Cloning and Expression of Drosophila melanogaster UDP-GlcNAc:?-3-D-Mannoside ? 1,2-N-Acetylglucosaminyltransferase I : Biological Chemistry

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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Turk, Boris / Wittinghofer, Alfred


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Cloning and Expression of Drosophila melanogaster UDP-GlcNAc:?-3-D-Mannoside ? 1,2-N-Acetylglucosaminyltransferase I

Mohan Sarkar / Harry Schachter

Citation Information: Biological Chemistry. Volume 382, Issue 2, Pages 209–217, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2001.028, June 2005

Publication History

Published Online:
2005-06-01

Abstract

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDPNacetylglucosamine: ?-3-Dmannoside ?-1,2-Nacetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxyterminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in ?ZAP II and two cDNAs lacking a start ATG codon were obtained. 5Rapid amplification of cDNA ends (5RACE) yielded a 2828 bp cDNA containing a fulllength 1368 bp open reading frame encoding a 456 amino acid protein with putative Nterminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the Nterminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an Nterminal (His)[6] tag. Protein purified by adsorption to and elution from nickel beads converted Man?1-6(Man?1-3)Man?octyl (M3-octyl) to Man?1-6(GlcNAc?1-2Man?1-3)Man?octyl. The K values (0.7 and 0.03 mM for M3-octyl and UDPGlc NAc respectively), temperature optimum (37 C), 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 searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GTAG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.

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