Dynamic Developmental Elaboration of N-Linked Glycan Complexity in the Drosophila melanogaster Embryo

Abstract

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/ Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wildtype or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing ␣-mannosidase II and core fucosyltransferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.Cell surface glycans mediate interactions between cells and define cellular identities within complex tissues at all stages of life (1-6). As embryonic cells differentiate and form organized tissues, glycan expression diversifies, generating glycosylation profiles that are specific for tissue and cell type (7-9). Mutations that affect oligosaccharide synthesis or processing result in neural deficits, skeletal/connective tissue abnormalities, anemia, compromised immune response, muscular dystrophy, or generalized failure to thrive (10 -14). The vital functions of cellular glycans and the pathophysiologic consequences of altered glycosylation emphasize the need for understanding the basic mechanisms that regulate glycan expression in intact organisms.The expanding characterization of glycosyltransferases in Drosophila melanogaster has begun to define the bounds of structural diversity in the glycan portfolio of the organism and has also generated new opportunities for genetically dissecting the mechanisms that control glycosylation. Loss-of-function mutations have been described in a handful...

Developmental Expression of the Neuron-specific N-Acetylglucosaminyltransferase Vb (GnT-Vb/IX) and Identification of Its in Vivo Glycan Products in Comparison with Those of Its Paralog, GnT-V

“…All brains for this study were immediately frozen at Ϫ80°C until glycomic analysis. The frozen whole brains were homogenized and delipidated by extraction twice for 3 h at room temperature on the rocker with a mixture of chloroform/methanol/water (4:8:3, v/v/v) as described (54,55). The emulsion was centrifuged at 2,800 ϫ g for 15 min at 4°C to remove the supernatant.…”

“…Sections from animals were matched using histological landmarks (beginning of the corpus callosum), and every 10th section was imaged and analyzed. Stereological analysis was carried out essentially as described (54).…”

Regulation of Notch signaling by glycosylation

“…The addition of Gal was required to obtain modulation of Notch signaling by Fringe. Gal has been found in Nlinked glycans of Drosophila embryos [25] and therefore a Gal-transferase that may potentially add Gal to O-fucose glycans is expected to exist in Drosophila. In mice lacking β4GalT-1, several Notch target genes involved in somitogenesis are poorly expressed at mid-gestation, and the majority of late term embryos have an extra lumbar vertebra [26].…”

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…Due to the wide variability in the N-glycans of various protists and invertebrates there is no single analytical workflow, but glycomic analyses of each species type very often have to be tailored to suit the properties of the glycans present. Thus, whereas N-glycans of nematodes, cestodes or trichomonads are either neutral or zwitterionic (31)(32)(33) , the protein-linked oligosaccharides of insects can be neutral, acidic or zwitterionic (34)(35)(36) and the N-glycans of oysters are neutral or sulphated (37) ; in the case of trematodes apparently only neutral glycans have been reported (38,39) .…”