Specific glycan expression is an essential characteristic of developing tissues. Molecular characterization of a mutation in Tollo that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. In wild-type embryos, Tollo is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. Neural differentiation and axon extension, to the extent that they are revealed by standard monoclonal antibody markers, are unaffected by loss of Tollo function. The ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors (Seppo, 2003).

Eukaryotic cells are enveloped within a complex coating of carbohydrate. Composed of the pendant oligosaccharide moieties of glycoproteins, glycolipids and proteoglycans, the glycocalyx constitutes the interface at which cells interact with each other and with their environment. Glycans mediate cellular recognition and adhesion, facilitate protein maturation, regulate protein activity through allostery and bioavailability, influence receptor ligation, and modulate transmembrane signaling. Specific oligosaccharide structural elements participate in each of these processes, requiring spatial and temporal regulation of glycan synthesis in developing and mature organisms. As a consequence of regulated expression, oligosaccharide structures are among the most discriminating markers for cellular differentiation in complex tissues. Despite extensive descriptions of specific oligosaccharide distributions, the cellular and molecular mechanisms by which cells achieve expression of their characteristic portfolio of surface glycans are largely unknown (Seppo, 2003 and references therein).

Antibodies raised against the plant glycoprotein, Horseradish Peroxidase (HRP), crossreact with an N-linked oligosaccharide epitope that is distributed throughout the Drosophila melanogaster nervous system and is also expressed in a small, well characterized subset of non-neural tissues (Jan, 1982; Snow, 1987). Two mutations abolish expression of the HRP epitope. In the first, designated nac, the epitope is absent in the larval, pupal and adult nervous system (Katz, 1988). The molecular nature of the nac mutation is unknown, but affected adults exhibit sensory afferent defasciculation and behavioral phenotypes (Whitlock, 1993; Phillis, 1993). The second mutation that abolishes HRP-epitope expression is carried on the TM3 balancer chromosome, an extensively rearranged form of the third chromosome (Snow, 1987). TM3 homozygotes do not express the HRP epitope in the embryonic nervous system but do produce the glycan in the expected non-neural tissues. Therefore, it is likely that the structural genes necessary for synthesis of the HRP epitope are intact and that the TM3 mutation alters a gene that regulates tissue-specific glycosylation (Seppo, 2003).

The TM3 locus that abolishes HRP-epitope expression has been characterized. The affected gene, which has been named 'tollo' encodes a member of the family of cell surface receptors with homology to the Toll protein (Toll-like receptors, TLRs). Genome sequence characterization re-identified the tollo locus, resulting in its designation as 'toll-8' (Tauszig, 2000). The founding member of the TLR family (Toll) was originally identified as a component of the signaling pathway that induces dorsal-ventral polarity in the Drosophila embryo. Subsequently, TLRs have also been shown to participate in innate immune responses in Drosophila and other organisms by transducing pattern recognition signals. The induction of tissue-specific glycosylation can now be added to the list of TLR functions (Seppo, 2003).

Nearly a half-century of molecular biochemistry has documented tissue-specific, cell-specific, stage-specific, and disease-specific oligosaccharide presentation. Despite this wealth of information, few descriptions exist of molecular mechanisms that control the specificity of glycan expression. Glycan synthesis has been modulated by manipulating various transmembrane signaling pathways, indicating that receptor-mediated events at the cell surface can influence oligosaccharide profiles. Although the surface receptors that transmit these signals have not been identified, the results of this study demonstrate that the Tollo/Toll-8 transmembrane protein influences cell-specific glycan expression in the Drosophila embryo (Seppo, 2003 and references therein).

The localization of Tollo expression to non-neural ectodermal cells and the rescue of neural-specific glycosylation by transgenic tollo both demonstrate that non-homologous cells modulate glycan expression in adjacent tissues. The close proximity of Tollo-expressing ectodermal cells to differentiating neurons is consistent with a molecular mechanism in which neural glycosylation is influenced by the activity of a neuronal surface receptor that directly binds ectodermal Tollo. Alternatively, the molecular activity of tollo may reside entirely within the ectodermal cell, exerting an indirect influence on neural glycosylation by propagating or attenuating instructive signals subsequently interpreted by local neurons. At present, the data cannot unambiguously distinguish whether the direct or indirect mechanism applies. However, the results of HRP-epitope rescue and Tollo misexpression studies indicate requirements that both models must satisfy (Seppo, 2003).

Neuronal synthesis of the HRP-glycan is rescued in Brd¹⁵/Brd¹⁵ embryos when Tollo is expressed in its wild-type ectodermal pattern (tollo-Gal4/UAS-tollo). However, ELAV-Gal4/UAS-tollo and rho-Gal4/UAS-tollo embryos fail to rescue the HRP epitope, despite driving misexpression in neurons and glia that would present Tollo to neuronal surfaces at developmental stages coincident with the normal Tollo expression pattern. Therefore, if tollo acts directly to alter neural glycosylation, ectodermal presentation of Tollo must be unique in comparison with expression in other cell types that also share contact with neurons; either the Tollo protein requires an ectodermal-specific post-translational modification for activity or an ectodermal co-factor is necessary for appropriate presentation to neurons. If tollo indirectly affects neural glycosylation by generating or influencing paracrine signals sensed by differentiating neurons, then the cellular context in which Tollo is expressed determines induction of the HRP epitope; either the relevant paracrine influence is specifically of ectodermal origin or a required tollo intracellular signaling pathway is absent from neurons and glia (Seppo, 2003).

The indirect mechanism is consistent with the function of other TLRs and is supported by two additional observations. (1) The discontinuous distribution of tollo mRNA in the neurogenic ectoderm of the ventral nerve cord indicates that tollo expression is limited to ectodermal cells contacting only a subset of the total differentiating neuron pool. Therefore, global CNS expression of the HRP epitope requires a signal unrestricted by the need for cell-cell contact. (2) HRP-epitope expression is rescued in PNS sensory neurons located near salivary glands that ectopically express Tollo, consistent with the generation of a locally active signal. Expression of the HRP epitope is not rescued in the CNS nor in more remote parts of the PNS by hsp70-Gal4/UAS-tollo, implying that temporal and physical barriers can limit Tollo activity (Seppo, 2003).

The unexpected, ectopic expression of the HRP epitope in the secretory epithelium of the salivary gland indicates that some developing tissues are only one signal away from assuming an altered glycosylation phenotype. At least within the salivary gland, this result also indicates that Tollo can act directly or can generate an autocrine signal that autonomously modulates glycosylation. For neural tissue, though, elaboration of the HRP epitope is a non-autonomous neuronal behavior that requires ectodermal Tollo expression. By analogy to Toll, soluble protein ligands (like Spätzle) are prime candidates for the Tollo activator, but the full diversity of TLR ligands has yet to be characterized in any organism (Seppo, 2003).

In plants and in the Drosophila adult, HRP-epitope structure has been demonstrated to contain an extensively trimmed high-mannose core carrying an alpha3-linked Fuc residue on the internal GlcNAc of the chitobiose. To generate the described Drosophila HRP epitopes, high-mannose oligosaccharides must first be trimmed to a Man₃GlcNAc₂ or Man₂GlcNAc₂ core. The core structure is then di-fucosylated (alpha3 and alpha6), requiring the activity of two distinct fucosyltransferases. Addition of Fuc alpha3 to the core requires previous and transient addition of GlcNAc to a terminal Man, yielding a di-fucosylated Man_2/3GlcNAc₂ oligosaccharide. Therefore, trimming mannosidases, two fucosyltransferases, an N-acetylglucosaminyltransferase and a hexosaminidase constitute the minimal set of processing activities required to generate an HRP epitope. Of these activities, addition of the alpha3 Fuc imparts antibody recognition to the oligosaccharide (Seppo, 2003).

A Drosophila fucosyltransferase that adds Fuc in alpha3 linkage to core GlcNAc has been characterized (Fabini, 2001). Designated 'FucTA', the enzyme exhibits in vitro acceptor specificity appropriate for synthesis of the HRP epitope and the gene maps to 71B2, 87 kb distal to tollo. Although this lies within the Brd¹⁵ deletion, combining tollo-Gal4 with UAS-tollo results in rescue of HRP epitope expression in Brd¹⁵ homozygotes. Therefore, glycan expression is rescued by Tollo/Toll-8 in a FucTA null background. The relevance of FucTA activity to HRP-epitope expression in the embryonic nervous system remains to be determined, but Drosophila requires alpha3 fucosyltransferase activity and the resulting capacity to synthesize the HRP epitope in multiple contexts. Mutants that lack the HRP epitope in larval and adult stages express the oligosaccharide embryonically and epitope expression in embryonic non-neural tissue is maintained in mutants that lack the embryonic neural oligosaccharide (Snow, 1987; Katz, 1988). Thus, multiple pathways, under independent control and active in different tissues and developmental stages, lead to synthesis of the HRP epitope (Seppo, 2003).

These results suggest superficially that loss of the HRP epitope is of relatively little consequence. However, the component of the HRP epitope structure that imparts antibody recognition may be distinct from the functional domain of the oligosaccharide. Therefore, mutations in genes such as tollo, which affect specific carbohydrate expression, may not immediately reveal oligosaccharide function. The nac mutant, which lacks larval, pupal and adult expression of the HRP epitope, exhibits grossly normal nervous system morphology (Katz, 1988; Phillis, 1993). Highly penetrant axon defasciculation errors are present in the nac adult but only become apparent when afferent projections arising from discrete subsets of dye-labeled sensory neurons in the wing margin are visualized at their entry point into the central nervous system (Whitlock, 1993). Until techniques of similar resolution are applied to embryos that lack the HRP epitope, the functional significance of loss of this tissue-specific glycan cannot be fully evaluated (Seppo, 2003).

In Drosophila, the HRP epitope is present on several neural proteins, many of which are also expressed in non-neural tissue where they lack the glycan (Desai, 1994; Snow, 1987; Sun, 1995; Wang, 1994). Thus, cells determine whether or not to construct the HRP epitope on a particular glycoprotein based on the tissue in which the protein is expressed, rather than on a signal intrinsic to the polypeptide. While tollo/toll-8 demonstrates that such tissue-specific glycan expression can be achieved through the activity of a Toll-like receptor, the correlation between Drosophila TLR expression and specific glycosylation patterns cannot be comprehensively assessed before glycan characterization in the Drosophila embryo is greatly expanded. Nonetheless, distributions of other TLRs exhibit spatial and temporal overlap with Tollo expression, raising the possibility that TLRs sculpt embryonic glycosylation patterns through combinatorial activation of glycosylation pathways in interacting domains of developing tissues (Seppo, 2003).

TLRs mediate pattern recognition (frequently glycan-based) as part of the innate, non-adaptive immune response in Drosophila and vertebrates. However, only a subset of Drosophila TLRs induce defensive responses. TLR family members appear divided into clans that function in innate immunity or that fulfill developmental needs (Tauszig, 2000). The capacity to control glycosylation could unite the TLR family in support of a common cause, to produce appropriate spatial and temporal patterns of cell-specific glycosylation. Expressed by immune cell types that participate in tissue surveillance, TLRs are positioned to locally influence cellular glycosylation in response to pathogen, thereby coupling innate detection of non-self patterns with expression of protective glycans on host cells. In addition, further analysis of the distribution and function of TLRs may indicate that the constitutive maintenance of diverse tissue glycan profiles is generally an active process in which glycan expression is continually renewed or responsively modified by TLR-mediated signaling. In mature tissues and in the embryo, the expression of glycans must be orchestrated to coincide with the appearance of relevant carbohydrate binding proteins that mediate cell adhesion and recognition. Therefore, broader mechanisms that impart specificity to cell-cell interactions are likely to be revealed with further characterization of the pathway by which tollo/toll-8 controls oligosaccharide expression (Seppo, 2003).

GENE STRUCTURE

cDNA clone length - 7210 bp

Bases in 5' UTR - 480

Exons - 1

Bases in 3' UTR - 2689

PROTEIN STRUCTURE

Amino Acids - 1346

Structural Domains

Motifs in the predicted protein define it as a member of the Toll-like Receptor family. Leucine-rich repeats and cysteine-rich domains are found in the extracellular portion of the molecule and a Toll homology (TH) domain is present in the C-terminal region. Based on the TH similarity and on the inability of Brd¹⁵/TM3 adults to reliably extricate themselves from their food, the gene was named 'tollo', a Finnish word roughly translated as 'stupid' (Seppo, 2003).

date revised: 23 May 2004

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