The Diversity of O-Linked Glycans Expressed during Drosophila melanogaster Development Reflects Stage- and Tissue-specific Requirements for Cell Signaling

Abstract

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the nonglycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by ␤-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Gal␤1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Gal␤1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAc␤1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAc␤1-3(GlcA␤1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcAextended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.

Golgi Glycosylation

“…Drosophila also expresses these O-glycans on EGF-like repeats of Notch and other proteins. However, O-fucose glycans in Drosophila may have a glucuronic acid attached to the O-fucose (Aoki et al 2008), and there is no evidence for the addition of Gal or sialic acid to the GlcNAc (Xu et al 2007). C. elegans has protein O-fucosyltransferase 1 (Loriol et al 2006), but no close homologue of Fringe, the transferase that adds GlcNAc to Fucose-O-EGF (Bruckner et al 2000;Moloney et al 2000).…”

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

“…In flies, O-fucose on Notch has not been seen elongated past the GlcNAc␤1-3Fuc-O-Ser/Thr disaccharide by galactosyltransferases or sialyltransferases (23). However, in an O-glycomics analysis of total protein extracts from fly embryos, Aoki et al (27) reported a novel GlcNAc␤1-3(GlcA␤1-4)fucitol glycan. Interestingly, this trisaccharide was observed in Fringe-rich tissues during embryonic and larval stages of fly development when Notch is known to function (27).…”

“…However, in an O-glycomics analysis of total protein extracts from fly embryos, Aoki et al (27) reported a novel GlcNAc␤1-3(GlcA␤1-4)fucitol glycan. Interestingly, this trisaccharide was observed in Fringe-rich tissues during embryonic and larval stages of fly development when Notch is known to function (27). Whether this O-fucose trisaccharide occurs on Notch remains to be resolved.…”

O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

“…The GlcNAc-␤1,3-Fuc disaccharide can be extended to a tetrasaccharide in mammals (16). A branched O-fucose trisaccharide (GlcUA-␤1,4-(GlcNAc-␤1,3)-Fuc) has been detected in total extracts of Drosophila larvae and wing discs, but it is not clear whether this modification is on EGF repeats of Notch (33). Elongation beyond the GlcNAc-␤1,3-Fuc disaccharide has not been detected on Drosophila Notch produced in S2 cells (34).…”