title: Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins.
abstract: Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785-796, 2017. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Ledi
authors.initials: L
authors.lastname: Liu
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Hiroe
authors.initials: H
authors.lastname: Wada
authors.affiliation: Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan.
authors.forename: Natsuki
authors.initials: N
authors.lastname: Matsubara
authors.affiliation: Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
authors.forename: Katsuto
authors.initials: K
authors.lastname: Hozumi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.|Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan.
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Itoh
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D000072100:Jagged-1 Protein / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D041681:NIH 3T3 Cells; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C424126:DLL4 protein, human; D055767:Immobilized Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008024:Ligands; D051880:Receptors, Notch
keywords: BINDING AFFINITY; BIOMATERIALS; DIFFERENTIATION; NOTCH PATHWAY; PROTEIN DOMAIN; PROTEIN IMMOBILIZATION; SIGNAL TRANSDUCTION