Abstract
Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis.
Highlights
The Notch signaling pathway mediates local interactions between adjacent cells and thereby regulates developmental processes in a wide variety of different tissues and species [1,2,3,4,5,6].PLOS ONE | DOI:10.1371/journal.pone.0123776 April 9, 2015DLL3 as a Substrate for FringeNotch receptors and their ligands, so-called DSL-proteins are transmembrane proteins with multiple EGF-like repeats of varying numbers in their extracellular domains [7,8,9]
Colocalization of DLL3 and LFNG in the Golgi apparatus, interaction of DLL3 and LFNG, and the presence of O-fucosylation consensus sites raises the possibility that DLL3 is modified by LFNG, the only fringe protein expressed in the presomitic mesoderm
Mutant DLL3 protein was present in completely ES-cell-derived embryos, and the stability of DLL3 and DLL3-S286A,T403V was similar in cultured cells in vitro, indicating that the inability of the mesoderm-specific promoter 2 (MSD2)::DLL3-S286A,T403V construct to rescue the pudgy phenotype cannot be attributed to absence of the mutant protein or obviously altered protein stability
Summary
Notch receptors and their ligands, so-called DSL-proteins (characterized by a conserved Cysteine-rich region found first in the Drosophila Delta, Serrate, and C. elegans lag-2 proteins) are transmembrane proteins with multiple EGF-like repeats of varying numbers in their extracellular domains [7,8,9]. The Notch protein is proteolytically processed and present as a non-covalently linked heterodimeric receptor at the cell surface [10,11]. Activation of Notch through different ligands can be modulated by Fringe proteins, glycosyltransferases that modify Notch in the trans-Golgi [19,20,21] and can accept ligands as substrates [22]. Little is known about how different ligands interact with various Notch receptors, and whether the signals elicited by these interactions are quantitatively or qualitatively different
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