Cloning and Expression of Drosophila melanogaster UDP-GlcNAc :alpha-3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST ) database with the amino acid sequence of human UDP-N-acetylglucosamine:al pha -3-D-mannoside beta -1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2 .4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carb oxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST c lone was used to screen a Drosophila cDNA library in lambda -ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of c DNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-termin al cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) dom ains. The protein showed 52% amino acid sequence identity to human GnT I. T his cDNA, truncated to remove the N-terminal hydrophobic domain, was expres sed in the baculovirus/Sf9 system as a secreted protein containing an N-ter minal (His), tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta -octyl (M3-octyl) to Ma n alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta -octyl. The K-m values (0.7 and 0.03 mM for M3-octyl and UDP-Glc-NAc respectively), temperature optimum (37 degreesC), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN se arches of the Berkeley Drosophila Genome Project database with the Drosophi la GnT I cDNA sequence as probe allowed localization of the gene to chromos omal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allo wed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.