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 non-glycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by β-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galβ1-3GalNAc) 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 Galβ1-3GalNAc, 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, (GlcNAcβ1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcβ1-3(GlcAβ1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended 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.
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
non-glycosaminoglycan,
O
-linked glycans expressed in
Drosophila
embryos. Through multidimensional mass spectrometric
analysis of glycans released from glycoproteins by β-elimination, we
detected novel as well as previously reported
O
-glycans that exhibit
developmentally modulated expression. The core 1 mucin-type disaccharide
(Galβ1-3GalNAc) 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 Galβ1-3GalNAc, 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, (GlcNAcβ1-3)fucitol, was not
detectable in whole embryos, mass spectrometry fragmentation and
exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as
GlcNAcβ1-3(GlcAβ1-4)fucitol. Consistent with the spatial
distribution of the Fringe function, the GlcA-extended 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.
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