Molecular Cloning and Characterization of Human GnT-IX, a Novel β1,6-N-Acetylglucosaminyltransferase That Is Specifically Expressed in the Brain

A novel β1,6-N-acetylglucosaminyltransferase (β1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5′-rapid amplification of cDNA end analysis and a further data base search. The open reading frame encodes a 792 amino acid protein with a type II membrane protein structure typical of glycosyltransferases. The entire sequence identity to human GnT-V is 42%. When pyridylaminated (PA) agalacto biantennary N-linked oligosaccharide was used as an acceptor substrate, the recombinant enzyme generated a novel product other than the expected GnT-V product, (GlcNAcβ1,2-Manα1,3-)[GlcNAcβ1,2-(GlcNAcβ1,6-)Manα1,6-]Manβ1,4-GlcNAcβ1,4-GlcNAc-PA. This new product was identified as [GlcNAcβ1,2-(GlcNAcβ1,6-)Manα1,3-][Glc-NAcβ1,2-(GlcNAcβ1,6-)Manα1,6-]Manβ1,4-GlcNAcβ1,4-GlcNAc-PA by mass spectrometry and 1H NMR. Namely, the new GnT (designated as GnT-IX) has β1,6GnT activity not only to the α1,6-linked mannose arm but also to the α1,3-linked mannose arm of N-glycan, forming a unique structure that has not been reported to date. Northern blot analysis showed that the GnT-IX gene is exclusively expressed in the brain, whereas the GnT-V gene is expressed ubiquitously. These results suggest that GnT-IX is responsible for the synthesis of a unique oligosaccharide structure in the brain.