Functional analysis of the atypical DSL protein DLL3
Final Report Abstract
Notch signal transduction is an evolutionary conserved essential signaling pathway that mediates cellto-cell communication between adjacent cells. 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 epidermal-growthfactor-like (EGF) repeats in their extracellular domains. In the mouse, there are three genes coding for Delta-type (DLL1, DLL3 and DLL4) DSL proteins. DLL1 and DLL3 have essential functions during somitogenesis and show overlapping expression in the presomitic mesoderm. However, null alleles of both Dll1 (Dll1lacZ) and Dll3 (Dll3pu) affect patterning processes in the presomitic mesoderm differently, suggesting that Dll1 and Dll3 have distinct, non-redundant functions during somite formation. In addition, the DLL1 and DLL3 proteins differ with respect to conserved amino acid residues in the DSL domain, the number of EGF repeats and a PDZ binding motif that is present in DLL1 but not in DLL3. Based on these observations we tested the idea that the observed phenotypic differences in Dll1 and Dll3 mutant embryos is caused at least in part by non-redundant functions, which are caused by biochemical non-equivalence of the DLL1 and DLL3 proteins. We replaced Dll1 with Dll3 by homologous recombination and analyzed mice expressing DLL3 instead of DLL1. DLL3 expressed from the Dll1 locus was functional (i.e. rescued the loss of endogenous DLL3), but did not rescue DLL1 function, demonstrating unambiguously that these proteins are functionally diverged. Further analyses showed that DLL3 resides in the Golgi apparatus (where also DLL1 is found) but is not present on the cell surface. The generation and analyses of chimeric DLL1/DLL3 proteins showed that the ICD of DLL1, and both spacing and sequence of the N-terminal region including the first EGF repeats are critical for DLL1 function, and the four proximal EGF repeats of DLL1 and DLL3 are functionally equivalent. Further biochemical characterization showed that DLL3 is O-fucosylated at EGF repeats 2 and 5, is further modified by glycosyltransferases of the FRINGE (FNG) family, and physically interacts with lunatic fringe (LFGN). DLL3 that cannot be O-fucosylated still interacted with Notch, LFNG and DLL1, and similar to wt DLL3 inhibited Notch in cis. However, in vivo O- fucosylation-deficient DLL3 failed to rescue the loss of endogenous DLL3. Thus, the cis-inhibitory activity of DLL3 on Notch1 that is observed in vitro might not fully reflect its essential functional property under physiological conditions in the PSM and modification of DLL3 by O-linked fucose is essential for its function during somitogenesis.
Publications
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(2007). Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo. The Journal of Cell Biology 178, 465–476. 1725–1736
Geffers, I., Serth, K., Chapman, G., Jaekel, R., Schuster-Gossler, K., Cordes, R., Sparrow, D. B., Kremmer, E., Dunwoodie, S. L., Klein, T., et al.
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(2015). O-fucosylation of DLL3 is required for its function during somitogenesis. PLoS ONE 10(4): e0123776
Serth, K., Schuster-Gossler, K., Kremmer, E., Hansen, B., Marohn-Köhn, B., and Gossler, A.
