Molecular Cloning and Expression of a Novel β-1,6-N-Acetylglucosaminyltransferase That Forms Core 2, Core 4, and I Branches

Abstract

Mucin-type O-glycans are classified according to their core structures. Among them, cores 2 and 4 are important for having N-acetyllactosamine side chains, which can be further modified to express various functional oligosaccharides. Previously, we discovered by cloning cDNAs that the core 2 branching enzyme, termed core 2 ␤-1,6-N-acetylglucosaminyltransferase-leukocyte type (C2GnT-L), is highly homologous to the I branching ␤-1,6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468 -478). Using these homologous sequences as probes, we identified an expressed sequence tag in dbEST, which has significant homology to C2GnT-L and IGnT. This approach, together with 5and 3 rapid amplification of cDNA ends, yielded a human cDNA that encompasses a whole coding region of an enzyme, termed C2GnT-mucin type (C2GnT-M). C2GnT-M has 48.2 and 33.8% identity with C2GnT-L and IGnT at the amino acid levels. The expression of C2GnT-M cDNA directed the expression of core 2 branched oligosaccharides and I antigen on the cell surface. Moreover, a soluble chimeric C2GnT-M had core 4 branching activity in addition to core 2 and I branching activities. A soluble chimeric C2GnT-L, in contrast, almost exclusively contains core 2 branching activity. Northern blot analysis demonstrated that the C2GnT-M transcripts are heavily expressed in colon, small intestine, trachea, and stomach, where mucin is produced. In contrast, the transcripts of C2GnT-L were more widely detected, including the lymph node and bone marrow. These results indicate that the newly cloned C2GnT-M plays a critical role in O-glycan synthesis in mucins and might have distinctly different roles in oligosaccharide ligand formation compared with C2GnT-L.Mucin-type glycoproteins are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine in a polypeptide (1). These attached O-glycans can be classified into several different groups according to the core structures (2). In many cells, core 1, Gal␤133GalNAc, is the major constituent of O-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides Gal␤133(GlcNAc␤136)GalNAc when core 2 ␤-1,6-N-acetylglucosaminyltransferase (C2GnT) 1 is present (3, 4). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Le x in mucin-type glycoproteins of blood cells can be formed only in core 2 branched oligosaccharides such as NeuNAc␣233Gal␤134(Fuc␣133) GlcNAc␤136(NeuNAc␣233Gal␤133)GalNAc␣3serine/ threonine (3, 5-7).Those sialyl Le x and sulfated sialyl Le x in O-glycans have been shown to be preferential ligands for P-and L-selectin (6 -10). It has been also shown that poly-N-acetyllactosamines can be extended from core 2 branches (3,5,7,11,12). Poly-Nacetyllactosamines provide the backbone for additional modifications such as sialyl Le x . Moreover, a linear poly-N-acetyllactosamine (Gal␤134GlcNAc␤133) n can ...

C2GnT-M is downregulated in colorectal cancer and its re-expression causes growth inhibition of colon cancer cells

“…In the present study, we showed that C2GnT-M expression was expressed at low levels in the majority of colorectal carcinomas and colon cancer cell lines compared with normal colon tissues. By using a cancer profiling array, we found that C2GnT-M was highly expressed in the normal gastrointestinal tract, for example, colon, rectum, and stomach, which was also found in a previous report (Yeh et al, 1999). Interestingly, there was no significant difference in C2GnT-M …”

“…The core 2 b-1,6-N-acetylglucosaminyltransferase (C2GnT) transfers GlcNAc to GalNAc of the core 1 acceptor structure Galb1-3GalNAca-Ser/Thr to form the core 2 branch in b-1,6 linkage. To date, three members of C2GnT have been cloned, leukocyte-type C2GnT (C2GnT-L or C2GnT1) (Bierhuizen and Fukuda, 1992), mucin-type C2GnT (C2GnT-M or C2GnT2) (Schwientek et al, 1999;Yeh et al, 1999), and thymusassociated C2GnT (C2GnT-T, C2GnT3) (Schwientek et al, 2000). In addition to the formation of the core 2 structure, C2GnT-M also exhibits activities to form the core 4 and I branch, as shown in Figure 1 (Schwientek et al, 1999;Yeh et al, 1999).…”

“…To date, three members of C2GnT have been cloned, leukocyte-type C2GnT (C2GnT-L or C2GnT1) (Bierhuizen and Fukuda, 1992), mucin-type C2GnT (C2GnT-M or C2GnT2) (Schwientek et al, 1999;Yeh et al, 1999), and thymusassociated C2GnT (C2GnT-T, C2GnT3) (Schwientek et al, 2000). In addition to the formation of the core 2 structure, C2GnT-M also exhibits activities to form the core 4 and I branch, as shown in Figure 1 (Schwientek et al, 1999;Yeh et al, 1999). Although C2GnT-M is highly expressed in the gastrointestinal tract, its physiologic or pathologic function in the gastrointestinal tract has not yet been studied.…”

A multipotential β-1,6-N-acetylglucosaminyl-transferase is encoded by bovine herpesvirus type 4

“…The most characterized ␤1,6GnTs encoded by the ␤1,6GnT gene family are the human (h) C2GnT-leukocyte-type (hC2GnT-L) (14), h-IGnT (15), and the h-C2GnT-mucin-type (h-C2GnT-M) (16). h-C2GnT-L and h-IGnT have core 2 and I branching activities, respectively.…”

The cis-Regulatory Element Gsl5 Is Indispensable for Proximal Straight Tubule Cell-specific Transcription of Core 2 β-1,6-N-Acetylglucosaminyltransferase in the Mouse Kidney

“…In addition, the formation of the core 2 structure is essential for further elongation of the carbohydrate chains. Three different core 2 ␤-1,6-GlcNAc transferases, termed core 2 GnT-I, -II, and -III, have been reported in humans (27)(28)(29). The mouse core 2 GnT is a homolog of human core 2 GnT-I.…”