Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch.
Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.
From the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
Beth M
BM
Harvey
From the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
Nadia A
NA
Rana
From the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
Hillary
H
Moss
the Program in Developmental Biology, Baylor College of Medicine, Houston, Texas 77030, and.
Jessica
J
Leonardi
the Program in Developmental Biology, Baylor College of Medicine, Houston, Texas 77030, and the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.
Hamed
H
Jafar-Nejad
From the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, rhalti@uga.edu.
Robert S
RS
Haltiwanger
D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D005643:Fucose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051880:Receptors, Notch / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; C497759:N protein, Drosophila; D010455:Peptides; D051880:Receptors, Notch; D005643:Fucose; D017351:N-Acetylglucosaminyltransferases; C090576:fng protein, Drosophila
EGF repeat; Fringe; Notch receptor; O-linked N-acetylglucosamine (O-GlcNAc); O-linked fucose; O-linked glucose; glycosylation; glycosyltransferase; mass spectrometry (MS)