Mucin synthesis

203

191

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 ...

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confidence: 99%

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confidence: 99%

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confidence: 99%

See 1 more Smart Citation

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

Yeh

Ong

Fukuda

1999

203

191

“…Surprisingly, these two genes were not similar in sequence to GnTV, which is responsible for formation of the ␤6GlcNAc branch in tetraantennary N-linked glycans (23). Studies of the kinetic properties and acceptor substrate specificities of ␤6GlcNAc-transferases involved in O-glycosylation from different cell lines and organs have suggested that multiple ␤6GlcNAc-transferases exist and that one of these would function in a manner that is distinct from the cloned C2GnT enzyme, in being capable of forming core 2, core 4, and possibly the branched I antigen structure (17,18). The existence of a homologous ␤6GlcNAc-transferase family containing C2GnT and IGnT suggests that additional ␤6GlcNAc-transferase activities may be encoded by homologous genes (22).…”

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confidence: 99%

Control of O-Glycan Branch Formation

Schwientek

Nomoto

Levery

et al. 1999

103

A novel human UDP-GlcNAc:Gal/GlcNAc␤1-3GalNAc␣ ␤1,6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-␣-D-glucosamine:acceptor ␤1,6-N-acetylglucosaminyltransferase (␤1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a ␤1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in ␤1,6Glc-NAc-transferases capable of forming core 2 O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

“…Two candidate branching reactions involving distinct ␤1,6-N-acetylglucosaminyltransferases (IGnT6) have been described in vitro (1). The "distally acting" dIGnT6 transfers a GlcNAc unit in the ␤1,6 linkage to the penultimate galactose residue at the growing end of the linear polylactosamine chain (Scheme 1) (2)(3)(4)(5)(6)(7)(8). By contrast, the "centrally acting" cIGnT6 transfers a GlcNAc residue in the ␤1,6 linkage to midchain galactose units of preformed as well as growing linear chains (3,9,10).…”

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confidence: 99%

Biosynthesis of Branched Polylactosaminoglycans

Leppänen¹,

Zhu²,

Maaheimo³

et al. 1998

Two types of ␤1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAc␤1-3Gal␤1-4GlcNAc␤1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAc␤1-3)Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Gal␤1-4GlcNAc␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc from the linear tetrasaccharide Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc-␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc and Gal␤1-4GlcNAc-␤1-3(GlcNAc␤1-6)Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc from the linear hexasaccharide Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAc␤1-3Gal␤1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.