Cloning and Expression of Drosophila melanogaster UDP-GlcNAc:?-3-D-Mannoside ? 1,2-N-Acetylglucosaminyltransferase I
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Mohan Sarkar
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.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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- Highlight: Glycobiology
- O-Glycosylation of the Mucin Type
- Glycoproteins from Insect Cells: Sialylated or Not?
- Congenital Disorders of Glycosylation: Glycosylation Defects in Man and Biological Models for Their Study
- Mitochondrial Single-Stranded DNA-Binding Proteins: in Search for New Functions
- Do Rodent and Human Brains Have Different N-Glycosylation Patterns?
- The Liver Flukes Fasciola gigantica and Fasciola hepatica Express the Leucocyte Cluster of Differentiation Marker CD77 (Globotriaosylceramide) in Their Tegument
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- Pathways of Mucin O-Glycosylation in Normal and Malignant Rat Colonic Epithelial Cells Reveal a Mechanism for Cancer-Associated Sialyl-Tn Antigen Expression
- 6-O-Sulfo De-N-Acetylsialyl Lewis X as a Novel High-Affinity Ligand for Human L-Selectin: Total Synthesis and Structural Characterization
- Segregation of Gangliosides GM1 and GD3 on Cell Membranes, Isolated Membrane Rafts, and Defined Supported Lipid Monolayers
- Structural Characterization of Fucose-Containing Oligosaccharides by High-Performance Liquid Chromatography and Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry
- Anencephaly: Structural Characterization of Gangliosides in Defined Brain Regions
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- Stimulation of Acid Sphingomyelinase Activity by Lysosomal Lipids and Sphingolipid Activator Proteins
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- Synthesis of Nucleotide-Activated Oligosaccharides by ?-Galactosidase from Bacillus circulans
- Elucidation of the Role of Functional Amino Acid Residues of the Small Sialidase from Clostridium perfringens by Site-Directed Mutagenesis
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- Experimental Regulation of STAT Gene Expression Reveals an Involvement of STAT5 in Interleukin-4-Driven Cell Proliferation
