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...

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