regulating cellular interactions with the extracellular matrix
(31).
The importance of fucose in modulating glycan functions is well demonstrated by the human genetic disease LAD II/CDGS IIc, which is characterized by the absent or reduced expression of fucosylated glycoconjugates on cell surfaces, including blood group H and Lewis antigens (32). Patients affected by this syndrome present with severe mental and growth retardation, facial and skeletal abnormalities and immunodeficiency characterized by recurrent bacterial infections. This latter finding has been related to a reduced leukocyte rolling on endothelial cells mediated by the selectins, which are unable to recognize their oligosaccharide ligands, impairing leukocyte recruitment into the inflamed tissues. Conversely, the specific causes that lead to the severe neurological, developmental, and morphologic defects need still to be clarified. The molecular basis of LAD II/CDGS IIc syndrome has been found in a defect in GDP-L-fucose import into the Golgi system (3336). The decreased GDP-L-fucose availability for the fucosyltransferases within the Golgi lumen explains the lower fucose content in glycoconiugates. However, the fact that some fucosylated antigens are undetectable (i.e. H antigen, selectin ligands), while LAD II cells incorporate low, but detectable levels of fucose into macromolecules, suggested that not all fucose-containing glycans are comparably decreased (36). The aim of the present study was the characterization of the residual fucose-containing glycoconjugates in cultured fibroblasts from a LAD II patient of Arab origin, in order to determine how the decreased availability of GDP-L-fucose inside the Golgi can affect their type and composition. Both terminal fucosylation of N-linked glycans and protein O-fucosylation are clearly involved in development and differentiation processes. Identification of the class of glycans that is most heavily affected in LAD II patients should provide clues to the role of these forms of fucosylation in the severe developmental defects observed in these patients.
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EXPERIMENTAL PROCEDURES |
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Tunicamycin TreatmentCells were grown in triplicate in 6-well plates and labeled for 18 h, using 50 µM [3H]fucose, as described above. Tunicamycin (0.5 µg/ml, from Sigma) was added together with the radiolabeled fucose. No toxicity, determined by MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, was observed in these incubation conditions. Incorporation of the radiolabel in macromolecules was determined after PTA (phosphotungstic acid)/HCl precipitation, as previously described (36). Protein concentration was analyzed by DC Protein Assay (Bio-Rad).
Cell FractionationMonolayers of labeled fibroblasts were
washed three times with ice-cold phosphate-buffered saline, harvested by
trypsinization, and washed again twice, to completely remove unbound
radioactivity. Cell pellets were then sequentially extracted at room
temperature, using CHCl3/CH3OH, to eliminate the lipidic
fraction, as the described procedure
(38). Pellets containing the
glycoproteins were then solubilized by heating for 20 min at 100 °C in 1%
SDS. The following steps followed the described procedure
(39), with slight
modifications. A Sephadex G-50 column (30 x 1 cm, mobile phase 50
mM formate + 0.1% SDS +
0.02% NaN3) was used to remove small contaminant molecules.
Protein-containing fractions (designed total glycoproteins), to be submitted
to analysis of N- and O-linked glycans, were pooled
together, and the volume was reduced by lyophilization. Aliquots (1.5 mg) of
the total glycoproteins were withdrawn in triplicate for fucose analysis. The
remaining glycoproteins were then precipitated using 8 volumes of acetone at
20 °C for 18 h. The precipitates were centrifuged at 12,000 x
g for 10 min, and the resulting pellets were resuspended in 0.5 ml of
1% SDS, to be used for glycan fractionation.
Determination of Total Fucose and [3H]Fucose-specific Activity in Total GlycoproteinsThe aliquots of total glycoproteins set apart in triplicate before acetone precipitation (see above) were used for the determination of total bound fucose content by HPAEC-PAD analysis. Proteins were acetone-precipitated in 1.5-ml screw tubes as described above, and the resulting pellets were directly hydrolyzed in 0.2 ml of 2 M trifluoroacetic acid at 100 °C for 4 h. Samples were then dried in a SpeedVac (Savant) and passed over a 0.5 ml Dowex 50 (proton form) column to remove amino acids and peptides. Samples were then dried again and further purified through a Zip Tip (C18) (Millipore) to remove any residual peptide contaminants. Samples were then subjected to HPAEC-PAD analysis, which was performed on a Dionex DX300 equipped with a pulsed amperometric detector (Dionex Corp., Sunnyvale, CA) using a Dionex PA-1 column under standard conditions for monosaccharide analysis (40). Fractions (0.25 min) were collected and radioactivity eluting in correspondence to fucose was determined by scintillation counting. The ratio between cpm and fucose pmol was used to calculate [3H]fucose-specific activity.
Release of N- and O-Linked Glycans from Total
GlycoproteinsThe solubilized proteins (6 mg) were treated with
PNGase F (Roche Applied Science) as described
(30,
39). The released
oligosaccharides were then separated from PNGase F-resistant glycoproteins on
a Sephadex G-50 column as described above. Fractions (1.0 ml) were collected,
and aliquots were analyzed by scintillation counting. The PNGase F-resistant
fractions were pooled, concentrated by lyophilization, and precipitated with
acetone. The residual glycoproteins were then subjected to alkali-induced
-elimination to obtain O-glycan release
(39). The released
oligosaccharides were then subjected again to a Sephadex G-50 column and 1.0
ml fractions were collected for further analysis. Fractions corresponding to
small oligosaccharides and monosaccharides were pooled and SDS was removed by
KCl precipitation. Samples were then desalted on a Dowex 50 column,
lyophilized, and residual boric acid was removed by repeated evaporation in
the presence of methanol. The samples were then chromatographed on a Superdex
peptide column, using water (0.5 ml/min) as mobile phase. Fractions (0.5 min)
were collected and analyzed by scintillation counting. Individual peaks
(monosaccharide and disaccharide) were analyzed by HPAEC analysis on an MA-1
column as described (40).
Radioactivity in the eluted fractions was determined by scintillation counting
and the retention time compared with that observed for unlabeled standards
(Fucitol, Glc
1,2Fucitol, Glc
1,3Fucitol, Glc
1,4Fucitol.
Disaccharides generously provided by Dr. Khushi Matta, Roswell Park Cancer
Center, Buffalo, NY).
Western Blotting and Immunoprecipitation of Notch1 ProteinFibroblasts from LAD II patient and two controls were seeded into 10 cm-diameter culture dishes and were incubated for 24 h in the presence of [3H]fucose. Immunoprecipitation and Western blotting experiments were performed as described (18). Briefly, after labeling cells were washed three times with cold Tris-buffered saline and lysed with the same buffer containing 1% Nonidet P-40 and protease inhibitors. The lysate was incubated overnight with Notch1-specific rabbit polyclonal Na antibody (41). The immunoprecipitated protein, untreated and treated with PNGase for 24 h (18), was analyzed by SDS-PAGE on a 412% gradient gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was analyzed by digital autoradiography using a Beta-imager 2000 instrument (Biospace, Paris, France). The radioactivity associated with individual proteins was determined with the specific Beta-Vision software provided by Biospace.
Notch1 presence in fibroblasts lysates before and after immunoprecipitation was analyzed by Western blotting experiments using Na antibody. The lysates were subjected to SDS-PAGE analysis on a 7.5% gel and, after separation, the proteins were transferred onto a PVDF membrane. Detection was performed with the ECL Plus detection system (Amersham Biosciences) according to the manufacturer's instructions.
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RESULTS |
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To test this hypothesis, cells were incubated in the presence of [3H]fucose and subjected to fractionation, in order to isolate and analyze N- and O-linked glycans. Aliquots of the recovered total glycoproteins were then used to determine radioactivity incorporated in glycoproteins (Fig. 2A) or subjected to hydrolysis followed by HPAEC-PAD analysis to determine total amount of bound fucose (Fig. 2B) and fucose-specific activity (Fig. 2C). As already observed for tunicamycin experiments, total radioactivity and the amount of fucose incorporated in macromolecules were decreased in LAD II samples (Fig. 2, A and B). [3H]Fucose final specific activity, determined by HPAEC-PAD analysis after acid hydrolysis of total glycoproteins, was slightly different between control and LAD II (Fig. 2C), possibly due to a different contribution of the salvage pathway to the cytosolic GDP-L-fucose pool in the two samples. The specific activity of [3H]fucose on total glycoproteins was then used to estimate the picomoles of total fucose bound to glycoproteins and in the different glycan fractions.
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The profiles of elution from G-50 column of [3H]fucose-labeled total glycoproteins after PNGase F treatment to release N-linked oligosaccharides are reported in Fig. 3A. Radioactivity eluting in correspondence to the oligosaccharide peak was highly decreased in LAD II samples, while the PNGase-resistant fraction was comparable between LAD II and controls (Fig. 3A). No differences in the elution profile were observed between LAD II and controls when [3H]glucosamine was used as label (Fig. 3B). This strongly suggests that the reduction observed is specific for fucose, and it is not accompanied by significant differences in N-glycan composition. Fucose content, estimated using [3H]fucose-specific activity in the pooled fractions containing the N-linked oligosaccharides, was markedly reduced in LAD II cells, while the amount of protein-bound, PNGase-resistant fucose was less affected in patient samples (Fig. 3C). This finding was in accord with the previous data obtained with tunicamycin.
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LAD II and Control Fibroblasts Contain Similar Levels of O-Fucose
SaccharidesLabeled proteins recovered from G-50 column were then
subjected to alkali-induced -elimination in the presence of
NaBH4, in order to release O-linked oligosaccharides and,
possibly, residual PNGase F-resistant N-linked oligosaccharides, and
analyzed again with a Sephadex G-50 column. [3H]fucose-labeled
material eluted in three main peaks, corresponding to residual proteins
resistant to
-elimination, high/medium molecular weight oligosaccharides
and small molecular weight oligosaccharides/monosaccharides, respectively
(Fig. 4A). While a
marked decrease of radioactivity was observed for the first two eluting peaks
in LAD II, the peak corresponding to the small oligosaccharides and
monosaccharides was comparable between LAD II and controls. Again, no
differences between controls and LAD II were observed in
[3H]glucosamine labeled samples
(Fig. 4B). When the
amount of fucose was estimated by using [3H]fucose-specific
activity, it revealed an increase in the amount of fucosylated material
corresponding to small oligosaccharides/monosaccharides from LAD II samples
compared with control samples (Fig.
4C).
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To characterize the size and composition of the third peak, which is
hypothesized to contain O-fucosylated species, the corresponding
fractions were pooled and subjected to gel filtration analysis on a Superdex
column. The profile of elution was comparable between controls and LAD II and
showed two species, corresponding to disaccharides and to monosaccharides,
respectively (Fig. 5, A and
B). HPAEC analysis of the monosaccharide indicated the
presence of fucitol, which is formed when O-fucose monosaccharide is
released by -elimination followed by NaBH4 reduction of the
aldehyde group (not shown). HPAEC profiles from the fractions containing the
disaccharide form are reported in Fig. 5,
C and D and show that the disaccharide
corresponds to Glc-
1,3-fucitol for both control and LAD II samples.
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O-Fucosylation of Notch1 Appears to Be Similar in LAD II and Control
FibroblastsTo specifically investigate whether
O-fucosylation on the EGF repeats of Notch is affected in LAD II,
fibroblasts were metabolically labeled with [3H]fucose and Notch
was immunoprecipitated using the Na antibody
(41). The expression of Notch1
in lysates of control and LAD II fibroblasts was verified by Western blot
experiments. A band migrating at 110 kDa was detected
(Fig. 6A), consistent
with the mass of the transmembrane/intracellular domain of Notch protein
(42), which is recognized by
Na antibody (18,
41). The amount of Notch1 in
LAD II fibroblasts was comparable to that detected in control cells. After
immunoprecipitation, the immunoreactive bands disappeared, confirming that all
Notch protein was immunoprecipitated (Fig.
6A).
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The immunoprecipitated proteins, either untreated or treated with PNGase F
to eliminate N-linked oligosaccharides, were separated by SDS-PAGE
and analyzed by autoradiography (Fig.
6B). Before PNGase F, bands of 200 kDa, which
correspond to the molecular weight of the extracellular domain of Notch1
(42), were observed both in
controls and LAD II samples. The amount of radioactivity detected in these
bands reflected [3H]fucose incorporation both as terminal
modification of N-linked glycans and as O-fucosylation and,
as expected, was severely decreased in LAD II cells, confirming the defective
incorporation in N-linked oligosaccharides. After PNGase F treatment,
together with a detectable shift in molecular mass due to removal of
N-linked oligosaccharides
(18), comparable levels of
PNGase F-resistant radioactivity (corresponding to O-fucosylated
species in Notch protein) were observed in both controls and LAD II cells.
Very similar results were obtained after labeling of the cells with
[14C]fucose and scintillation counting of the gel slices following
immunoprecipitation and SDS-PAGE separation of the bands (not shown). These
results demonstrate that, while [3H]fucose incorporation as
terminal fucosylation of N-linked glycans is severely affected, the
O-fucosylation on EGF repeats on Notch1 protein is not significantly
reduced in LAD II fibroblasts. The same conclusion holds for total
O-fucosylation.
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DISCUSSION |
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O-fucosylation has been described so far in both EGF-like repeats
and TSRs, and at least two different protein O-fucosyltransferases
are responsible for modification of these repeats. O-FucT-1, which
modifies EGF repeats, has been cloned and characterized
(4344);
conversely, nothing is as yet known about the enzyme(s) responsible for
fucosylation of TSRs. However, preliminary observations suggest that
O-FucT-1 will not modify TSRs and that a separate enzymatic activity
exists.2 Since total
levels of the disaccharide Glc-1,3-Fuc-O(S/T), which is characteristic
of TSRs (10,
29), are normal in LAD II
cells, it can be assumed that O-fucosylation of this type of repeats
is in general not affected. Conversely, we were consistently unable to
identify by chromatographic methods O-fucose elongated to the
tetrasaccharide form, which, together with O-fucose monosaccharide,
is a typical of modification of EGF-like repeats of proteins such as Notch.
However, the amount of [3H]fucose-labeled tetrasaccharide bound to
Notch can be expected to be very low and below the detection limits by
chromatographic methods. The data obtained after immunoprecipitation of
[3H]fucose-labeled Notch1 confirmed a significant reduction in the
incorporation of this sugar in N-linked glycans, while comparable
amounts between LAD II and control cells were observed after PNGase treatment
of immunoprecipitated Notch proteins. This indicates that
O-fucosylation of EGF-like repeats is also not affected in LAD II
fibroblasts.
A potential explanation for the difference between addition of terminal
fucose and O-fucosylation could rely in a different affinity for
GDP-L-fucose, the protein fucosyltransferases having a lower
Km for the nucleotide-sugar compared with the
other fucosyltransferases that add fucose as terminal modification of the
oligosaccharide chains. For instance, O-FucT-1, which modify EGF
repeats, has a Km for GDP-L-fucose of
5 µM, while several fucosyltransferases involved in
terminal fucosylation of N-glycans have nearly 10-fold higher
Km values
(4344).
A similar phenomenon has been described in a mutant line of MDCK cells, which
is severely defective for UDP-galactose transport into the lumen of Golgi
vesicles (45). In these cells,
galactosylation of keratan sulfate as well as of glycoproteins and glycolipids
is highly decreased, while that of heparan sulfate and chondroitin 4-sulfate
is unaffected. This suggested that the Km for
UDP-galactose of the galactosyltransferases involved in the biosynthesis of
the linkage region of proteoglycans is significantly lower than that of the
other galactosyltransferases and that UDP-galactose availability may be able
to modulate proteoglycan expression
(45).
Another possible explanation for the normal levels of O-fucosylation in LAD II could derive from a different localization of the transferases. It is in fact known that terminal fucosylation of N-glycans is a late event in the formation of the protein-bound oligosaccharides and the fucosyltransferases responsible for this fucose transfer are mainly localized in trans-Golgi and trans-Golgi network. Conversely, the subcellular site of protein O-fucosylation is not yet clear. The membrane-bound localization of the protein O-fucosyltransferase identified so far and the presence of mostly high mannose type oligosaccharides on it suggest that this protein is probably localized in either the endoplasmic reticulum or the cis-Golgi region (43). Other than localization of GDP-L-fucose transport activity to the Golgi apparatus (46), no information is presently available on the distribution of the GDP-L-fucose transporter within the Golgi system. It is not known if transport occurs through only one protein species or other isoforms that can be expressed at different Golgi sites.
Since data were obtained in fibroblasts from a LAD II patient with a
specific point mutation in the GDP-L-fucose transporter, it cannot
be excluded that in other tissues or in patients bearing other mutations
different fucosylation patterns can occur. However, the extensive decrease of
fucose as terminal modification of N-linked glycans observed in our
study is in agreement with an almost absent expression of blood group H
antigen and of Lewis ligands for the selectins in blood cells
(32). Thus, the severe
impairment of growth and development affecting these LAD II patients may be
related to a generalized defect of selectin/ligand interaction or of other
mechanisms involving recognition of fucose as terminal modification, rather
than to a bulk defect in O-fucosylation. Further studies are required
to understand if a limited GDP-L-fucose supply is also able to
differentially affect the activity of the several 1,2,
1,3/4,
and
1,6 fucosyltransferases and if specific types of terminal fucose
linkages are more affected than other ones in N-glycans from LAD II
fibroblasts. For instance, genetic ablation of FUT8 (responsible for addition
of
1,6-linked fucose to the N-linked core GlcNAc) appears to
be semi-lethal. Mice die a few weeks after birth and display LAD II-like
characteristics, thus suggesting that core fucosylation is essential for
proper
development.3
The mechanisms that underlie and control the production of fucosylated glycans are not yet completely understood. The results obtained in the present study give some insights into them, indicating that GDP-fucose transport into the Golgi lumen is able to regulate the biosynthesis of fucosylated glycoconjugates. The importance of understanding these processes derives from the fact that both the enzymes involved in the biosynthesis of GDP-L-fucose and the subsequent transport of this metabolite into the Golgi represent distinctive targets for the pharmacological modulation of the expression of fucose-containing structures (i.e. SLex). This approach perhaps may be used in therapy in order to disrupt selectin-mediated intercellular adhesion processes (i.e. during inflammation, graft rejection, tumor metastasis). A possible drawback of such therapies could be represented by an impaired production of other classes of glycans, such as O-fucosylated glycoproteins, which could be essential for other cellular functions, thus causing unwanted side effects. Our results indicate that, by choosing the appropriate target for a pharmacological treatment, it should be possible to modulate selectively the expression of fucose as a terminal modification of N-linked glycans, leaving O-fucosylation unaffected.
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FOOTNOTES |
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** Supported by CNR Target Project Biotechnology, by CNR Agenzia 2000, and by CNR/MURST Project (Legge 95/95). To whom correspondence should be addressed: Dept. of Experimental Medicine, Viale Benedetto XV, 1 16132 Genova, Italy. Tel.: 39-010-3538151; Fax: 39-010-354415; E-mail: tonetti{at}unige.it.
1 The abbreviations used are: EGF, epidermal growth factor-like; LAD II/CGDS
IIc, Leukocyte Adhesion Deficiency type II/Congenital Disorder of
Glycosylation IIc; TSR, thrombospondin type 1 repeat; PNGase F, protein
N-glycosidase F; HPAEC-PAD, high pH anion exchange
chromatography-pulsed amperometric detection.
2 Y. Luo and R. S. Haltiwanger, unpublished data.
3 N. Taniguchi, personal communication (to R. S. H.).
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ACKNOWLEDGMENTS |
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REFERENCES |
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