Pat Whiteman

The CD46-Jagged1 interaction is critical for human TH1 immunity

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (TH1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4+ T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate TH1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients.

Solution Structure and Dynamics of a Calcium Binding Epidermal Growth Factor-like Domain Pair from the Neonatal Region of Human Fibrillin-1.

Fibrillin-1 is a mosaic protein mainly composed of 43 calcium binding epidermal growth factor-like (cbEGF) domains arranged as multiple, tandem repeats. Mutations within the fibrillin-1 gene cause Marfan syndrome (MFS), a heritable disease of connective tissue. More than 60% of MFS-causing mutations identified are localized to cbEGFs, emphasizing that the native properties of these domains are critical for fibrillin-1 function. The cbEGF12–13 domain pair is within the longest run of cbEGFs, and many mutations that cluster in this region are associated with severe, neonatal MFS. The NMR solution structure of Ca2+-loaded cbEGF12–13 exhibits a near-linear, rod-like arrangement of domains. This observation supports the hypothesis that all fibrillin-1 (cb)EGF-cbEGF pairs, characterized by a single interdomain linker residue, possess this rod-like structure. The domain arrangement of cbEGF12–13 is stabilized by additional interdomain packing interactions to those observed for cbEGF32–33, which may help to explain the previously reported higher calcium binding affinity of cbEGF13. Based on this structure, a model of cbEGF11–15 that encompasses all known neonatal MFS missense mutations has highlighted a potential binding region. Backbone dynamics data confirm the extended structure of cbEGF12–13 and lend support to the hypothesis that a correlation exists between backbone flexibility and cbEGF domain calcium affinity. These results provide important insight into the potential consequences of MFS-associated mutations for the assembly and biomechanical properties of connective tissue microfibrils.

Structural Consequences of Cysteine Substitutions C1977Y and C1977R in Calcium-binding Epidermal Growth Factor-like Domain 30 of Human Fibrillin-1

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29–31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29–30, but not cbEGF30–31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.

Molecular basis for Jagged-1/Serrate ligand recognition by the Notch receptor.

Background: The site of Jagged/Serrate ligand recognition by Notch is unknown. Results: Two critical residues involved in an intramolecular hydrophobic interaction across the central β-sheet of EGF12 form a ligand-binding platform. Conclusion: The ligand-binding region is adjacent to a Fringe-sensitive residue involved in modulating Notch activity. Significance: The results have implications for understanding receptor/ligand recognition, Notch regulation by O-glycosylation, and the development of paralogue-specific antibodies. We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications.

Biophysical characterisation of fibulin-5 proteins associated with disease.

FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments. Limited proteolysis, NMR spectroscopy and chromophoric calcium chelation experiments showed that the G267S and S227P substitutions cause long-range structural effects consistent with protein misfolding. Cellular studies using fibroblast cells further demonstrated that these recombinant forms of mutant fibulin-5 were not present in the extracellular medium, consistent with retention. In contrast, no significant effects of I169T and G202R substitutions on protein fold and secretion were identified. These data establish protein misfolding as a causative basis for the effects of G267S and S227P substitutions in AMD and CL, respectively, and raise the possibility that the I169T and G202R substitutions may be polymorphisms or may increase susceptibility to disease.

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

Summary The Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4.

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N‐terminus of Notch ligands, which has both lipid‐ and receptor‐binding properties. We present novel structures of human ligands Jagged2 and Delta‐like4 and human Notch2, together with functional assays, which suggest that ligand‐mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand‐dependent Notch signalling in different physiological contexts.

Bacterial expression and in vitro refolding of limited fragments of the Notch receptor and its ligands.

Prokaryotic expression of limited fragments of the Notch receptor and its ligands followed by in vitro refolding has been used for the production of the significant amounts of protein required for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy. As an illustration of the protocol for the production of these EGF-containing constructs we have focused on a limited fragment of human Notch 1 that contains three calcium-binding EGF domains, hNotch-111-13. Following characterization by the methods described here, this construct has been shown to be functionally competent in a range of assays and the structure has been solved by X-ray crystallography and NMR.

Abstract C128: Development of therapeutic anti-Jagged1 monoclonal antibodies

Background: The Notch pathway is an evolutionarily conserved cell-signaling system that plays an important role in both physiologic and pathologic conditions such as embryonic development and cancer. In the latter, Notch signaling has been reported to promote tumor growth by regulating different aspects of tumor biology such as cell survival, proliferation, angiogenesis, and stemness. Because of its importance, targeting of this pathway has been attempted via both small molecules (γ-secretase inhibitors; GSIs) and neutralizing antibodies (against individual Notch receptors or DLL4 ligand) but limitations still hamper the clinical use of such therapeutics, generally due to pathway complexity and toxicities caused by complete pathway inhibition (GSIs). Targeting Notch ligands such as Jagged1 (JAG1), offers the opportunity to selectively block specific elements of the pathway important in tumor biology thus avoiding normal tissue toxicities. Methods: Structural studies defined a region of JAG1 (DSL + EGF1-3) that bound Notch1, which was used as an immunogen. Using classical hybridoma technology we have generated and subsequently characterised a panel of monoclonal antibodies (mAbs) against the JAG1 ligand, both in vitro (cell signaling and cell biology assays) and in vivo (tumor xenografts in both mice and rats). Results: Four functional blocking mAbs recognised a unique JAG1 epitope within its DSL domain binding interface with Notch1, effectively blocking ligand-receptor interaction in vitro. Importantly, our mAbs were cross-reactive with rat Jag1 but not the murine orthologue. These were able to inhibit endogenous JAG1-induced signaling in tumor and stromal cells (eg. vascular smooth muscle cells). JAG1 mAb treatment reduced in vitro breast cancer 3D growth, exhibiting reduced expression of important genes such as HES1, IL6 and decreased numbers of cancer stem cells. Treatment did not affect JAG2-induced growth, or signaling mediated by other Notch ligands (eg. DLL4), confirming mAb specificity. Importantly, JAG1 mAb treatment in vivo inhibited Notch signaling and tumor growth in cancer xenograft models in two different host animals. Host body weight, blood tests and histological analysis detected no toxicity. Conclusions: We generated neutralizing mAbs able to inhibit JAG1-induced signaling both in vitro and in vivo. The ability of JAG1 blockade to impair tumor growth without toxicity indicates it has the potential to make a contribution to the current arsenal of cancer therapeutics. This mAb treatment will be subjected to further in vivo testing, both alone and in combination with other therapeutic approaches, to further expand our knowledge of its clinical potential and mechanism of action. Funding and conflicts of interest This work is supported by Cancer Research UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We have filed a patent application, WO/2014/111704, claiming therapeutic use of the JAG1 antibodies. Citation Format: Massimo Masiero, Demin Li, Pat Whiteman, Carol Bentley, Jenny Greig, Tasneem Hassanali, Sarah Watts, Stephen Stribbling, Jenna Yates, Ji-Liang Li, Susan Mary Lea, Penny Ann Handford, Adrian Llewellyn Harris, Alison Hilary Banham. Development of therapeutic anti-Jagged1 monoclonal antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C128.