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

Abstract

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 A, and horseradish peroxidase reaction. Recently it was shown that these mucous cells secrete glycoproteins having GlcNAc␣134Gal␤3R at nonreducing terminals of the carbohydrate moieties. Herein we describe the expression cloning of a cDNA encoding a human ␣1,4-N-acetylglucosaminyltransferase (␣4GnT), a key enzyme for the formation of GlcNAc␣134Gal␤13R. 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␣134Gal␤3R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAc␣134Gal␤3R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The ␣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 ␣4GnT revealed that ␣1,4-linked GlcNAc residues are transferred most efficiently to core 2 branched O-glycans (Gal␤13 4GlcNAc␤136(Gal␤133)GalNAc), forming GlcNAc␣13 4Gal␤134GlcNAc␤136(GlcNAc␣134Gal␤133)GalNAc. Transfection of ␣4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that ␣4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the ␣4GnT is a glycosyltransferase that forms ␣1,4-linked GlcNAc residues, preferentially in O-glycans.

Exploring the Utility of Carbohydrate Associated Transferase Activities as Potential Tumor Markers for Human Gastric Cancer

“…1, 3 Galactosidase treatment was subjected to PNA-agarose affinity chromatography and it showed > 90% non-binding to this column. On the contrary to these results, Nakayama et al (1999) without showing data just mentioned in their paper that their cloned human 1, 4 Nacetylglucosaminyltransferase acted only on the Gal moiety of mucin core 2 trisaccharide. We find that [6-3 H] GlcNAc linkage to 1, 6 linked GlcNAc in mucin core 2 is completely resistant to Jack bean -N-acetyl hexosaminidase which has a broad specificity cleaving nonreducing terminal 1-2, 3, 4 or 6 linked GlcNAc and GalNAc residues (Li and Li 1970).…”

“…The 1,4 GlcNAc-capped O-glycans expressed by gastric gland mucous cell-derived mucin prevents colonization of H pyroli. Thus, the differential expression of distinct O-glycans in stomach provides therapeutic potentialities based on specific carbohydrate modulation (Nakayama et al 1999;Reis et al 2000 andKobayashi et al 2009). Werther et al (1996) examined the frequency of sialyl Tn expression and its prognostic value in gastric cancer by immunohistochemical analysis of 340 gastric tumors and found that sialyl Tn expression is a marker of gastric cancer progression suggesting that cancer associated mucins play a role in the malignant behavior of the tumor.…”

Automated Motif Discovery from Glycan Array Data

“…As shown in Figure 6, for HPA binding there are eight binding glycans that possess terminal a-linked-GalNAc, which is consistent with prior studies of its binding specificity (Wu, 1991). However, GLYMMR also identified five binding glycans possessing terminal a-linked-GlcNAc, which is a relatively rare glycan in animals (Nakayama et al, 1999;Zhang et al, 2001), and was not previously described to be well recognized by HPA. If glycans with this latter structural feature had not been on the array, this motif would not have been discovered.…”

“…Motifs a and b are related and clearly consistent with the known specificity for terminal a-GalNAc, but motif c terminates in a-GlcNAc. This second glycan recognition by HPA has largely been overlooked, since a-GlcNAc occurs rarely in mammals, and has so far only been found in O-glycans of human gastric mucins (Nakayama et al, 1999;Zhang et al, 2001). HPA was recently described as a member of a group of invertebrate lectins that are involved in innate immunity in the snail, where it participates in the protection of fertilized eggs by agglutinating a variety of microorganisms (Sanchez et al, 2006).…”

Development of Novel Glycosyl Donors for 1,2-<i>cis</i> Glycosylation Reaction for Amino Sugar and Synthesis of anti-<i>Helicobacter pylori</i> Oligosaccharide

“…Unfortunately, these hypotheses have yet to be proven-investigations have been restrained due to limited amounts of the anti-H. pylori hexasaccharide. At present, the hexasaccharide is available only via recombinant glycoprotein CD43, involving a-1,4-N-acetyl glucosaminyl transferase in Chinese hamster ovary cells (28,29). Availability of the hexasaccharide and its derivatives would allow detailed structure-activity relationship and biological studies of the inhibition mechanism.…”