Expression cloning of a human α1,4-N-acetylglucosaminyltransferase that forms GlcNAcα1→4Ga1β→R, a glycan specifically expressed in the gastric gland mucous cell-type mucin

Among mucus-secreting cells, the gastric gland mucous cells, Brunner's glands, accessory glands of pancreaticobiliary tract, and pancreatic ducts exhibiting gastric metaplasia are unique in that they express class III mucin identified by paradoxical Con A staining composed of periodate oxidation, sodium borohydride reduction, Con ii and horseradish peroxidase reaction, Recently it was shown that these mucous cells secrete glycoproteins having GlcNAc alpha 1 --> 4Gal beta --> R at nonreducing terminals of the carbohydrate moieties, Herein we describe the expression cloning of a cDNA encoding a human alpha 1,4-N-acetylglucosaminyltransferase (alpha 4GnT), a key enzyme for the formation of GlcNc alpha 1 --> 4Gal beta 1 --> R. COS-1 cells were thus cotransfected with a stomach cDNA library and a leukosialin cDNA. Transfected COS-1 cells were screened by using monoclonal antibodies specific for GlcNAc alpha 1 --> 4Gal beta --> R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAc alpha 1 --> 4Gal beta --> R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The alpha 4GnT gene is located at chromosome 3p14.3, and its transcripts are expressed in the stomach and pancreas. An in vitro GlcNAc transferase assay by using a soluble alpha 4GnT revealed that alpha 1,4-linked GlcNAc residues are transferred most efficiently to core 2 branched O-glycans (Gal beta 1 --> 4GlcNAc beta 1 --> 6(Gal beta 1 --> 3)GalNAc), forming GlcNAc alpha 1 --> 4Gal beta 1 --> 4GlcNAc beta 1 --> 6(GlcNAc alpha 1 --> 4Gal beta 1 --> 3)GalNAc. Transfection of alpha 4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that alpha 4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the alpha 4GnT is a glycosyltransferase that forms alpha 1,4-linked GlcNAc residues, preferentially in O-glycans.