Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA
; Accepted on April 3, 2000.
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Abstract |
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Key words: Drosophila/glycosaminoglycan/ N-linked oligosaccharide/glycosphingolipid/lectin/glycosyltransferase/heparan sulfate/HRP-epitope
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Introduction |
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In other model organisms, such a monumental undertaking would generate an encyclopedic tome well beyond the intended scope of these reviews. However, random mutational analysis has only rarely identified Drosophila genes directly related to complex carbohydrate structure or function, and the biochemical characterization of Drosophila glycans has historically been limited by the availability of relatively small quantities of tissue. Fortunately, over the last 5 years a handful of developmental phenotypes identified in mutagenesis screens have been ascribed to loss-of-carbohydrate function (Table I). In addition to generating increased interest in Drosophila glycobiology, these mutants provide genetic inroads, through the analysis of their interactions with known and still undiscovered loci, toward broadening the range of identified genes that contribute to glycan function. Furthermore, molecular characterization of the mutagenized genes has highlighted the value of identifying Drosophila homologues of known glycosyltransferases and stressed the need for generating a database of expressed Drosophila oligosaccharides. Such a structural foundation would not only inform the analysis of genetic lesions, but would also provide critical guidance in deciphering the biosynthetic capacity soon to be revealed by the sequence of the Drosophila genome.
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Glycan function |
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Lectin staining of Drosophila embryos and tissues has demonstrated spatial enrichment and dynamic variation in carbohydrate expression (Fristrom and Fristrom, 1982; Callaerts et al., 1995
; D'Amico and Jacob, 1995
). ConA and WGA reveal, respectively, high-mannose or more processed N-linked oligosaccharides in diverse and overlapping sets of tissues, primarily ectodermal derivatives, throughout development. For example, while the neuroepithelium is devoid of significant WGA staining, ConA strongly stains axons. ConA also stains membrane associated with the acrosomal region of Drosophila sperm (Perotti and Pasini, 1995
). Other lectins, possessing preference for Gal- or GalNAc-terminated structures (BS-I, LAA, APA, PNA, ACA, TL, BPA), present significant temporal variation in staining that correlates with organogenesis in mesodermal, ectodermal and imaginal tissues. A limited set of fucose-binding plant lectins (UEA-I, TPA) stain embryos weakly. Neither the identity of the recognized oligosaccharide structure nor the nature of the relevant aglycone has been defined for any of these lectin-binding ligands.
Evidence for the existence of sialylated glycoconjugates has been mostly negative among invertebrate species (Dennis et al., 1987). However, the staining of Drosophila early embryonic cell types, including migrating germ cell precursors, with sialic acidbinding lectins (LPA and LFA) is neuraminidase-sensitive (Roth et al., 1992
; D'Amico and Jacob, 1995
). In later embryonic stages, LFA demonstrates both neural and general ectodermal staining and Western blot analysis with a poly(
2,8)sialic acid (PSA) specific monoclonal antibody detects PSA immunoreactivity between 1418 h of embryogenesis. Since this developmental period correlates with active nervous system development, it has been proposed that PSA expression modifies axonal and cell adhesion molecule function during Drosophila neural development as has been demonstrated in mammalian systems (Acheson et al., 1991
). Another glycan proposed to mediate cell adhesion in vertebrates, the L2/HNK-1 epitope, has also been immunologically visualized in neural, non-neural, and imaginal tissue of Drosophila larvae (Dennis et al., 1991
). In Drosophila, the aglycone bearing the L2/HNK-1 epitope is unknown, although both glycoproteins and nonsulfated glycolipids are cross-reactive in another dipteran, Calliphora vicina (Dennis et al., 1988
).
A fortuitously identified antibody probe offers another opportunity to study tissue-specific oligosaccharide expression in Drosophila. Antibodies raised against the plant glycoprotein, horseradish peroxidase (HRP), stain the entire Drosophila nervous system (Snow et al., 1987; Katz et al., 1988
). The antisera recognizes a non-peptide epitope shared by several Drosophila membrane glycoproteins, some of which are broadly distributed but only recognized by HRP antibodies when expressed in the nervous system (Desai et al., 1994
; Wang et al., 1994
; Sun and Salvaterra, 1995a
,b). Recognition is attributable to a carbohydrate moiety based on sensitivity to trifluoromethanesulfonate and periodate oxidation and on resistance to boiling in SDS with ß-mercaptoethanol (Snow et al., 1987
). Antibody binding is also abolished by preabsorbing the antiserum with HRP protein, HRP glycopeptides, or glycopeptides prepared from another plant glycoprotein, pineapple stem bromelain (PSB), which is also recognized by anti-HRP antisera. The structure of the recognized N-glycan in both HRP and PSB is of the form Man
1,6(Xylß1,2)Manß1,4GlcNAcß1,4(Fuc
1,3)GlcNAc with the
1,6Man and
1,3Fuc residues being essential for antibody binding (Ishihara et al., 1979
; Kurosaka et al., 1991
). The cross-reacting epitope in Drosophila, however, remains essentially uncharacterized although it is found on both neutral and acidic oligosaccharides (Seppo et al., 2000
).
Genetic analysis of tissue-specific glycosylation
Two Drosophila mutations, nac and tollo, interfere with HRP-epitope expression and affect neural development. Mutations in the nac locus abolish epitope expression in the adult nervous system. While nac homozygotes are viable and were initially described as normal when raised at room temperature, they display abnormal wing and eye facet morphology when forced to develop at 18°C (Katz et al., 1988). Furthermore, even when reared at room temperature, visualization of the pathways that individual sensory axons take into the central nervous system in nac mutants reveals significant defasciculation and inappropriate routing (Whitlock, 1993
). Neither the nature of the affected gene nor the mechanism by which it modulates carbohydrate expression is currently known.
Independent of the nac locus, a genetic lesion on the TM3 balancer chromosome abolishes HRP-epitope expression in the embryonic nervous system. Isolation of the relevant TM3 mutation identified a gene designated tollo, which encodes a putative cell-surface transmembrane receptor of the Toll/18-Wheeler family, indicating that expression of the HRP-epitope is induced by specific signaling events. While TM3 homozygotes do not develop past embryogenesis (due to multiple chromosomal rearrangements unrelated to tollo), heterozygotes bearing a noncomplementing tollo deletion in combination with the TM3 chromosome are viable despite lacking the HRP-epitope. tollo mutants are also characterized by displaced sensory organ precursors in the peripheral nervous system and axon defasciculation (Seppo et al., 2000).
In both nac and tollo the correlation between HRP-epitope expression and notable defects in neural development implies a developmental role for neural specific glycosylation in Drosophila. However, the glycosylation defects in both mutations affect an epitope recognized by an antibody, which may or may not be the functionally relevant structure; aberrant glycans may possess residual functionality. Nonetheless, both mutations control glycosylation in a tissue- and time-dependent manner, providing an opportunity to genetically dissect mechanisms that regulate glycan expression.
Genes predicted to modify glycosaminoglycan structures also modulate morphogen signaling
It has recently been demonstrated that glycan function contributes to normal Drosophila development even before fertilization. The embryonic dorsalventral axis is established by asymmetric activation of a proteolytic cascade in the perivitelline space surrounding the embryo. The spatially restricted proteolytic processing of several zymogens results in the generation of a soluble ligand (Spätzle) for an embryonic membrane receptor (Toll) which, when engaged, induces development of characteristic ventral embryonic structures through differential activation of transcription (Morisato and Anderson, 1995; Belvin and Anderson, 1996
). Genetic analysis originally identified 12 genes that affect dorsalventral patterning and placed 9 of the 12, all expressed in germline cells, into a series from zymogen cleavage to transcription factor activation. Of these 9, however, none exhibit spatial restriction in expression. The remaining three, designated pipe, nudel, and windbeutel, are expressed by follicle cells surrounding the developing egg in the Drosophila egg chamber and became candidates for imparting spatial information (Hong and Hashimoto, 1995
; Nilson and Schüpbach, 1998
; Sen et al., 1998
). Only the expression of Pipe, however, is spatially restricted. Cloning of pipe revealed significant similarity to vertebrate heparan sulfate 2-O-sulfotransferase and has suggested that spatially restricted modification of heparan sulfate proteoglycan structure generates a pre-pattern within the follicular epithelium that leads to localized protease activation (Nilson and Schüpbach, 1998
; Sen et al., 1998
). A potential target for such modification is Nudel, a secreted protein which contains sites appropriate for glycosaminoglycan addition and exhibits electrophoretic behavior consistent with extensive glycosylation (Hong and Hashimoto, 1995
; LeMosey et al., 1998
). To date, however, neither Pipe enzymatic activity nor sulfation of relevant substrates, such as Nudel glycans, has been demonstrated.
Before the cloning of pipe it was already apparent that glycosaminoglycans function at multiple stages in Drosophila development. Classic genetic screens to identify loci that control embryonic morphology yielded novel protein morphogens such as Wingless (Wg) and Hedgehog (Hh), as well as Drosophila homologues of vertebrate growth factors such as transforming growth factor-ß/bone morphogenetic protein (decapentaplegic, Dpp, in Drosophila) and fibroblast growth factors (Branchless, Bnl, in Drosophila) (Nusslein-Volhard and Wieschaus, 1980; Perrimon et al., 1986
; Schüpbach and Wieschaus, 1986
; Nusse and Varmus, 1992
; Tabata and Kornberg, 1994
; Lecuit et al., 1996
; Nellen et al., 1996
; Sutherland et al., 1996
). The characterization of phenotypes associated with mutant morphogen alleles led to the initiation of screens for interacting genes that subsequently identified relevant receptors and downstream signaling components (Perrimon et al., 1989
; Ruberte et al., 1995
; Beiman et al., 1996
; Bhanot et al., 1996
; Chen and Struhl, 1996
; Gisselbrecht et al., 1996
; Stone et al., 1996
). In addition, interaction screens revealed that mutant alleles of genes encoding Drosophila homologues of glycosaminoglycan synthetic or processing enzymes also affect morphogen signaling.
The first glycan-related gene to be reported, a Drosophila homologue of UDP-glucose dehydrogenase (UDP-GDH), was identified based on its ability to enhance weak wg phenotypes or, on its own, to phenocopy wg or hh mutations. Mutations in UDP-GDH, originally identified by three groups and now designated sugarless, are embryonic lethal and affect early cuticular patterning in a manner similar to wg (Binari et al., 1997; Häcker et al., 1997
; Haerry et al., 1997
). To assess UDP-GDH function later in development, the generation of mutant clones in imaginal discs can be induced by mitotic recombination, resulting in a mosaic tissue. In wing discs the result is wing structure loss that resembles the wg mutant phenotype. In the eye disc, where Dpp expression is dependent on Hh signaling, clonal loss of Sugarless results in reduced Dpp expression and a smaller eye (Haerry et al., 1997
). In the whole embryo, cuticular phenotypes, resembling wg and hh mutations, could be rescued by injection of heparan sulfate or UDP-GlcA into embryos before cellularization of the syncytial blastoderm (Binari et al., 1997
). In addition, processing of endogenous Syndecan in sugarless mutants yields protein that is more homogenous and possesses greater electrophoretic mobility than that of the proteoglycan expressed in a wild-type background, consistent with the expected function of UDP-GDH in generating UDP-GlcA for heparan, chondroitin, or dermatan synthesis (Haerry et al., 1997
). However, direct demonstration of structurally altered glycosaminoglycan in UDP-GDH mutants or of enzymatic activity associated with the wild-type protein is not yet available. Furthermore, whether reduced UDP-GDH imposes relevant constraints on the synthesis of other glycan classes, notably GlcA-bearing Drosophila glycolipids (see below), remains to be determined.
Controlled diffusion is crucial for spatially limiting induction by Hh early in embryogenesis. Hh strongly influences cell fate within 810 cell diameters in the anterior-posterior axis, a range of action partially resulting from protein processing steps that include the addition of a cholesterol moiety (Porter et al., 1996). Mutations in the tout velu (ttv) gene, originally isolated in a screen for altered cuticle patterning, were subsequently shown to block Hh movement in mosaic wing discs. ttv encodes a Drosophila member of the EXT/EXT-like family of proteins, genes associated with a human disorder (multiple exotoses syndrome) characterized by bony outgrowths and increased incidence of chondrosarcomas and osteosarcomas (Bellaiche et al., 1998
; The et al., 1999
). While vertebrate EXT1 and EXT2 have been implicated both in heparin synthesis and also in the Golgi-targeting of EXT hetero-oligomeric complexes, the function of the extended family of EXT-like proteins is less characterized (McCormick et al., 1998
, 2000). The high homology of EXT-like proteins suggests that they may possess related glycosyltransferase activities, but it is currently unclear whether they participate in GAG synthesis (J.Esko, personal communication; Kitagawa et al., 1999
).
While an enzymatic activity for Ttv has not yet been demonstrated, ttv mutant embryos exhibit markedly reduced (but not eliminated) staining, relative to wild type, when probed with monoclonal antibody 3G10 which recognizes a heparinase-generated epitope (The et al., 1999). It is proposed that residual staining represents the activity of a second molecularly identified Drosophila EXT family member. That more than one EXT or EXT-like gene should exist in Drosophila is also predicted from the observation that Wg and FGF signaling are independent of ttv despite their own apparent modulation by heparan sulfate (Lin et al., 1999
; The et al., 1999
). However, comparison of heparan-derived disaccharide profiles in wild-type and ttv mutant tissues demonstrates reduction of heparan sulfate to undetectable levels in the ttv mutant (Toyoda et al., 2000
). Therefore, it is currently unclear whether the dissection of Wg and FGF signaling modulation from Hh signaling modulation reflects qualitative or quantitative differences in relevant glycosaminoglycan composition. Nonetheless, if separate morphogen signaling pathways utilize their own glycosaminoglycan initiation or polymerization enzymes, all chains of unmodified heparan repeats must not be created equal in Drosophila.
Multiple signaling pathways converge on the glycosaminoglycans of a single proteoglycan
Functional analysis of a Drosophila glypican homologue has recently converged with the characterization of putative glycosaminoglycan modifying enzymes. Originally identified as a gene responsible for cell cycle progression in a limited set of dividing cells, dally encodes a Drosophila glypican (Nakato et al., 1995). The dally mutant phenotype exhibits characteristics of both Dpp and Wg signaling defects and also demonstrates tissue- and stage-specific genetic interactions with dpp and wg (Jackson et al., 1997
; Tsuda et al., 1999
). Its similarity to vertebrate glypican predicted that Dally might carry heparan sulfate chains. Demonstration of heparan addition to epitope-tagged Dally expressed in an insect cell line has been accomplished by cleavage with heparin lyase II and nitrous acid (but not chondroitinase ABC) which cause tagged Dally to alter its electrophoretic mobility from a broad, high molecular weight distribution to a simpler, more rapidly migrating profile. Furthermore, posttranslational modification of endogenous Dally is greatly reduced in sugarless (UDP-GDH) mutants (Tsuda et al., 1999
). The same genetic screen for modifiers of Wg signaling that yielded sugarless identified another putative glycosaminoglycan modifying gene that was designated sulfateless because its sequence predicts significant similarity to vertebrate heparan sulfate N-deacetylase/N-sulfotransferase (NDST). Dally post-translational modification is also altered in the sulfateless mutant background and, consistent with the central importance of NDST and UDP-GDH to heparan synthesis, both sulfateless and sugarless affect FGF signaling (Lin and Perrimon, 1999
; Lin et al., 1999
).
While it is not surprising that the processing of a glypicans glycans would be dependent on the activity of UDP-GDH and NDST, it is significant that this dependence was first suggested by genetic interactions and similarities in developmental phenotypes observed between mutations in the product (Dally), the enzymes and two signaling pathways (wg and dpp). The convergent dependence of two morphogen signaling pathways on the glycosaminoglycan chains of a single protein presents an opportunity to investigate the generation of specificity in glycan-modulated signaling. Since Dally differentially affects signaling through the Wg and Dpp pathways in a stage- and tissue-dependent fashion, it has been suggested that this Drosophila glypican acts as a regulatory component of a multiprotein morphogen/receptor complex (Lin and Perrimon, 1999; Tsuda et al., 1999
). Signaling specificity would be dictated by the complement of glycosaminoglycan structural motifs carried by a population of Dally molecules.
The extent to which signaling specificity can be encoded within glycosaminoglycan structure will directly depend on the diversity of postsynthetic modifications expressed in the organism. A significant advance toward defining this diversity was recently achieved by the analysis of chondroitin and heparan sulfate disaccharide profiles in Drosophila (and Caenorhabditis elegans) tissues (Yamada et al., 1999; Toyoda et al., 2000
). Drosophila heparan sulfate disaccharide diversity is comparable to the disaccharide heterogeneity of vertebrates but greater than that of Caenorhabditis elegans. Ovarian, embryonic, larval, and adult tissues all possess mono-, di-, and tri-sulfated forms of N-, 2-O- and 6-O-sulfated disaccharides in varying ratios; the analytic technique precluded detection of 3-O-sulfate. Non-sulfated and 4-O-sulfated chondroitin disaccharides were also determined to exist. Therefore, the stage is set for correlating glycosaminoglycan structural alteration with developmental function in defined genetic mutations. However, the characterization of glycans on individual proteoglycan core populations, a level of resolution likely to be important for biological specificity, has not yet been achieved but should be aided by the genomic identification of potential modifying enzymes that may demonstrate genetic interactions with core protein mutants.
Endogenous lectins have been identified by genetic, molecular, biochemical, and genomic approaches
Recognition by binding proteins provides one mechanism by which cell-surface carbohydrate expression can be harnessed to drive specificity in developmental processes. While the full diversity of lectins is far from characterized, two major classes of previously described animal lectins, C-type and galectin, are represented in Drosophila. One Drosophila galectin has been identified but only as an expressed sequence tag (EST). Therefore, neither the function nor the diversity of galectins in Drosophila can currently be assessed (Cooper and Barondes, 1999). With regard to the Siglec family, the scarcity of sialic acid, the absence of myelin and the lack of a lymphocyte-based cellular immune response in Drosophila make it unlikely that true homologues will be found. Nevertheless, the diversity of Drosophila membrane proteins possessing immunoglobulin repeats suggests that the identification of immunoglobulin-type lectin homologues with divergent carbohydrate binding properties remains a possibility.
The diversity of C-type lectins in Drosophila is more completely characterized. The first C-type lectin identified in Drosophila was isolated and purified from pupal extracts as a functional, but not structural, homologue of a previously characterized C-type lectin that is induced upon injury or infection of Sarcophaga peregrina larvae (Takahashi et al., 1985). The Drosophila version is Gal-specific and is also induced upon larval injury, indicating that it functions in innate immunity. While constitutive expression in late larval and early pupal stages was also reported, embryonic expression was not detected (Haq et al., 1996
). The second identified Drosophila C-type lectin was cloned as the gene affected in the furrowed mutation, named for deep invaginations in the adult eye that result from altered ommatidial morphology in partial loss-of-function alleles (Leshko-Lindsay and Corces, 1997
). The Furrowed protein is most similar to vertebrate selectins in its domain structure, containing multiple complement binding repeats in addition to a C-type lectin domain (CTLD). Expressed in embryonic, larval, pupal, and adult stages, Furroweds binding specificity remains undefined. Finally, a recent survey of the partially completed Drosophila genome sequence has identified 21 potential genes containing CTLDs (Theopold et al., 1999
). For comparison, analysis of the completed Caenorhabditis elegans genome yielded at least 125 proteins with CTLDs, 19 of which show maximal conservation of residues required for calcium ligation and carbohydrate binding (Drickamer and Dodd, 1999
).
Emerging families of Drosophila proteins interact with carbohydrate
Other Drosophila proteins proposed or demonstrated to interact with carbohydrates defy current lectin classification schemes. DS47 is an abundant chitinase-like protein secreted into the hemolymph and was the first of a family of related proteins now called imaginal disc growth factors (IDGF) to be identified (Kirkpatrick et al., 1995; Kawamura et al., 1999
). Since the IDGFs all lack active site residues essential for hydrolytic activity, it is unlikely that they are N-acetylglucosaminidases although they may retain carbohydrate binding activity. While DS47 was originally proposed to function as a receptor for foreign glycans in the innate immune response, the more recently identified members of this family have strong growth-promoting activities that act synergistically with insulin to stimulate the proliferation of imaginal disc cells in culture.
The Drosophila Fringe protein also possesses molecular similarity to an enzyme family, in this case to several galactosyltransferases that function in bacterial lipooligosaccharide synthesis (Yuan et al., 1997). Drosophila Fringe is a secreted protein genetically demonstrated to modulate signaling activities that establish compartment boundaries during imaginal disc development (Panin et al., 1997
; Dominguez and de Celis, 1998
; Klein and Arias, 1998
). Although enzymatic activity has yet to be demonstrated for Drosophila or vertebrate Fringe or Fringe-like molecules, the possibility remains that upon secretion they retain functionally relevant carbohydrate binding activity.
While carbohydrate binding activity has not so far been demonstrated for the IDGFs or Fringe, the Drosophila Gliolectin protein was initially identified based on its ability to impart carbohydrate-mediated adhesion to an otherwise non-adherent cell line (Tiemeyer and Goodman, 1996). Gliolectin is expressed by a subset of glial cells at the midline of the developing nervous system, where it is positioned to facilitate early axon pathfinding across the embryonic midline. In vitro, Gliolectin preferentially binds a subset of Drosophila glycosphingolipids whose common element is a non-reducing terminal GlcNAcß,3Hexose (Gal or Man). The characterization of physiologically or developmentally relevant ligands for Gliolectin or any other Drosophila lectin is lacking and would greatly benefit from a broader understanding of endogenous oligosaccharide diversity.
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Conserved biosynthetic and processing components |
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The biosynthesis of N-linked oligosaccharides in Drosophila begins conventionally with the formation of a precursor oligosaccharide linked to dolichol. Although synthesis of mannosyl-, glucosyl-, and oligosaccharyldolichol has been demonstrated in Drosophila cell lines, little chitobiosyldolichol was detected, suggesting that intermediates are rapidly processed to the full-size oligosaccharide donor (Parker et al., 1991; Sagami and Lennarz, 1987
). Transfer of oligosaccharide to protein is probably accomplished as in other species since a Drosophila gene with similarity to one component of the yeast and human oligosaccharyltransferase complex has been described (Stagljar et al., 1995
). As in vertebrate species, the lipid-linked precursor oligosaccharide carries three
-linked Glc residues (Glc3Man9GlcNAc2). Therefore, following trimming by Glucosidase I, Drosophila glycoproteins carrying appropriate N-linked oligosaccharides are substrates for quality control mediated by calnexin or calreticulin and UDP-glucose:glycoprotein glucosyltransferase (Tanimura et al., 1979
; Parker et al., 1995
; Christodoulou et al., 1997
).
The potential for processing N-linked oligosaccharides, at least to hybrid structures, is apparent in cloned genes and genomic sequence
For the subsequent processing of N-linked oligosaccharides, Drosophila possesses homologues of both Mannosidase I and II (Foster et al., 1995; Kerscher et al., 1995
). A null mutation in Mannosidase I (mas-1) has very little phenotypic effect; mildly altered wing and eye structures are highly penetrant while peripheral nervous system defects were observed only rarely (Kerscher et al., 1995
). Since oligosaccharides ranging from Man8GlcNAc2 through Man5GlcNAc2 were found in the mas-1 null mutant, it is likely that redundant or alternate processing pathways exist in Drosophila just as they have been demonstrated in the mouse for loss of Mannosidase II (Chui et al., 1997
; Roberts et al., 1998
). The Drosophila Mannosidase II homologue has been demonstrated to possess appropriate hydrolytic specificity and has been immunocytochemically localized to the Golgi in the embryo (Rabouille et al., 1999
). Beyond mannose trimming, evidence for N-linked processing is currently circumstantial or lacking (Oriol et al., 1999
). A negative report suggests that Drosophila lacks GlcNAc-TI or at least lacks a homologue that can be identified by low-stringency Southern analysis with a mouse probe (Kumar et al., 1992
). However, cell lines derived from other insect orders, primarily lepidoptera, possess both GlcNAc-TI and GlcNAc-TII activities (Altmann et al., 1993
).
For purposes of this review, we prospected the currently available Drosophila EST database for identified sequence similarities to glycoprotein processing enzymes (Rubin, 1996). This effort revealed Drosophila ESTs with similarity to vertebrate
1,6 fucosyltransferase, ß1,3 galactosyltransferase, ß1,4 galactosyltransferase, ß1,2 GlcNAc transferase (GlcNAc-TI), ß1,4 GlcNAc transferase, O-linked GalNAc transferase and O-linked GlcNAc transferase. The dependence of N-linked oligosaccharide diversity on GlcNAc-TI activity led us to look more closely at this particular Drosophila EST similarity. A single EST (Berkeley Drosophila Genome Project clot identifier 5653:1) with similarity to vertebrate GlcNAc-TI identifies a partial Drosophila cDNA (GenBank accession GM01211) that maps to a 65 kb stretch of sequenced Drosophila genomic DNA (GenBank accession 6437050). Within this stretch of genomic DNA lies a gene that predicts a protein product with 49% amino acid identity (70% similarity) to human GlcNAc-TI. Therefore, while the direct chemical demonstration of hybrid or complex oligosaccharides has not yet been achieved in Drosophila, new motivation for identifying such structures and new opportunities for studying the regulation of glycan synthesis will likely be provided by the expanded description of Drosophila synthetic and processing enzymes made available by completion of the genome sequence.
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Chemically defined structures |
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While an acidic component was detected, but not characterized, in preparations of larval membrane glycans, the identification of sialic acid in Drosophila embryos by lectin cytochemistry, immunohistochemistry and carbohydrate chemistry provides supportive evidence for protein sialylation (see above) (Williams et al., 1991; Roth et al., 1992
). The chemical identity of the sialic acid released from lyophilized embryo preparations by mild acid hydrolysis was confirmed to be N-acetylneuraminic acid by gas-liquid chromatographymass spectrometry. Overall, the emerging outlines of the glycoprotein glycan profile in Drosophila suggests that the majority of oligosaccharides are likely to be high-mannose structures. However, the presence of appropriate enzyme sequences within the genome and the sensitive detection of specific oligosaccharide moieties both leave open the possibility that more highly processed glycans are expressed in restricted amounts and in limited contexts.
Glycosphingolipids are enriched in N-acetylhexosamine residues frequently derivatized with phosphoethanolamine
The observation that glycosphingolipids are enriched in detergent-insoluble membrane microdomains prepared from vertebrate cells has now been extended to the cell membranes of Drosophila embryos. In the first characterization of Drosophila glycosphingolipid-enriched membrane rafts, glucosylceramide (GlcßCer) and Manß4GlcßCer demonstrated enrichments of ~3-fold over phospholipid and more complex glycolipids were also enriched although not characterized or quantitated (Rietveld et al., 1999).
Another study has characterized eight of 12 major glycosphingolipids, accounting for more than one-half of the ceramide-linked carbohydrate in Drosophila (Seppo et al., 2000). While glycosphingolipids with sialic acid are not detectable, both zwitterionic and acidic structures contribute to the total pool of glycosphingolipid oligosaccharide (Table II). Zwitterionic lipids possess phosphoethanolamine (PEtn) linked to one or more GlcNAc residues and comprise a family of serially related structures. The longest characterized zwitterionic glycolipid, an octaosylceramide, designated Nz28, has the structure GalNAcß,4(PEtn-6)GlcNAcß,3Galß,3GalNAc
,4GalNAcß,4(PEtn-6)GlcNAcß,3Manß,4GlcßCeramide. Heptaosyl- (Nz7), hexaosyl- (Nz6), pentaosyl- (Nz5) and tetraosyl- (Nz4) forms of Nz28, sequentially truncated from the non-reducing terminus, possess only one phosphoethanolamine moiety. The major acidic lipid, designated Az29, possesses two phosphoethanolamine moieties and a GlcA linked to a Gal-extended Nz28. Two other acidic glycolipids, Az9 and Az6, exhibit one phosphoethanolamine moiety and the same hexose and N-acetylhexosamine composition as Az29 and Nz6, respectively.
The fully extended Drosophila glycolipid core oligosaccharide differs from that of other dipterans in the linkage at a single glycosidic bond, a distinction implying structural and biosynthetic consequences (Wiegandt, 1992; Seppo et al., 2000
). Furthermore, acidic species account for a larger proportion of the total glycosphingolipid pool and phosphoethanolamine substitution of GlcNAc is more complete in the Drosophila embryo than in the glycolipids characterized from the larvae and pupae of other dipterans. The extent to which these divergent characteristics reflect interspecies variation between dipterans or stage-specific glycosphingolipid expression during dipteran development remains unknown.
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Concluding remarks |
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The lead phenotypes described in this review promise to provide a crack in the dam, potentially initiating a torrent of insight into carbohydrate expression and function. Armed with new genes and new hypotheses, Drosophila glycobiology can utilize techniques developed over the last two decades that increasingly permit targeted assessment of gene function. Some of the more powerful strategies provide for the spatial and temporal control of transgenic expression, the analysis of otherwise lethal mutations in mosaic tissue patches and the generation of targeted loss-of-function by RNA interference or P-element excision (Spradling and Rubin, 1982; Xu and Rubin, 1993
; Brand et al., 1994
; Kennerdell and Carthew, 1998
; Spradling et al., 1999
). The full development of glycobiology in Drosophila has, however, been stunted by the lack of characterized glycan structures. This deficit is being remedied one oligosaccharide at a time as analytic techniques of increasing sensitivity become available. Completion, annotation, and analysis of the Drosophila genome, coupled to continued elucidation of the organisms glycome, promises to significantly catalyze further understanding of carbohydrate structure and function.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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