Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling

Abstract

SUMMARYMutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch

“…7C). Adding an extra copy of Notch results in additional wing vein material in the adult wings called the Confluens phenotype (45,46). Because N ϩ/Ϫ ; Notch gt-FH /ϩ females did not exhibit the Confluens phenotype (Fig.…”

“…Although determining the efficiency of glucosylation and xylosylation provides additional insight to investigating the role of O-glucosylation on Notch activity, there are still unanswered questions. The exact molecular mechanisms of how O-glucosylation promotes Notch activity (29,45,53) and xylosylation of the O-glucose inhibits Notch activity (33) are yet to be determined.…”

“…The Notch genomic transgene with a 3XFLAG-2XHA (FH) epitope tag was generated by gap repair mutagenesis as described previously (45,62). The FH sequence was inserted between EGF36 and the LNR repeats into the Notch-attB-P[acman]-Ap R construct (45).…”

“…The FH sequence was inserted between EGF36 and the LNR repeats into the Notch-attB-P[acman]-Ap R construct (45). The precise location and sequence of the tag is as follows:…”

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

“…We also studied the genetic interaction between O-fut1 R245A knock-in and rumi, which encodes an O-glucosyltransferase that adds O-glucose to the EGF-like repeats of N (27)(28)(29)(30). Based on the presented results, we propose a model showing how multiple O-glycosylation sites in EGF-like repeats might affect the function of N.…”