Biochem. J. (2007) 402
(105–115) (Printed in Great Britain)
Towards abolition of immunogenic structures in insect cells: characterization of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core a1,3-fucosyltransferase and the first insect Lewis-histo-blood-group-related antigen-synthesizing enzyme
Dubravko RENDI
*, Jaroslav KLAUDINY†, Ute STEMMER*, Julia SCHMIDT*, Katharina PASCHINGER* and Iain B. H. WILSON*
1*Department für Chemie, Universität für Bodenkultur, Muthgasse 18, A-1190 Wien, Austria, and †Department of Glycobiology, Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia
Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of a1,3-fucosylation: the modification by a1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcb1,4(Fuca1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active a1,3-FucTs (a1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core a1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Lex [Galb1-4(Fuca1-3)GlcNAc] epitope in vitro and is the first Lewis-type a1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed a1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance.
Key words: allergy, anti-(horseradish peroxidase), fucosyltransferase (FucT), honey-bee (Apis mellifera), Lewis X, N-glycan.
Abbreviations used: AMPD, 2-amino-2-methyl-1,3-propanediol; BCIP, 5-bromo-4-chloroindol-3-yl phosphate; EST, expressed sequence tag; FucT, fucosyltransferase; GalGal, Galb1-4GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-Asn; bGNbGN, GalNAcb1-4GlcNAcb1-2Mana1-6(GalNAcb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-Asn; GnGn, GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-Asn; GnGnF6, GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4(Fuca1-6)GlcNAcb1-Asn; MM, Mana1-6(Mana1-3)Manb1-4GlcNAcb1-Asn; HRP, horseradish peroxidase; anti-HRP, antiserum raised against HRP; LacdiNAc, N-acetylgalactosaminyl-b1,4-N-acetylglucosamine (GalNAcb1-4GlcNAc); LacNAc, galactosyl-b1,4-N-acetylglucosamine (Galb1-4GlcNAc); lacto-N-fucopentaose III, Galb1-4(Fuca1-3)GlcNAcb1-3Galb1-4Glc-PA; Lex, Galb1-4(Fuca1-3)GlcNAc; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; NBT, Nitro Blue Tetrazolium; ORF, open reading frame; RP-HPLC, reverse-phase HPLC; RT, reverse transcriptase; pyridylaminated lacto-N-neo-tetraose, Galb1-4GlcNAcb1-3Galb1-4Glc-PA; pyridylaminated lacto-N-tetraose, Galb1-3GlcNAcb1-3Galb1-4Glc-PA.
1To whom correspondence should be addressed (email iain.wilson@boku.ac.at).
The DNA sequences described in this paper will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AM279411 (FucTA), AM279412 (FucTB) and AM279413 (FucTC).
Received 26 June 2006/27 September 2006; accepted 10 October 2006
Published as BJ Immediate Publication 10 October 2006, doi:10.1042/BJ20060964
The Biochemical Society, London ©2007
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