Molecular cloning and characterization of cDNA coding for β1,2N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum

In plants as well as in animals beta 1,2N-acetylglucosaminyltransferase I (GlcNAc-TI) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from oligomannosidic N-glycans to complex or hybrid type N-linked oligosaccharides, Employing degenerated primers deduced from known GlcNAc-TI genes from animals, we were able to identify the cDNA coding for GlcNAc-TI from a Nicotiana tabacum cDNA library. The complete nucleotide sequence revealed a 1338 base pair open reading frame that codes for a polypeptide of 446 amino acids. Comparison of the deduced amino acid sequence with that of already known GlcNAc-TI polypeptides revealed no similarity of the tobacco clone within the putative cytoplasmatic, transmembrane, and stem regions. However, 40% sequence similarity was found within the putative C-terminal catalytic domain containing conserved single amino acids and peptide motifs, The predicted domain structure of the tobacco polypeptide is typical for type II transmembrane proteins and comparable to known GlcNAc-TI from animal species. In order to confirm enzyme activity a truncated form of the protein containing the putative catalytic domain was expressed using a baculovirus/insect cell system, Using pyridylaminated Man(5)- or Man(3)GlcNAc(2) as acceptor substrates and HPLC analysis of the products GlcNAc-TI activity was shown. This demonstrates that the C-terminal region of the protein comprises the catalytic domain, Expression of GlcNAc-TI mRNA in tobacco leaves was detected using RT-PCR, Southern blot analysis gave two hybridization signals of the gene in the amphidiploid genomes of the two investigated species N,tabacum and N,benthamiana.