Cell surface carbohydrates were predicted to play a role in cell-cell communication and signal transduction over 30 years ago. This prediction was based on the observation that a carbohydrate-rich coat, or glycocalyx, composed of an incredibly complex array of carbohydrate structures surrounds essentially every cell in the body. The presence of such structural complexity at the cell surface, the site of cell-cell communication and signal transduction, led to the proposal that complex carbohydrates assist in the equally complex task of communication between a cell and the outside world. Nonetheless, only a few examples demonstrating a role for carbohydrates in cell-cell communication or signal transduction have been uncovered in the ensuing decades. One reason for the slow appearance of data supporting the hypothesis relates to the technical difficulty of determining oligosaccharide structures, but recent advances in analysis of oligosaccharide structures have led to a number of confirmations of the predictions made so long ago. This article describes the role of glycosylation in one particular signal transduction event, the role of O-fucose modification in the function and regulation of the Notch receptor.
|Notch Signal Transduction|
The genetic locus for Notch was identified in the early part of the
20th century in one of the early genetic screens performed
in Drosophila. Notch mutants show an X-linked lethal phenotype,
and the locus derived its name from the fact that the females have a
small notch in their wings. The gene for Notch was identified in 1985
and shown to encode a large (>300 kDal) cell surface receptor (for
an excellent review on Notch, see ref. 1).
Notch participates in a variety of developmental events, specifically
what are known as cell fate decisions. The most detailed studies on
Notch function have been performed in Drosophila where Notch
functions have been shown to be necessary for the formation of numerous
organs, including the neuronal system, vasculature, eyes, legs and wings.
Notch homologues have been identified in all metazoans, with four in
mammals (Notch1-4). Notch plays numerous roles in development in vertebrates,
and defects in Notch signaling result in a variety of human diseases,
including several types of cancer and developmental disorders such as
Alagille syndrome, spondylocostal dystoses, and CADASIL (Cerebral Autosomal
Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy).
Recent work has demonstrated a link between the pathogenesis of multiple
sclerosis and Notch activation. The number and magnitude of the developmental
events involving Notch receptor signaling is staggering.
Fig. 1 Notch signaling
Notch activation is regulated at numerous levels3,4. For instance, expression of ligand in the same cell as Notch can exert an inhibitory effect on signaling from an adjacent cell, referred to as cell-autonomous inhibition by ligand. A number of modulators of Notch activity have been identified, including Wingless, Scabrous, Numb, Deltex and Disheveled. A novel regulator, Fringe, was initially discovered during a mutant screen for genes involved in boundary formation during wing development in Drosophila. Subsequent work showed that it functions by regulating the Notch pathway, inhibiting signaling from Serrate while potentiating signaling from Delta. Interestingly, Fringe's effects are cell autonomous with respect to Notch (i.e. Fringe needs to be expressed in the same cell as Notch to function) and Fringe is a secreted protein. These observations led to the suggestion that Fringe may mediate its effects by either binding to or post-translationally modifying the extracellular domain of Notch. The fact that Fringe showed weak sequence similarity to several bacterial glycosyltransferases raised the possibility that Fringe functions by altering the structure of the carbohydrate chains on Notch.
|Role of Glycosylation in Notch Signaling|
|The extracellular domain of Notch is modified with several forms of glycosylation including O-fucose and O-glucose5. These unusual forms of O-linked glycosylation occur on the hydroxyl groups of serine or threonine residues at consensus sequences within epidermal growth factor (EGF)-like repeats6. An EGF-like repeat is a protein motif defined by the presence of six conserved cysteine residues forming three disulfide bonds, and the consensus sequences for O-fucose and O-glucose are found in the context of these cysteines. For instance, the O-glucose site occurs between the first and second conserved cysteine of an EGF-like repeat at the sequence C1XSXPC2, while the O-fucose site is found between the second and third conserved cysteines at the sequence C2X4-5S/TC3 (Fig. 2). The Notch extracellular domain is composed of up to 36 tandem EGF-like repeats (e.g. mammalian Notch1 and Notch2, Drosophila Notch), many of which contain consensus sequences for O-fucose and O-glucose saccharides (Fig. 3). The locations of many of the sites for glycosylation are evolutionarily conserved, suggesting that the sugars play an important role in the biology of Notch.|
Fig. 2 Some EGF-like
repeats are modified with O-fucose and/or O-glucose saccharides.
Fig. 3 Many of the EGF-like
repeats in the extracellular domain of Notch contain sites for O-fucose
and/or O-glucose modifications.
Although little is known about the O-glucose modifications,
a number of studies suggest that the O-fucose modifications are
essential for Notch function. Reduction in expression levels of protein
O-fucosyltransferase I (O-FucT-1), the enzyme responsible
for addition of O-fucose to EGF-like repeats, by RNA interference
in Drosophila results in Notch-like phenotypes7.
Detailed studies have demonstrated that reduction of O-FucT-1
levels affects Notch function in many different developmental contexts,
suggesting O-fucose is essential for all aspects of Notch signaling.
Genetic ablation of O-FucT-1 in mice results in a phenotype more
severe than any single Notch knockout, suggesting O-fucose
modifications are essential for all Notch isoforms8.
In addition, cells with defects in GDP-fucose synthesis (the fucose-donor
for all fucosyltransferases) do not support Notch signaling9,10.
These results strongly suggest that Notch must be modified with O-fucose
|Unanswered Questions and Future Directions|
|Although it is now clear that the O-fucose modifications
on Notch are essential for its function and for Fringe to modulate its
activity, the molecular mechanisms for how these saccharides affect Notch
are not well understood13,14.
It is not known why O-fucose is required for Notch function. Preliminary
studies show that Notch is expressed at the cell surface in the absence
of O-fucosylation suggesting that O-fucose is not required
for proper cell-surface expression7.
Thus, the sugars could participate directly in binding to the ligands,
or they may play a role in causing Notch to assume a certain conformation
necessary for ligand binding and/or proteolysis. In addition, the mechanisms
by which the Fringe-mediated alterations in O-fucose structures
modulate Notch signaling are not known. One complication for all of the
available models is that Fringe has opposite effects on signaling from
the two classes of ligands, potentiating signaling from Delta ligands
while inhibiting signaling from Serrate/Jagged ligands. This suggests
that at least two mechanisms may be at play. The change in O-fucose
structure could have a direct effect on ligand binding, or it may induce
a conformational change in Notch that indirectly alters ligand binding
or susceptibility to TACE. Alternatively, another protein may be involved,
and the alterations in O-fucose structure could affect the ability
of Notch to interact with this protein. Several proteins are known to
interact with the EGF-like repeats of Notch and affect signaling (e.g.
Scabrous, Wingless). In addition, ligand expressed in the same cell as
Notch can exert an inhibitory effect. Alterations in the interactions
of Notch with any of these proteins, mediated by a change in O-fucose
structure, could provide a mechanism for Fringe action. Interestingly,
O-fucose modifies EGF-like repeats involved in ligand binding and
in the Abruptex region (Fig. 3). Abruptex
mutations result in hyperactivatible forms of Notch; thus the region in
which they occur is believed to be inhibitory. The presence of O-fucose
saccharides at these sites may offer some clues to the function of these
sugars. More work needs to be done to determine which of these mechanisms
is at play with each of the ligands.
In addition to not knowing the molecular mechanisms of how the O-fucose glycans affect Notch function, little is known about the function of the O-glucose glycans on Notch. Notch is modified with O-glucose in both the monosaccharide and trisaccharide (proposed structure: Xyl-1,3-Xyl-1,3-Glc-1-O-Ser/Thr) forms5. The sites of O-glucose on Notch have not yet been mapped, but preliminary data from our laboratory indicates that the predicted sites are in fact modified. The O-glucose sites on Notch are as highly conserved across evolution as the O-fucose sites (Fig. 3), suggesting an equally important role for these modifications in Notch biology. Enzymatic activities have been identified for the protein O-glucosyltransferase responsible for addition of O-glucose to EGF-like repeats15 and for the two predicted xylosyltransferases16,17, but a great deal of work remains to identify these enzymes and their biological function.
|Perspectives and Summary|
|The proposal that cell surface glycans participate in cellular communication is being realized. The past decade has seen numerous examples of specific biological events being controlled or regulated by cell surface glycoconjugates. The involvement of sialyl Lewis x structures in recruitment of leukocytes to sites of inflammation, the role of heparin sulfate proteoglycans in Wnt, hedgehog and TGF signaling pathways, the role of O-mannose saccharides in binding of -dystroglycan to the extracellular matrix, and the modulation of NCAM interactions by alterations in polysialic acid are all clear examples of the role cell surface glycans play in modulating significant biological events. The role of O-fucose on Notch is a new addition to this ever-growing list. The discovery that Fringe is a glycosyltransferase resulted directly from the ability to compare genomes from a variety of species. The next decade should be an exciting time, as more putative glycosyltransferases are shown to synthesize sugar structures essential for a variety of biological functions.|
|May 13, 2003 / Copyright (c) Glycoforum, All Rights Reserved.|