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J. Biol. Chem., Vol. 276, Issue 30, 28058-28067, July 27, 2001

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Identification of Core 1,3-Fucosylated Glycans and Cloning of the Requisite Fucosyltransferase cDNA from Drosophila melanogaster

POTENTIAL BASIS OF THE NEURAL ANTI-HORSERADISH PEROXIDASE EPITOPE^*

Gustáv Fabini, Angelika Freilinger, Friedrich Altmann, and Iain B. H. Wilson

From the Glycobiology Division, Institut für Chemie, Universität für Bodenkultur, Muthgasse 18, A-1190 Wien, Austria

Received for publication, January 22, 2001, and in revised form, May 29, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core 1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both 1,3- and 1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding 1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core 1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core 1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF⁶; K_m 11 µM) over the same structure lacking a core fucose residue (GnGn; K_m 46 µM). The Drosophila core 1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycoproteins from plants and invertebrates are often highly immunogenic and many antibodies (both IgG and IgE) directed against them bind to core 1,3-fucose and/or 1,2-xylose residues of their N-linked oligosaccharides (1-7); since these modifications are present across plant species and one or the other modification is present in a number of invertebrates, these antibodies are highly cross-reactive. Indeed, polyclonal antibodies to horseradish peroxidase (anti-HRP)¹ have been used for nearly two decades to specifically stain neurons, and their growth pathways, in Drosophila melanogaster (8-11); similar staining has also been described in grasshopper (11), whereas in Caenorhabditis elegans 10% of neurons are stained by this antibody (12). A number of proteins in Drosophila, such as an Na⁺,K⁺ATPase christened Nervana, a receptor tyrosine phosphatase, and cell adhesion molecules (e.g. fasciclin I and II, neurotactin and neuroglian), have been found to bind anti-HRP (9-11, 13, 14); however, no data on their glycosylation have been described. A number of Drosophila nac (neurally altered carbohydrate) mutants have been described, which are defective in anti-HRP staining of adult flies and display wing morphological defects (15) and, when under cold stress, some minor behavioral and eye developmental abnormalities (9). Neither the affected gene(s) nor the underlying biochemical defect(s) have been identified.

As compared with the amount of information known on the genetics and genome of Drosophila, relatively little is known about the structures of its glycoconjugates; there exists one report from 1991 on the N-linked oligosaccharides of larval membrane glycoproteins in which only data on oligomannosidic and core 1,6-fucosylated N-glycans and no data on glycan structures that could explain anti-HRP binding were presented (16). However, subsequently, core 1,3-linked fucosylated glycans were found in insects (e.g. on bee venom glycoproteins) (17-19). Thus, since 1,2-xylose has to date not been found in this class of organism, we still considered it reasonable to hypothesize that anti-HRP binding in Drosophila was due to core 1,3-fucosylation of N-linked oligosaccharides.

In the present paper, we report the specific inhibition of anti-HRP staining of Drosophila embryonic nervous system by a neoglycoconjugate carrying core 1,3-fucose. We also describe the first analysis of the N-glycans of adult flies to be published and demonstrate the presence of N-glycans carrying both core 1,3- and 1,6-linked fucose. Furthermore, we describe the isolation of a Drosophila cDNA encoding a fucosyltransferase activity that creates in vitro an epitope for anti-HRP antibody.²

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Immunostaining of Whole-mount Drosophila Embryos-- D. melanogaster embryos (Canton S) were collected and staged at 25 °C. After dechorionation in 50% bleach for 3 min, the embryos were thoroughly rinsed in water and fixed for 20 min in 4% paraformaldehyde in PBS/n-heptane (1:1), followed by devitellinization in n-heptane/methanol (1:1). Fixed embryos were washed three times with methanol and then equilibrated in 0.1% Triton X-100 in PBS. The embryos were blocked at 4 °C overnight in 0.1% Triton X-100, 0.1% BSA, and 5% normal goat serum in PBS and incubated with rabbit anti-HRP (1 µg/ml, Sigma) or anti-bee venom antibodies (20 µg/ml, Sigma) for 90 min at room temperature. After vigorous washing, embryos were probed with FITC-conjugated (1:200, Sigma) or biotin-conjugated goat anti-rabbit IgG (1:300, Vector Laboratories, CA). In the latter case, the embryos were stained using a Vectastain Elite peroxidase kit with diaminobenzidine as a substrate (0.07 mg/ml) in the presence of 0.012% hydrogen peroxide, mounted in 70% glycerol (v/v) in PBS, and visualized by light microscopy. FITC-labeled samples were observed by using a Bio-Rad MRC 600 confocal scanning microscope with 488 nm excitation.

For anti-HRP staining inhibition studies, fucosylated and chemically defucosylated bromelain glycopeptides cross-linked to bovine serum albumin (BSA-MUXF³ and BSA-MUX),² respectively, were prepared as described (6) and estimated to contain 0.1 nmol of GlcNAc/µg of conjugate (equivalent to an average of 3.5 glycans incorporated/BSA molecule). The chemical defucosylation to yield MUX glycopeptide was estimated by sugar content analysis to have been 95% effective. These neoglycoconjugates in the concentration range 0.004-40 µM in terms of GlcNAc (or 0.04-400 µg/ml) were allowed to preabsorb the rabbit anti-HRP antibodies (1 µg/ml) for 1 h at room temperature before applying as primary antibody in whole-mount embryo stainings.

Preparation of N-Glycans from Adult Flies-- Three grams of frozen whole flies (Canton S strain) were denatured in 20 ml of stirred boiling water (10 min). After cooling, formic acid (to attain a final concentration of 5% (v/v)) and 1 mg of pepsin were added. Proteolysis was allowed to proceed overnight at 37 °C. The resulting extract was centrifuged; the pellet was washed and the washings added to the first supernatant. The supernatant was applied to a Dowex AG50W×2 column (1.5 × 50 cm), and the column was washed with 50 ml of 2% (v/v) acetic acid. Glycopeptides were eluted with 0.4 M ammonium acetate, pH 6, and orcinol-positive fractions were pooled and concentrated prior to gel filtration (Sephadex G25 medium; elution with 1% (v/v) acetic acid). The resultant glycopeptide fractions were lyophilized and dissolved in 250 µl of 100 mM citrate-phosphate, pH 5, and incubated at 95 °C for 10 min to inactivate any remaining proteases. The N-glycans were released using peptide:N-glycosidase A (37 °C, 24 h). The sample was then acidified by adding two volumes of 5% (v/v) acetic acid and applied to a 2-ml Dowex AG50W×2 column. The column was washed with 2% (v/v) acetic acid, and the unretained orcinol-positive fractions were subject to gel filtration (Sephadex G15; elution with 1% (v/v) acetic acid) and then to reversed-phase chromatography (200 µl of Lichroprep; elution with 5% (v/v) acetic acid). The material retained on the 2-ml Dowex column was subject to a second round of peptide:N-glycosidase A digestion, in order to release remaining anti-HRP binding components. Lyophilized N-glycans were dissolved in water and subject to either MALDI-TOF analysis (see below) or pyridylamination (17, 20, 21). The final yield of N-glycans was estimated as being 10-15 nmol, as judged by determination of the amino sugar content (22) and assuming two GlcNAc residues/N-glycan.

Enzyme-linked Immunoassays of Drosophila Peptides-- An aliquot (1%) from each stage of the (glyco)peptide and glycan purification was removed and diluted 1:100 in water. Equal amounts of diluted (glyco)peptide or glycan (0.5 mg/ml bromelain glycopeptides were used as a control) and a freshly prepared aqueous solution containing 0.5 mg/ml each of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysucciminde were added to wells of Cova-Link ELISA strips (Nunc; 50 µl of each solution/well). The strips were incubated for 90 min at 37 °C and then blocked and incubated with antibodies as previously described (6), except that 1:2500 (~7 µg/ml) anti-HRP or anti-bee venom were used as primary antibodies.

Matrix-assisted Laser Desorption-Ionization Mass Spectrometry-- Aliquots of 0.8 µl of underivatized or pyridylaminated N-glycans were applied to a flat sample platen and dried immediately under mild vacuum, followed by addition of 1 µl of matrix (2% (w/v) 2,5-dihydroxybenzoic acid in 30% (v/v) acetonitrile or 0.03 M 1-hydroxyisoquinoline, 0.1 M 2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile) and drying once more (23, 24). MALDI-TOF mass spectra were acquired on a Dynamo (Thermo BioAnalysis, Hemel Hempstead, United Kingdom) linear time-of-flight mass spectrometer with a dynamic extraction setting of 0.1. External mass calibration was performed with pyridylaminated N-glycans or with a partial dextran hydrolyzate. On-plate -fucosidase digestion was performed using 0.2 milliunits of bovine kidney fucosidase in 10 mM ammonium acetate, pH 5, with incubation in a "wet chamber" at 37 °C.

Reversed-phase HPLC of Pyridylaminated N-Glycans-- After crude gel filtration to remove excess 2-aminopyridine, pyridylaminated oligosaccharides were fractionated by reverse-phase chromatography on an ODS column (0.4 × 25 cm), based on a previously published method (17, 21), at a flow rate of 1.5 ml/min. The starting buffer was 0.1 M ammonium acetate, pH 4.0, and a gradient increasing at 1% per min 30% (v/v) methanol was applied. Columns were calibrated daily in terms of glucose units with a pyridylaminated partial dextran hydrolysate (3-20 glucose units). Selected fractions, or the entire N-glycan pool, were subject to exo- or endoglycosidase digestions as follows: Canavalia ensiformis (jack bean) -mannosidase (200 milliunits in 20 µl of 50 mM sodium acetate, 0.1 mM zinc chloride, pH 4.2), C. ensiformis -hexosaminidase (5 milliunits in 20 µl of 0.1 M sodium citrate, pH 5.0), bovine kidney -fucosidase (4 milliunits in 20 µl of 0.1 M sodium citrate, pH 5.0), and endoglycosidase H (2 milliunits in 20 µl of 0.1 M citrate-phosphate, pH 5.0).

Cloning and Expression of cDNAs Encoding Drosophila Fucosyltransferase Homologues-- Sequences encoding two putative 1,3-fucosyltransferases, FucTA (Flybase entry CG6869 mapped to 3L; 71B2-3) and FucTB (CG4435 mapped to 2L; 30B5), were found by homology searching of the assembled gene products in the Drosophila genome data base (www.flybase.org). The open reading frame for FucTC was assembled from sequenced genomic fragments (nucleotides 10042-11851 of GenBank^TM accession no. AE003066; as yet unmapped) using the GeneMark program available at the EMBL Outstation homepage (www.ebi.ac.uk). The cDNAs encoding soluble forms of three putative fucosyltransferases (amino acid positions FucTA: 29-503; FucTB: 26-444; FucTC: 56-425) were amplified from D. melanogaster (wild type strain Canton S) total RNA by RT-PCR. The primers were designed with relevant restriction sites (in bold) at the 5' ends for cloning as follows: FucTA, 5'-CGGGAATTCAAGGAGCGCGAAATATGGAAG-3' and 5'-CCGGGGTACCTCAGTCGTCGCTGGAGTCG-3'; FucTB, 5'-CGCGCTGCAGGATCGGAAAATATCATTAACTACG-3' and 5'-CCGGGGTACCTTAAGTATTTGAACTATTACTGC-3'; FucTC, 5'-CGGGAATTCAAAGTCACTCAGTCACCGC-3' and 5'-CCGGGGTACCTCACAAACGTATTCGGCTTTG-3'.

EcoRI-KpnI-digested PCR products for FucTA and FucTC and the PstI-KpnI-digested PCR fragment for FucTB were cloned into pPICZ expression vectors (Invitrogen, The Netherlands). The integrity and reading frames of the constructs, as well as 5' and 3' ends of full-length RT-PCR products, were confirmed by DNA sequencing using an ABI PRISM Big Dye Terminator Sequencing Ready Mix and ABI PRISM 310 genetic analyzer (PerkinElmer Life Sciences, Applied Biosystems). Nucleic acid sequences were analyzed using the DNAStar suite of programs and NCBI BLAST and NCBI BLAST2 programs (www.ncbi.nlm.nih.gov).

Plasmid DNA (10 µg) was then linearized with MssI or SacI (MBI Fermentas, Germany) prior to transformation by electroporation into Pichia pastoris GS115 according to instruction manual version C (Invitrogen). Transformants were screened for integration into the Pichia genome by PCR using AOX1-specific primers. On average, 5-10 selected positive transformants were grown overnight to A₆₀₀  14 at 30 °C in 5 ml of BMGYC medium (100 mM potassium phosphate, pH 6.0, 1% (w/v) yeast extract, 2% (w/v) peptone 140, 1% (w/v) casamino acids, 1.34% (w/v) yeast nitrogen base, 4 × 10⁵% (w/v) biotin, 1% (v/v) glycerol). The cells were washed once with 1.34% (w/v) yeast nitrogen base and then resuspended in 10 ml of BMMYC medium (composition as for BMGYC, except that 1% (v/v) methanol substitutes for glycerol) for induction of expression. Over a 72-h period, aliquots of supernatant were removed every 24 h and supplementary methanol added to maintain a final concentration of 1% (v/v).

Assay of Fucosyltransferase Activity-- Culture supernatants were concentrated 10-fold using Vivaspin concentrators (Vivascience, Gloucestershire, United Kingdom), the buffer replaced with 50 mM MES, pH 6.0, 5 mM MnCl₂ and then assayed for core 1,3-fucosyltransferase activity using dabsyl-GnGn (see Fig. 1 for an explanation of oligosaccharide structures), which is a dabsylated tetrapeptide with the sequence Gly-Glu-Asn-Arg derived from Pronase digestion of asialo-agalacto bovine fibrin. The standard 20-µl assay mixture, based on that described previously (25), consisted of 5 µl of 10-fold concentrated supernatant and final concentrations of 100 mM MES, pH 6.0, 1 mM AMP, 50 mM N-acetylglucosamine, 1 mM GDP-Fuc, 10 mM MnCl₂, and 0.05 mM dabsyl-GnGn. After incubation for 5 or 24 h at 37 °C, an aliquot of the assay mixture was diluted 10-fold with water. Then, 0.8 µl of diluted assay mixture was mixed on the sample platen with 0.8 µl of 1% (w/v) -cyanohydroxycinnaminic acid in 70% (v/v) acetonitrile and allowed to dry. MALDI MS spectra were then acquired with external mass calibration performed with a mixture of tryptic peptides derived from the peanut allergen Ara h 1. 

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Structures of N-glycans referred to in this study.

The enzyme was also assayed using dansyl-GnGn or dansyl-GnGnF⁶, which are dansylated dipeptides (Asn-Ser) derived from IgG carrying an asialoagalacto-N-glycan with or without prior fucosidase digestion (26). Samples were injected after incubation onto an RP-HPLC column (5-µm Hypersil ODS, 4 × 250) and eluted isocratically in 9% acetonitrile buffer in 50 mM potassium phosphate, pH 2. The glycopeptide substrate and product were detected by fluorescence. Elution positions were compared with those of dansyl-GnGn left untreated, incubated with recombinant Arabidopsis core 1,3-fucosyltransferase (27) (generating standard GnGnF³), or incubated with an extract of chicken heart containing 1,6-fucosyltransferase activity (prepared as described in Ref. 28 in order to generate GnGnF⁶). The exact concentration of acceptor substrate was determined by analysis of the GlcNAc and amino acid content (22).

In addition, dansyl glycopeptides derived from IgG were galactosylated in vitro with bovine milk 1,4-galactosyltransferase to generate GalGal and GalGalF⁶. Dansylated MM and MMF⁶ glycopeptides were generated by hexosaminidase treatment of GnGn and GnGnF⁶ derived from IgG. The subsequent fucosyltransferase assays were performed as follows; 2 µl of concentrated FucTA supernatant was incubated with 0.2 nmol of dansylated glycopeptide and 10 nmol of GDP-Fuc in a final volume of 20 µl. For K_m determinations, dansyl glycopeptides derived from IgG (GnGn and GnGnF⁶) were used with 2 µl of concentrated FucTA supernatant (16-h incubation time).

Preparation and Analysis of Fucosylated Transferrin-- Human transferrin (5 mg; Sigma) was digested with 0.2 units of sialidase from Clostridium perfringens (Sigma) in 1 ml of 50 mM sodium acetate, pH 5.0, at 37 °C overnight. After addition of 2 units of -galactosidase from Aspergillus oryzae (29), the sample was incubated overnight to yield "GnGn-transferrin." Fucosylation of the glycomodified transferrin was performed as described above for glycopeptide, except that 10 µg of GnGn-transferrin was used as the substrate and Triton X-100 was added to a final concentration of 0.08% (w/v). An aliquot containing ~3 µg of partially 1,3-fucosylated transferrin (termed "GnGnF³-transferrin") was then incubated with 4 µl of 50 mM sodium citrate, pH 5.0, and 1 milliunit of -N-acetylglucosaminidase from Streptococcus pneumoniae at 37 °C over night to obtain MMF³-transferrin. In all reactions a chicken heart extract possessing 1,6-fucosyltransferase activity, either heat-denatured (as a control) or native (to produce, e.g. MMF⁶-transferrin), was added.

Various transferrin glycopreparations were separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The nitrocellulose sheets were blocked for 1 h with 3% nonfat dried milk in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and subsequently incubated for 1 h with rabbit anti-horseradish peroxidase antibody (Sigma) diluted 1:2000 in blocking buffer. After washing the nitrocellulose membrane three times with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, bound antibodies were detected by alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma), followed by color detection using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate.

The N-glycans of the variously modified transferrin samples were analyzed by mass spectrometry. Briefly, 4 µg of protein were incubated with 0.2 µg of pepsin in 20 µl of 5% (v/v) formic acid overnight at 37 °C. The digest was dried in a Speed-Vac evaporator. Enzymatic release, clean-up, and analysis by MALDI-TOF MS of N-glycans were performed as described (23).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Re-evaluation of the Drosophila Anti-HRP Carbohydrate Epitope-- Previously published data on the characterization of plant epitopes reacting with anti-HRP antibody (3, 6), as well as data indicating that the plant glycoprotein bromelain inhibited anti-HRP binding to Drosophila (9), led us to hypothesize that core 1,3-fucose is the key element recognized by anti-HRP in neural tissue of the fruit fly. To test this we used neoglycoconjugates of bromelain glycopeptide cross-linked to bovine serum albumin, BSA-MUXF³ and BSA-MUX (6), to compete for binding to anti-HRP on Drosophila whole-mount embryo preparations. The core 1,3-fucosylated neoglycoconjugate BSA-MUXF³ had the capacity to inhibit binding of anti-HRP to the complete nervous system of Drosophila embryo (Fig. 2B), whereas the (at least 95%) defucosylated form (BSA-MUX) showed no or little inhibitory effect (Fig. 2C). The total abolition of neural staining with the anti-HRP antibody (1 µg/ml) was achieved at a concentration of 40 µg/ml BSA-MUXF³ (i.e. 4 µM in terms of GlcNAc).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Immunostaining of whole-mount Drosophila embryos. Whole-mount embryos were stained with either anti-HRP (A-D) or anti-bee venom (E) antibodies and examined by light microscopy using biotin-labeled secondary antibody (A-C) or by fluorescence microscopy using FITC-labeled secondary antibody (D and E). Laterally viewed embryos are oriented with anterior to the left and ventral to the bottom. In the preabsorption experiment (A-C), the anti-HRP staining of the complete embryonic nervous system (A) is totally blocked upon incubation of anti-HRP antibody with BSA-MUXF3 (B), whereas incubation with defucosylated neoglycoconjugate BSA-MUX (C) did not lead to a significant inhibition of the anti-HRP staining. Interestingly, anti-bee venom fluorescent staining (E) results in an apparently identical pattern as that with anti-HRP (D).

ELISA was also used to examine the interaction of adult Drosophila extract with anti-HRP. The antibody was preincubated with 2 µM (in terms of GlcNAc) of either BSA-MUXF³ or BSA-MUX; the percentage of blank-corrected inhibitions were respectively 85% and 30%. (In considering these figures, it should be borne in mind that 5% residual fucose was still present on BSA-MUX.) In addition, since free glycans are not able to cross-link to the Cova-Link strips, the demonstrated lack of an ELISA reading after peptide:N-glycosidase A digestion of Drosophila glycopeptides was concluded to indicate that the anti-HRP epitope was indeed present on the N-glycans prepared for the later structural analyses.

Since the N-glycan structures of two honeybee venom components previously elucidated (17, 19) indicated the presence of core 1,3-fucose (but not 1,2-xylose) and since an anti-bee venom phospholipase antiserum has been previously shown to be partly directed against N-glycan components cross-reacting with plant glycoproteins (4), we decided to also probe Drosophila whole-mount embryos with anti-whole bee venom antiserum. The immunofluorescence pattern of the nervous system is indistinguishable from that of anti-HRP, although somewhat less intense (Fig. 2, panels D and E). Also, ELISA indicated that the interaction of anti-bee venom with crude adult Drosophila extract, as well as peptic peptides, was 70% inhibited by 20 µg/ml BSA-MUXF³ (i.e. 2 µM in terms of GlcNAc content), whereas the interaction of anti-HRP with these extracts was 70% inhibited by 25 µg/ml bee venom phospholipase (also 2 µM in terms of GlcNAc).

Taken as a whole, these results suggest that core 1,3-fucose is a sugar moiety indispensable for the recognition of the Drosophila neural carbohydrate epitope by anti-HRP antibodies. This observation is strongly supported by the data reported below on the detection of core 1,3-fucosylated N-glycan structures in Drosophila and isolation of the requisite fly fucosyltransferase cDNA, the recombinant enzyme having the potential to modify in vitro N-glycan substrates lacking core 1,3-fucose and so create de novo the epitope recognized by anti-HRP antibody.

Analysis of Drosophila N-Glycans-- In order to test whether the fly contains core 1,3-fucosylated structures, we prepared N-glycans from adult Drosophila. Previously, the only published data on Drosophila N-glycans were acquired after hydrazinolysis of membrane glycoproteins from third instar larvae and indicated a full range of oligomannosidic structures, as well as some MMF⁶ (16). Our MALDI-TOF MS analysis of the glycans from adults (Fig. 3A and Table I) also shows the presence of oligomannose structures (Man₉, for instance, accounts for 20% of the total N-glycans), but in this case a simple glycan (m/z 1080.0) with one deoxyhexose residue (presumably fucose) is the major structure (36% of the total); low quantities of glycans with one additional GlcNAc residue, with and without fucose, were also detected. Most significantly, however, about 1% of the total N-glycans were found to carry two deoxyhexose residues. To examine their nature further, the N-glycans were subject to a number of analyses, including glycosidase digestion either of the entire underivatized N-glycan pool (with subsequent MALDI-TOF MS analysis) or of the entire pyridylaminated N-glycan pool (with subsequent RP-HPLC analysis), as well as fractionation by HPLC of the pyridylaminated N-glycans (Fig. 3B) and MALDI analysis of each HPLC fraction (see Table I).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Map of neutral N-glycans of adult Drosophila. A, MALDI-TOF spectrum of underivatized N-glycans (with annotated m/z values for [M + Na]+ ions, see Table I for interpretation); B, RP-HPLC chromatogram of pyridylaminated N-glycans (with annotated glucose units of the external standards and, in bold, fraction numbers). Peaks indicative of the presence of MUF3F6 and MMF3F6 are marked with an asterisk.

The major RP-HPLC fraction (fraction 12) contained a species with an m/z value indicative of a monofucosylated Man₃ structure. Digestion with bovine -fucosidase of the complete pool of pyridylaminated Drosophila glycans showed a shift in the RP-HPLC retention time of the major peak from 13 to 7.5 g.u. (the former, relatively late, retention time being in agreement with previous analyses of core 1, 6-fucosylated glycans (17-19), the latter being the same retention time as MM, the expected product). Similarly, on-plate -fucosidase digestion of the underivatized whole N-glycan pool with subsequent analysis by MALDI indicated a shift in the m/z value of the major peak from 1080.0 to 933.8, suggestive of the removal of one fucose residue. The MALDI, RP-HPLC, and digestion data, therefore, indicate that MMF⁶ is the major N-glycan in whole adult flies. Among the oligomannosidic structures, MALDI-TOF MS of the HPLC fractions demonstrated there were single isomers of Man₃, Man₄, Man₉, and a probable GlcMan₉ and two or three isomers each of Man₆, Man₇, and Man₈. The peaks containing Man_5-9 were sensitive to endoglycosidase H, as judged by digestion of the whole pyridylaminated N-glycan pool (data not shown).

As determined by MALDI-TOF MS, HPLC fraction 9 contained four structures with m/z values characteristic of Man₃, Man₄, and difucosylated forms of Man₂ and Man₃. Fraction 9 was therefore re-applied to reversed phase before and after -fucosidase digestion. From the undigested sample, it was possible to separate subfractions 9A and 9B (Fig. 4A, upper chromatogram), whereas, similarly to the results of -fucosidase digestion of the endoglycosidase-H treated entire pyridylaminated N-glycan pool, -fucosidase digestion caused a shift in the retention time of the peak corresponding to subfraction 9B from 8.0 g.u. to 5.0 and 5.2 g.u (Fig. 4A, lower chromatogram). The species at 5.0 and 5.2 g.u. have the same retention time as MMF³ and MUF³, structures previously analyzed by the same method in this laboratory from bee venom and ragweed pollen glycoproteins (17, 19, 21). Furthermore, whereas subfraction 9A contained Man₃ and Man₄ as judged by MALDI-TOF analysis, subfraction 9B contained species with m/z values compatible with the presence of MUF³F⁶ and MMF³F⁶ (Fig. 4B; respective m/z values of 1140.8 and 1303.4 as compared with theoretical values of 1142.1 and 1304.2). Since (a) the structures present in subfraction 9B have m/z values compatible with difucosylation, (b) previous data indicate that the core 1,3-linked fucose is relatively resistant to bovine -fucosidase, and (c) difucosylated structures from bee venom exhibit similar RP-HPLC behavior (17, 19), we conclude that ~1% of the N-glycans from adult Drosophila carry two core fucose residues (see Table I).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Analysis of fraction 9. A, RP-HPLC of fraction 9 and of -fucosidase-treated fraction 9 (showing glucose units of the external standards); B, MALDI-TOF spectrum of subfraction 9B.

Characterization of Recombinant Drosophila Core 1,3-Fucosyltransferase-- Searching the newly sequenced Drosophila genome with mammalian Lewis and plant core 1,3-fucosyltransferases indicated the presence of three homologous genes. The probable reading frames were analyzed and primers for RT-PCR were designed, and the entire open reading frames of FucTA, FucTB, and FucTC were isolated. Subsequently, cDNA fragments encoding the soluble forms of these proteins (i.e. excluding the predicted cytoplasmic and transmembrane domains) were expressed in the yeast P. pastoris. The media of the yeast cells were then assayed for fucosyltransferase activity by MALDI-TOF MS using dabsylated glycopeptides carrying GnGn (specific for core fucosyltransferases) or GalGal (i.e. GnGn with two non-reducing terminal 1,4-galactose residues; also suitable for Lewis^x-generating 1,3-fucosyltransferases) N-glycans as substrates (see Fig. 1 for structure of GnGn). In these studies, in which the appearance of new peaks 146 m/z units larger than substrate peak and its laser-induced breakdown product was considered indicative of the transfer of fucose, GnGn was found to be the most suitable substrate for one of the three novel 1,3-fucosyltransferase fly homologues, named FucTA (Fig. 5A). Some activity of FucTA toward GalGal was also detected; subsequent sequential -galactosidase and -N-acetylhexosaminidase digestion indicated that the fucose was transferred to the core and not to the antennae (data not shown). On the other hand, no activity was found in the media of FucTB or FucTC transformants with either of the aforementioned substrates. Interestingly, prefucosylation of GnGn with chicken heart extract (GnGnF⁶) resulted in a superior conversion of this substrate by FucTA than with GnGn (Fig. 5B), whereas no activity was detected for any of the three Drosophila fucosyltransferase homologues when disaccharides lacto-N-biose (Gal1,3GlcNAc; substrate for Lewis^a-generating enzymes) or N-acetyllactosamine (Gal1,4GlcNAc; substrate for Lewis^x-generating enzymes) were used as substrates in a radioactive assay using GDP-L-[¹⁴C]fucose. The activity in the medium of FucTA-transformed Pichia was ~10 milliunits/liter, as judged by assays using GnGnF⁶ as substrate.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Analysis of fucosyltransferase reaction products by MALDI-TOF and RP-HPLC. MALDI-TOF spectra of fibrin-derived dabsylated GnGn glycopeptide incubated in the presence of concentrated culture supernatant from P. pastoris expressing Drosophila FucTA either without (A) or with (B) prior incubation with chicken heart extract. RP-HPLC chromatograms of IgG-derived dansylated GnGn (C and D) and GnGnF6 (E and F) glycopeptides incubated in the presence (D and F) and absence (C and E) of concentrated culture supernatant from P. pastoris expressing Drosophila FucTA.

To examine the substrate preference of FucTA further, an RP-HPLC method using fluorescently-labeled dansylated (rather than dabsylated) GnGn and GnGnF⁶ glycopeptides derived from IgG as acceptors was employed. Under this method, as shown by a previous study (26) and by control experiments with recombinant Arabidopsis thaliana core 1,3-fucosyltransferase, the appearance of a new peak of lower retention is indicative of the addition of an 1,3-linked fucose residue (Fig. 5, C-F; compare chromatograms C and E with the respective chromatograms D and F after incubation with the recombinant Drosophila enzyme). Again, since the concentration of the glycopeptide acceptors was identical, the results of these assays suggested a preference of the enzyme for GnGnF⁶ over GnGn (compare F with D). More exactly, when the acceptor concentration was varied and the results analyzed by means of a Hanes' plot, the K_m values for GnGnF⁶ and GnGn were found to be 11 and 46 µM, respectively, suggestive of a preference for the 1,6-fucosylated acceptor (see also Table II). In addition, a variety of dansylated galacto-, agalacto-, and ahexosaminylglycopeptides from IgG were tested as substrates. Galactosylated variants were either generated from asialofibrin or by galactosylation in vitro of IgG glycopeptides to yield GalGal and GalGalF⁶, and the percentage conversion was compared by analysis of RP-HPLC peak areas. The comparisons show that 1,6-fucosylated glycans were better, and 1,4-galactosylated glycans worse, substrates. On the other hand, glycopeptides lacking any terminal GlcNAc residues (i.e. carrying MM or MMF⁶) were not substrates at all. It was also demonstrated that, for GnGn or GalGal, the nature of the peptide portion (IgG or fibrin) of the dansylated glycopeptide had no effect on the percentage conversion (data not shown).

Complete sequencing of the cDNA encoding FucTA indicated that this enzyme is a protein with a predicted length of 503 amino acids (Fig. 6) with a putative N-terminal transmembrane domain (residues 11-28), a feature well known in other Golgi glycosyltransferases. A comparison with the annotation of Flybase entry CG6869 indicates that our FucTA cDNA is shorter than predicted, due to the presence of an extra mini-intron of 66 nucleotides. Other small changes encountered between the nucleotide sequences of the experimentally obtained and predicted cDNAs may of course be due to differences in the strain of Drosophila used. Compatible with the enzymatic data, alignments (Fig. 7) indicate that FucTA is probably more closely related to plant core 1,3-fucosyltransferases (e.g. as judged by the presence of a AXF(I/V)SNCXARNXRLQ motif and its greater size, 503 residues) than FucTB and FucTC (which are characterized also by shorter sequences of 444 and 425 residues, respectively, and may be closer to the mammalian Lewis-type 1,3/4-fucosyltransferases). The overall identity of Drosophila homologues with mammalian and plant 1,3-fucosyltransferases is not significantly high (about ~ 20%); however, comparisons of the regions of homology displayed in Fig. 7 revealed, on average, 32% amino acid identity. Typical 1,3-fucosyltransferase motifs (I/V)DXYG, YKFXLAFENS, DY(I/V)TEK, and CXXC as described previously (30, 31) were detected in all aligned sequences. Interestingly, FucTA has a unique N terminus (up to residue 163 of 503) displaying no homology to FucTB, FucTC, plant, or mammalian fucosyltransferases. In contrast, other than the cytoplasmic, transmembrane, and stem regions, which tend to be divergent anyway, it is the C-terminal region of plant core fucosyltransferases that accounts for their greater length.

View larger version (63K): [in this window] [in a new window]   Fig. 6.   cDNA and protein sequence of D. melanogaster core 1,3-fucosyltransferase (FucTA). The nucleotide and deduced amino acid sequence comprising 503 amino acids of FucTA is shown. The putative transmembrane domain (residues 17-28) is underlined. Consensus asparagine-linked glycosylation sites are double underlined, and splice sites are in bold.

View larger version (58K): [in this window] [in a new window]   Fig. 7.   Alignment of FucTA with other Drosophila, plant and mammal FucTs. Conserved amino acid residues are depicted as black boxes. Dashed lines represent gaps required for alignment of the sequences. In addition to the typical 1,3-fucosyltransferase consensus sequences YKFXLAFENS, DY(I/V)TEK, and CXXC found in all homologues, a long stretch of identity AXF(I/V)SNCXARNXRLQ, which may be characteristic for core 1,3-fucosyltransferases is also noted.

Anti-HRP Binding of 1,3-Fucosylated Glycoproteins-- In order to probe anti-HRP binding of the MMF³F⁶ structure more closely, we decided to create de novo the same structures as found in the course of this study in fly in a protein-bound form suitable for Western blotting. Human transferrin was chosen as a carrier for generation of the epitope in vitro, since it has predominantly simple biantennary N-glycans attached at two different asparagine residues, which after desialylation and degalactosylation are suitable substrates for core fucosyltransferase, and since it is a protein found neither in plants nor insects. Even though the properties of anti-HRP have been much studied in the past using plant or insect glycoproteins (6, 32), neoglycoconjugates (6), or, at relatively high concentration, pyridylaminated HRP oligosaccharides (3, 5), all these studies have relied on use of chemical or enzymatic treatments to destroy the epitope rather than show that a recombinant enzyme can create it.

Using recombinant Arabidopsis core 1,3-fucosyltransferase and chicken heart extract (possessing 1,6-fucosyltransferase activity) as controls, we could demonstrate that human asialoagalacto-transferrin (GnGn-transferrin) is modified in vitro by the Drosophila enzyme to generate singly fucosylated (GnGnF³) and, in combination with chicken heart extract, doubly fucosylated N-glycans (GnGnF³F⁶). With subsequent -hexosaminidase digestion, the same difucosylated structure as one of those found in adult Drosophila, i.e. MMF³F⁶, was created de novo. All preparations of modified human transferrin were checked by analysis of its N-glycans before and after incubation with fucosyltransferase.

Control and modified transferrin samples were subject to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-HRP antibody. We could clearly demonstrate that core 1,3-fucosylation with either the Drosophila or Arabidopsis enzymes (Fig. 8, lanes 5-8), and not 1,6-fucosylation (Fig. 8, lane 4), of the human transferrin enabled its recognition by anti-HRP antibodies. The prior hexosaminidase treatment was found to greatly enhance the reactivity of anti-HRP antibodies in a Western blot (data not shown); thus, the preference of the antibody reflects the essential lack of terminal GlcNAc residues on both horseradish peroxidase (3, 33) and, as is shown in the present study, Drosophila glycoproteins.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Anti-HRP antibody recognizes core 1,3-fucosylated glycoproteins. Human transferrin (lane 1) and its modified N-glycoforms (lanes 2-8) were probed with rabbit anti-HRP antibody by Western blotting. Human asialoagalactotransferrin (GnGn-transferrin) was incubated with either -N-acetylhexosaminidase alone (lane 2; MM), heat-denatured chicken heart extract (lane 3; also MM), native chicken heart extract (lane 4; MMF6), Drosophila FucTA and denatured chicken heart extract (lane 5; MMF3), Drosophila FucTA and native chicken heart extract (lane 6; MMF3F6), Arabidopsis core 1,3-fucosyltransferase and denatured chicken heart extract (lane 7; MMF3) or Arabidopsis core 1,3-fucosyltransferase and native chicken heart extract (lane 8; MMF3F6). After incubations with native and/or denatured fucosyltransferase preparations, lanes 3-8 were treated with -N-acetylhexosaminidase as described under "Experimental Procedures." Note that only transferrin modified by core 1,3-fucosyltransferases was recognized by anti-HRP antibody.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study we have applied various approaches to examine the possible molecular basis of neural anti-HRP staining in Drosophila. We have demonstrated that (a) core 1,3-fucosylated neoglycoconjugates are able to specifically abolish the anti-HRP staining of the nervous system in whole-mount Drosophila embryos; (b) staining of embryos with anti-bee venom antiserum, which contains antibodies binding to 1,3-fucosylated glycoproteins (4, 6), yields the same pattern as anti-HRP staining; (c) adult flies contain core 1,3-fucosylated N-glycans of a type also found in other insect glycoproteins (bee venom and Mamestra brassicae cells) (17-19) and share the feature of core 1,3-fucosylation with Schistosoma (34), Haemonchus (35), and plants (24, 33); (d) the fly genome encodes a core 1,3-fucosyltransferase that would be, presumably, responsible for the formation of the detected core 1,3-fucosylated glycans; (e) the recombinant fly core 1,3-fucosyltransferase can be used to modify human transferrin in order to create de novo a core 1,3-fucosylated N-glycan structure found in adult flies (i.e. MMF³F⁶), a modification correlating with the acquisition of binding to anti-HRP. Our data, therefore, strongly suggest that N-glycan core 1,3-fucosylation is the key post-translational process modifying Drosophila neural glycoproteins and creating the epitope recognized by the anti-HRP antiserum. Studies by others also indicate that the cross-reaction to fly neuronal tissue is due to carbohydrate (as suggested by experiments showing abolition of binding after periodate oxidation) and moreover due to an N-glycan (as indicated by the inhibition observed after preabsorption of anti-HRP with HRP glycopeptides or with inactivated bromelain) (9, 11). However, we are the first to study this inhibition using neoglycoconjugates with both native and defucosylated forms of the bromelain glycopeptide.

The following points also suggest that it is relatively unlikely that any other glycomodification is responsible for anti-HRP binding in Drosophila. First, 1,2-linked xylose (the only other obvious candidate for binding to anti-HRP) has not been found on the N-glycans from any insect, in this or any other study; neither is there any detectable homologue of the plant 1,2-xylosyltransferase in fly (although, of course, it is theoretically possible that a functional evolutionary convergence has occurred). Second, our results indicate that peptide:N-glycosidase A treatment of the glycopeptide preparation removed all the anti-HRP cross-reactive material as determined by ELISA. Third, the low level (~1%) of 1,3-fucosylation in adult flies is compatible with previous reports (8-11), indicating that the anti-HRP epitope is restricted to a small fraction of the cells (i.e. only neurons and male reproductive tissue). Fourth, use of other methods (i.e. hydrazinolysis) on Drosophila larvae, which also exhibit anti-HRP staining, failed to result in the detection of any other structure (not even difucosylated) that could conceivably bind anti-HRP (16). Fifth, two mutations that eliminate anti-HRP binding of Drosophila embryos (in the deficiency mutant Brd¹⁵ and the TM3 chromosomal balancer; Refs. 9, 11, 36, and 37) map closely to the same region as the FucTA gene. Indeed, the location of the estimated breakpoints in Brd¹⁵ (71A1-2 and 71C1-2) would suggest that the FucTA gene (mapped to 71B2) is absent in this mutant, whereas in TM3 (which has multiple breakpoints including 71C) it is, at least, a remarkable coincidence that anti-HRP binding is abolished. Certainly, we will be examining these chromosomal aberrations in our ongoing studies.

The substrate preference of FucTA is of interest, since relatively few N-glycans are present in the preparation that carry non-reducing terminal GlcNAc or Gal residues; indeed, like many N-glycans from plant and invertebrate sources, the vast majority of the N-glycans have non-reducing terminal mannose residues, even if the glycans are core fucosylated, and so could not act as substrates for the FucTA core 1,3-fucosyltransferase. Indeed, all core 1,3- and 1,6-fucosyltransferases described to date do not utilize mannose-terminating N-glycans, whether they be of insect, plant, or mammalian origin (25, 38, 39). This raises the possibility that, even though the prior action of, at least, GlcNAc-TI is required, the Drosophila glycans have become processed in the Golgi in vivo or degraded during extraction. The former scenario is very likely since a membrane-bound Golgi -hexosaminidase has been previously found in insects (40), whereas the latter is more unlikely since, in contrast to native fly extracts, the boiled fly extract used for the glycan preparation contained no detectable -hexosaminidase activity. The other aspects of the substrate preference of FucTA are also worthy of comment; the enzyme can significantly, uniquely in comparison to other core fucosyltransferases (25, 27, 38), utilize glycopeptides, which have both antennae galactosylated, whereas it has a bias toward 1,6-fucosylated N-glycans. These substrate preferences may, of course, reflect that galactosylated glycans are only barely present in the fly and possibly became irrelevant during evolution as a `NO-GO' signal, whereas 1,6-fucosylated glycans account for about 40% of the total and perhaps became a positive signal for the enzyme. Indeed, all 1,3-fucosylated glycans in the fly are also 1,6-fucosylated; in this connection, it is also noteworthy that the insect and mammalian 1,6-fucosyltransferase activities previously characterized cannot utilize core 1,3-fucosylated acceptors (41).

Many genes encoding glycosyltransferase homologues can be identified in Drosophila, including core 1,6-fucosyltransferase and GlcNAc-TII; however, considering that the GlcNAc-TI has recently been described (42), the core 1,3-fucosyltransferase described in the present study is, therefore, only the second Drosophila Golgi glycosyltransferase to be characterized. The cloning of its cDNA, the localization of the corresponding gene and the determination of the structure of 1,3-fucosylated glycans gives us both genetic and biochemical means to define further its potential role in the biosynthesis, expression, and biological significance of the anti-HRP epitope in Drosophila.

	ACKNOWLEDGEMENTS

We thank Dr. Florian Rüker and Dr. Jan Mucha for their respective advice related to expression in Pichia and cloning in general, Dr. Regina Voglauer and Peter Bencúr for help with confocal microscopy, Drs. Christian Schlötterer and Wolfgang Miller for fly strains, and Professor Herta Steinkellner and colleagues for DNA sequencing. Alexandra Spiess and Eva Hartmann performed initial experiments on fucosylation of transferrin as part of their undergraduate biochemistry projects.

	FOOTNOTES

^* This work was supported in part by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung Grant P13810-GEN (to I. B. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AJ302045 (FucTA), AJ302046 (FucTB), and AJ302047 (FucTC).

Recipient of a Glycoscience Research Award from Neose Technologies, Inc. To whom correspondence should be addressed. Tel.: 43-1-36006-6065; Fax: 43-1-36006-6059; E-mail: iwilson@edv2.boku.ac.at.

Published, JBC Papers in Press, May 29, 2001, DOI 10.1074/jbc.M100573200

² Abbreviations of N-glycan structures (GnGn, MMF⁶, etc.) are explained in Fig. 1.

	ABBREVIATIONS

The abbreviations used are: HRP, horseradish peroxidase; MALDI, matrix-assisted laser desorption-ionization; TOF, time-of-flight; MS, mass spectrometry; BSA-MUX, conjugate of bovine serum albumin with defucosylated bromelain glycopeptides; BSA-MUXF³, conjugate of bovine serum albumin with native bromelain glycopeptides; g.u., glucose unit(s); BSA, bovine serum albumin; HPLC, high performance liquid chromatography; RP, reverse phase; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction; FITC, fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid; FucT, fucosyltransferase; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; dabsyl, 4-dimethylamino- azobenzene-4'-sulfonyl.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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