Distinct contributions of β4GalNAcTA and β4GalNAcTB to Drosophila glycosphingolipid biosynthesis
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- Stolz, A., Haines, N., Pich, A. et al. Glycoconj J (2008) 25: 167. doi:10.1007/s10719-007-9069-5
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Drosophila melanogaster has two β4-N-acetylgalactosaminyltransferases, β4GalNAcTA and β4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcβ,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that β4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the β4GalNAcT trisaccharide acceptor structure GlcNAcβ,3Manβ,4GlcβCer, are lethal. In contrast, flies doubly mutant for the β4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in β4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcβ,3Manβ,4GlcβCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the β4GalNAcT single mutants we show that β4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In β4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in β4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that β4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both β4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcβ,3Manβ,4GlcβCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.
The Drosophila melanogaster genome encodes three members of the β1,4galactosyltransferase (β4GalT) family . In mammals seven β4GalT homologs exist; six of these catalyse the formation of lacNAc (Galβ,4GlcNAc) on various glycolipid and glycoprotein acceptor structures [2, 3]. The remaining galactosyltransferase acts on xylose residues and is involved in glycosaminoglycan linker region biosynthesis . An ortholog of this enzyme catalyses the same reaction in Drosophila [5, 6]. The two other Drosophila β4GalT family members have no correlation to a specific mammalian enzyme and have been shown to encode N-acetylgalactosaminyltransferases (GalNAcTs) that synthesise the lacdiNAc (GalNAcβ,4GlcNAc) structural element [1, 7, 8]. Both enzymes are typical type II transmembrane proteins, but only β4GalNAcTA has been found to have clear activity in vitro [1, 7]. β4GalNAcTB, on the other hand, requires a cofactor for optimal activity  (manuscript in preparation).
The lacdiNAc structural element is found in mammalian glycans, but restricted to a very limited number of proteins and synthesised by protein specific GalNAc transferases  only distantly related to the β4GalT family [10, 11]. In invertebrates, lacdiNAc is more abundant and found on both glycoproteins and glycolipids . In Caenorhabditis elegans, both glycoproteins and glycosphingolipids (GSLs) carry the disaccharide structure [13, 14], which is synthesised by Ceβ4GalNAcT, able to catalyse the transfer of GalNAc on both types of glycoconjugates . Several insect species have lacdiNAc containing N-glycans [16, 17] and enzymes able to act on glycoprotein acceptors have been identified [18, 19]. In contrast, in Drosophila, lacdiNAc has, despite intensive analyses of glycoproteins , only been found on GSLs of the arthro-series , which represent the common glycolipid series of arthropods and nematodes. These GSLs are characterised by a core structure with mannose linked to glucosylceramide [22, 23]. LacdiNAc occurs in the initial GSL structure, GalNAcβ,4GlcNAcβ,3Manβ,4GlcβCer and can also be found in more elongated structures .
The biological function of glycolipids in Drosophila has been demonstrated by mutants lacking the mannosyltransferase (egghead, egh) or the GlcNAc transferase (brainiac, brn) [24–27]. These mutants have very similar lethal developmental phenotypes and show defects in epithelial morphogenesis during oogenesis and embryogenesis [28, 29]. In contrast to this, mutants in the β4GalNAc transferases, show non-lethal, rather mild behavioural and morphological phenotypes, differing for the two transferases [1, 30, 31]. Drosophila mutants for β4GalNAcTA display an abnormal locomotion phenotype, indicating a role for this enzyme in the neuromuscular system [1, 31], whereas a small proportion of homozygous β4GalNAcTB mutant flies exhibit abnormal oogenesis due to defective epidermal growth factor receptor signalling between the oocyte and follicle cells . Flies doubly mutant for the β4GalNAcTs are viable  and these flies can be maintained as a homozygous stock over many generations indicating fertility is not significantly compromised (N. Haines and K.D. Irvine, unpublished). The lethality of egh and brn mutants compared to the viability of β4GalNAcT double mutants suggests two alternative possibilities: that the essential functions of GSLs can be fulfilled by the trisaccharide GSL structure, or that the β4GalNAcTs do not function in GSL synthesis and that this role is carried out by additional uncharacterized enzymes.
To resolve this issue and to determine the contribution of the two different β4GalNAcTs for extending the GSL trisaccharide in Drosophila we have carried out an analysis of GSL structures generated in the single and double β4GalNAcT mutants. We find that in double mutants no GSLs larger than the trisaccharide product of Brainiac are synthesised, demonstrating that the β4GalNAcTs are indeed required for GSL synthesis in vivo. We find that β4GalNAcTB is the prominent enzyme for lacdiNAc formation on glycolipids: the β4GalNAcTB mutant shows a reduction in lacdiNAc containing structures with the accumulation of GalNAc transferase acceptor structures. In contrast, the GSL profile of the β4GalNAcTA mutant is essentially identical to that of the wild-type flies.
2 Material and methods
2.1 Extraction, purification and preparation of Drosophila GSLs
Drosophila melanogaster flies of wild-type strain (Oregon R) and knock out strains β4GalNAcTA4.1, β4GalNAcTBGT and the double mutant β4GalNAcTA4.1; β4GalNAcTBGT  were collected and frozen. 1,5 g of frozen material were extracted by the method of Folch . Therefore, the flies were disrupted in a Dounce homogenizer in 3 vol (4 ml per g wet weight) of ice-cold, deionized water. After sonification of the suspension 4 vol of methanol were added and again homogenized, followed by the addition of 8 vol of chloroform, homogenization and sonification. The 8:4:3 (chloroform/ methanol/ water) extract was vigorously shaken and then centrifuged to remove insoluble material. After centrifugation the upper phase was collected, dried under a nitrogen-stream and re-dissolved in 3:47:48 (chloroform/methanol/water). Salt and hydrophilic contaminants were removed from extractions by reverse-phase chromatography (Sep-Pak® Plus C18 columns, Waters Corporation, Milford, MA, USA) . The column was equilibrated with 5 column volume of 3:47:48 and the sample was applied. Subsequently, the column was washed twice with 10 column volumes of water. The glycolipids were eluted with 10 column volumes of 10:10:1 and dried under a nitrogen-stream. For further analysis the samples were dissolved in chloroform/methanol/water (30:60:8).
2.2 High-performance thin-layer chromatography
Glycolipid preparations corresponding to 50 mg of flies were spotted onto nanosilica-gel 60 plates (Nano-Durasil-20, Macherey-Nagel, Düren, Germany) and developed in running solvent composed of chloroform/methanol/0.25% aqueous KCl (5:4:1). GSLs were visualised chemically by 0.5% orcinol(w/w)/62.5% methanol/10% H2SO4-staining.
2.3 Mass spectrometry
Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of extracted GSLs was performed on an Ultraflex II TOF/TOF MS (Bruker Daltonics, Bremen, Germany) in the reflector negative-ion mode using 2,5-dihydroxybenzoic acid (20 mg/ml in 30% acetonitrile; Bruker) as matrix and in the reflector positive-ion mode using 6-aza-2-thiothymine (5 mg/ml in water; Sigma) as matrix for sample preparation. Fragment-ion spectra were acquired by laser-induced decay in the LIFT mode as described previously .
3.1 Small glycospingolipids accumulate in the β4GalNAcTB and β4GalNAcT double mutant
3.2 GSL structures from wild-type and β4GalNAcTA mutant are identical
Registered GSLs species
Registered mass (m/z)
Besides the zwitterionic GSLs with PE-moieties, wild-type and β4GalNAcTA mutant exhibited acidic and neutral GSLs, which were likewise analyzed by MALDI-TOF/TOF-MS (data not shown). A group of glucuronic acid-containing GSLs were detected (Table 1), which is in accordance with previous results for Drosophila and other dipterans [21, 22]. Furthermore, the neutral GSLs GalNAcα,4GalNAcβ,4GlcNAcβ,3Manβ,4GlcβCer (N5) and GlcNAcβ,3Galβ,3GalNAcα,4GalNAcβ,4GlcNAcβ,3Manβ,4GlcβCer (N7) were registered (Table 1).
3.3 Precursor structures accumulate in β4GalNAcTB and β4GalNAcTA/TB double mutants
The analysis of GSLs structures in Drosophila β4GalNAcTA and β4GalNAcTB mutants show that in the absence of both enzymes GSLs are not further extended after the transfer of GlcNAc by Brainiac. This result demonstrates that the two described β4GalNAc transferases are the only enzymes able to catalyze the synthesis of lacdiNAc on Drosophila glycolipids. Furthermore, the accumulated precursor structure carries no PE modification on the terminal GlcNAc residue, suggesting that PE is transferred co-ordinately with the transfer of GalNAc or after the action of the GalNAc transferases. The detection of the neutral GSL species GalNAcα,4GalNAcβ,4GlcNAcβ,3Manβ,4GlcβCer (N5) and GlcNAcβ,3Galβ,3GalNAcα,4GalNAcβ,4GlcNAcβ,3Manβ,4GlcβCer (N7) in wild-type flies and both single mutants indicates that modification of GlcNAc with PE probably occurs independently, after the transfer of GalNAc.
In this study a new GSL structure, related to the octasaccharide with two PE groups, having an additional branching hexose, was detected. Branched structures have not been described in insects, but based on characterized GSL structures in C. elegans  the hexose residue could be a β6-linked glucose residue. Alternatively, one of the Drosophila β3-galactosyltransferases acting on GalNAc residues  could branch the GSL structure in this position.
The loss of complex GSLs in the β4GalNAcT double mutant flies suggests that extended glycan structures with and without terminal glucuronic acid residues are of limited importance for Drosophila development. The absence (in the double mutant) results in a mild phenotype , at least compared to the lethal phenotype of brainiac and egghead mutants . Considering these differences, the trisaccharide ceramide product of Brainiac by itself is already sufficient to prevent the occurrence of the brainiac phenotype.
Nevertheless, β4GalNAcTA mutants show defects in behaviour and in the neuromuscular system [1, 31], whilst a small proportion of β4GalNAcTB mutants display a defect in epithelial morphogenesis . These different phenotypes indicate that the enzymes have distinct functional roles.
We have found that glycolipid biosynthesis in the β4GalNAcTA mutant is not significantly different from wild-type. However, as elongated GSLs are still detectable in the β4GalNAcTB single mutant, but not in the double β4GalNAcTA; β4GalNAcTB mutant, β4GalNAcTA seems able to transfer GalNAc to GSL and may contribute to the generation of these structures in wild-type flies. The phenotypes ascribed to the β4GalNAcTA mutants could therefore be due to loss of GSL structures. Perhaps β4GalNAcTA plays a role in generating GSLs in only a limited number of cells rather than significantly contributing to the overall glycolipid profile of the complete fly. Arguing against this is the finding that both β4GalNAcTA and β4GalNAcTB are widely expressed in Drosophila  suggesting that β4GalNAcTA function is unlikely to be defined by its expression pattern. Another explanation would be that the main function of β4GalNAcTA is not in glycolipid biosynthesis but in the generation of a structure thus far not identified.
β4GalNAcTB can correct the behaviour phenotype of the β4GalNAcTA mutant when ubiquitously expressed . This suggests that β4GalNAcTB is capable of generating the structures that are normally synthesised by β4GalNAcTA and its failure to do so in the β4GalNAcTA mutant is because it is not expressed in the required cells. Although β4GalNAcTB clearly plays a major role in glycolipid synthesis it remains uncertain if the ability of this enzyme to rescue the β4GalNAcTA phenotype is due to its ability to generate lacdiNAc-containing GSLs. On the other hand, over-expression of β4GalNAcTA cannot completely overcome the β4GalNAcTB mutant phenotype . This correlates with our observation that in the β4GalNAcTB mutant the overall GSL biosynthesis defect is much more severe than in the β4GalNAcTA mutant. This may indicate that β4GalNAcTA is a less efficient in glycolipid biosynthesis than β4GalNAcTB.
Both β4GalNAcTs show genetic interaction with egghead and brainiac , the phenotypes of both mutants become more severe in combination with a double egghead/brainiac heterozygotes. This suggests that these enzymes all contribute to the generation of the same glycan structure in Drosophila and that the mutant phenotypes arise directly from loss of GSL. However, β4GalNAcTA and β4GalNAcTB are not functionally exchangeable and, considering the minor role we have identified for β4GalNAcTA in generating GSL structures, it thus remains possible that these enzymes synthesize lacdiNAc in other glycoconjugates than GSLs; loss of such structures could underlie aspects of the mutant phenotypes.