Regulation of signal transduction by glycosylation

Abstract

The incredible diversity found in cell‐surface glycoconjugate structures led researchers over 30 years ago to propose that complexity in carbohydrates must play a role in cellular communication. Recent studies from a number of laboratories have confirmed this hypothesis, demonstrating that cell‐surface glycoconjugates play significant roles in signal transduction events. One striking example is the effect of O‐fucose modifications on the Notch‐signalling pathway. Notch is a cell‐surface receptor that is essential for proper development. The extracellular domain of Notch contains up to 36‐tandem epidermal growth factor‐like (EGF) repeats, many of which are predicted to be modified at putative consensus sequences with O‐fucose and O‐glucose saccharides. Genetic alterations (by knockout or RNAi methodologies) in the enzyme responsible for the addition of O‐fucose to Notch, protein O‐fucosyltransferase‐1, result in severe, embryonic lethal phenotypes resembling Notch mutants. Thus, O‐fucosylation appears to be essential for proper Notch function. Elongation of the O‐fucose monosaccharide by the β1,3‐N‐acetylglucosaminyltransferase, Fringe, modulates Notch function, either increasing or decreasing response from ligands depending on context. Although it is now clear that O‐fucose modifications affect Notch signalling, the molecular mechanism by which this occurs is not known. As an initial step in understanding how O‐fucose glycans affect Notch function, we are mapping O‐fucose modifications to specific sites on Notch. Already, we have demonstrated that O‐fucose modifies one of the EGF repeats involved in ligand binding, suggesting that the sugars may play a role in Notch–ligand interactions. Experiments to test the role of O‐fucose modifications at specific sites are in progress. We have also found that Fringe modifies O‐fucose on some EGF repeats but not others. Our initial analyses suggest that the basis of this specificity is encoded within the sequences of the individual EGF repeats. Site mapping has also confirmed the presence of O‐glucose saccharides on Notch. The evolutionarily conserved, predicted O‐glucose sites on Notch are as numerous as those for O‐fucose, suggesting that the O‐glucose modifications will play an equally important role in Notch biology. We have recently identified an enzymatic activity capable of catalyzing the addition of O‐glucose to EGF repeats. Purification of the protein O‐glucosyltransferase is underway. These and other results will be discussed.