No abstract

Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.

The glycan symbol nomenclature proposed by Harvey et al. in these pages has relative advantages and disadvantages. The use of symbols to depict glycans originated from Kornfeld in 1978, was systematized in the First Edition of “Essentials of Glycobiology” and updated for the second edition, with input from relevant organizations such as the Consortium for Functional Glycomics. We also note that >200 illustrations in the second edition have already been published using our nomenclature and are available for download at PubMed.

Notch receptor signaling regulates cell growth and differentiation, and core components of Notch signaling pathways are conserved from Drosophila to humans. Fringe glycosyltransferases are crucial modulators of Notch signaling that act on epidermal growth factor (EGF)-like repeats in the Notch receptor extracellular domain. The substrate of Fringe is EGF-O-fucose and the transfer of fucose to Notch by protein O-fucosyltransferase 1 is necessary for Fringe to function. O-fucose also occurs on Cripto and on Notch ligands. Here we show that mouse embryos lacking protein O-fucosyltransferase 1 die at midgestation with severe defects in somitogenesis, vasculogenesis, cardiogenesis, and neurogenesis. The phenotype is similar to that of embryos lacking downstream effectors of all Notch signaling pathways such as presenilins or RBP-Jκ, and is different from Cripto, Notch receptor, Notch ligand, or Fringe null phenotypes. Protein O-fucosyltransferase 1 is therefore an essential core member of Notch signaling pathways in mammals

No abstract

O -linked N -acetylglucosamine ( O -GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O -GlcNAc transferase (OGT). O -GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O -GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O -GlcNAcylation in AD has been impeded by the difficulty in characterization of O -GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O -GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O -GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O -GlcNAc. Overall, 458 O -GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O -GlcNAcylation and phosphorylation. This study produced the most comprehensive O -GlcNAc proteome of mammalian brain tissue with both protein identification and O -GlcNAc site assignment. Interestingly, we observed O -β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O -GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1- O -Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3- N -acetylglucosaminyltransferases.

Abstract The Symbol Nomenclature for Glycans (SNFG) is a community-curated standard for the depiction of monosaccharides and complex glycans using various colored-coded, geometric shapes, along with defined text additions. It is hosted by the National Center for Biotechnology Information (NCBI) at the NCBI-Glycans Page (www.ncbi.nlm.nih.gov/glycans/snfg.html). Several changes have been made to the SNFG page in the past year to update the rules for depicting glycans using the SNFG, to include more examples of use, particularly for non-mammalian organisms, and to provide guidelines for the depiction of ambiguous glycan structures. This Glycoforum article summarizes these recent changes.

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewisx and sialyl-Lewisx determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.