Notch receptor comes under scrutiny in new study
A study has revealed for the first time molecular details of a receptor which is part of a signalling pathway crucial to many fundamental biological processes.
The findings, published in PNAS (1), come from the lab of Professor Penny Handford in the department, in collaboration with colleagues at Stony Brook and the Sir William Dunn School of Pathology in Oxford.
Notch signalling at the dorsal-ventral compartment border during Drosophila wing development
Their work provides insight at the molecular level into how interactions between the receptor, Notch1, and its ligands are affected by sugar modifications.
The Notch signalling pathway is highly conserved across multicellular organisms. It plays an important role in cell-cell communication and is essential in many aspects of development, cell fate determination and tissue patterning and homeostasis.
Notch receptors on one cell interact with ligand on another, triggering release of the intracellular domain of Notch from the membrane and its translocation to the nucleus where it activates gene expression. Mammals have four Notch receptors and five ligands (Jagged and Delta-like), Drosophila has a single receptor and two ligands, Delta and Serrate.
Whilst many details are known about the downstream activation of Notch, very little is known about the behaviour of the ligand-binding extracellular domain of Notch which contains multiple tandem epidermal growth factor-like (EGF) modules. Residues within the EGF domains are targets for O-glycoslation, sugar modifications that involves a series of enzyme-catalysed additions.
One particular set of enzymes called Fringe adds GlcNAc onto O-fucose monosaccharides to make the disaccharide. This appears to increase signalling via Delta but inhibit signalling through Serrate, at least in Drosophila. The molecular basis for these effects is unknown but may involve changes to the ligand-binding region in Notch, which is localised to EGF domain 12.
Diagramatic representation of EGF domains 12 and 13 of hN1, with cysteines highlighted in yellow and residues responsible for coordination of calcium highlighted in red. O-fucose and –glucose glycans added in this study are indicated
Previous experience working on the structurally related fibrillin protein, also rich in EGF domains, meant that the Handford lab was well placed to study the impact of glycosylation on ligand binding to Notch.
'We had the tools to make these proteins and decided to target Notch and look at the molecular properties of some of the domains,' explains Professor Handford, whose DPhil student, Paul Taylor, carried out much of the work. 'We wanted to look at how these modifications affect the ligand-binding function of the molecule.'
The researchers purified a prokaryotically expressed and refolded fragment of human Notch 1 (hN1). The fragment of EGF domains 11-13 encompasses the core ligand-binding site, which is adjacent to a glycosylation site. In collaboration with glycobiology expert Professor Haltiwanger at Stony Brook, they enzymatically added the sugars stepwise to obtain purified fragments with different, specific modifications.
To explore the impact of sugars on ligand binding, both in a cellular context and at the level of molecular interactions, they developed a flow cytometry binding assay and used SPR.
'We found that when you modify this region, you see that both ligand families [Delta-like 1 and Jagged] bind tighter,' says Professor Handford. 'Modification by Fringe enzyme is what is having the impact on binding – it's the disaccharide having an effect. If we add trisaccharide and tetrasaccharide, then we don't see any additional increase in binding.'
Interestingly, within the Delta family there are differences, with the unmodified DLL4 having a high intrinsic affinity for Notch in the absence of sugar modification.
As the group had enough material to take the work a step further, it decided to look at the high resolution structure of glycosylated Notch fragment using crystallography. They did this in collaboration with Professor Susan Lea at the Sir William Dunn School of Pathology.
'We knew where the ligand binds on Notch,' says Professor Handford. 'Our work showed us where the sugar additions lie'. The modifications did not appear to induce a conformational change locally or globally in the overall structure of the fragment, but instead appeared to have an impact on the ligand-binding area.
'We believe that Fringe addition has a direct effect by extending the patch that the ligand can bind to, and that is novel,' she comments, but points out that they would have to solve the crystal structure of the complex to prove this.
Structure of the O-fucosylated variants of hN1 EGF domain 12 showing b) the disaccharide X-ray structure highlighting contacts between residues and the O-fucose and GlcNAc, and c) the unmodified and disaccharide X-ray structures highlighting residues contributing to Jagged1 binding (red). Residue T466 is highlighted in yellow, together with the disaccharide
Although the work can explain the increased signalling observed with Delta, it does not explain why Serrate-mediated signalling is inhibited. For this, the group suggests that other glycosylated regions of Notch may be involved.
Professor Handford comments that this is the first study to use this complementary set of techniques to systematically explore the impact of sugar modifications on Notch at a molecular level – something that has been made possible by combining the expertise of her group with that of the other two groups.
'These proteins are difficult to produce because they are very disulphide-rich and are sticky, so binding assays need careful optimisation,' she says. This is her group's expertise.
'I met Bob Haltiwanger at a Notch meeting and suggested the collaboration. He and his postdoc Hideyuki Takeuchi added the sugars. I was already working with Susan Lea, so once we had the functional data she encouraged us to try crystallisation with her postdoc, Devon Sheppard.'
Encouraged by the success of this project, the group has already started to investigate another EGF region of Notch that is implicated in being regulated by glycosylation.