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β-1,3-N-acetylglucosaminyltransferase

Glycosylation is one of the most common post-translational modifications of proteins. Glycans are involved in various structural and functional roles in membrane and secreted proteins. The oligosaccharides in eukaryotic cells are primarily classified based on the nature of the linkage to proteins or lipids. There are about five classes of glycans produced namely, N-linked glycans (Asp/Arg); O-linked glycans (Ser/Thr/Tyr); Phospho-glycans (P-Ser); C-linked glycans (Trp) and glypiation (GPI anchor) (1).

Glycosyltransferase

The glycans are enzymatically synthesized by the stepwise reactions of multiple glycosyltransferases in the cell. More than 100 glycosyltransferases are localized in the endoplasmic reticulum and Golgi apparatus and are involved in the involved in the glycan synthesis on proteins, lipids and proteoglycans (2). The glycosyltransferases were grouped into functional subfamilies based on similarities of sequence, their enzyme characteristic, donor specificity, acceptor specificity and the specific donor and acceptor linkage (3). The Glycosyltransferase sequence comprises about 330-560 amino acids long and share the same type II transmembrane protein structure with four functional domains: a short cytoplasmic domain, a targeting / membrane anchoring domain, a stem region and a catalytic domain (4). Mammals utilize only 9 sugar nucleotide donors for glycosyltransferases such as UDP-glucose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid. Other organisms have an extensive range of nucleotide sugar donors (1).

β-1,3-N-acetylglucosaminyltransferase

β-1,3-N-acetylglucosaminyltransferase (β3GnT) (EC 2.4.1.149) is a group of enzymes belongs to the family glycosyltransferases. The systematic name of this enzyme class is UDP-N-acetyl-D-glucosamine:β-D-galactosyl-1,4-N-acetyl-D-glucosamine-β-1,3-N-acetyl-D-glucosaminyltrans-ferase. This enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to Gal in the Galβ1-4 Glc (NAc) structure with β-1,3 linkage. These enzymes were grouped into GT family 31, 49 in CAZy database. The enzyme has 2 substrates namely, UDP-N-acetyl-D-glucosamine and β-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R and its 3 products are UDP, N-acetyl-β-D-glucosaminyl-1,3-β-D-galactosamine and D-glucosaminyl-R. This enzyme participates in 4 metabolic pathways: keratan sulfate biosynthesis, glycosphingolipid biosynthesis -neo-lacto series, glycan structures - biosynthesis 1, and glycan structures - biosynthesis 2 (5). The motif analysis in Manic Fringe (5) showed some of the important functional domains predicted to be common among the β3GnT enzymes. The first Motif was a structural motif necessary for maintaining the protein fold. The second, DXD motif represented in many glycosyltransferases is involved in the binding of the nucleotide-sugar donor substrate, both directly and indirectly through coordination of metal ions such as magnesium or manganese in the active site. A glycine-rich loop was the third motif found at the bottom of the active site cleft. This loop is likely to play a role in the recognition of both the GlcNAc portion of the donor and the substrate. The last motif was a conserved Aspartate in the catalytic domain (6).

The variety of β1,3-linked GlcNAc residues in different types of glycoconjugates and cells at varying stages of differentiation suggests the presence of various β3GnT with different substrate specificities and expression profiles in human tissues. There are about 9 different β3GnT enzymes have been identified so far, in which the β3GnT9 was identified recently and yet to be characterized.

The β3GnT1 (iGnT) was the first enzyme to be isolated when cDNA of a human β-1,3-N-acetylglucosaminyltransferase essential for poly-N-acetyllactosamine synthesis was studied (7). The poly-N-acetyllactosamines synthesized by iGnT provide critical backbone structures for the addition of functional oligosaccharides. It is widely distributed in various tissues and is structurally different from other β3GnTs. It has been reported recently that β3GnT1 is involved in attenuating prostate cancer cell locomotion by regulating the synthesis of laminin-binding glycans on -DG (8). The β3GnT2 enzyme is mainly responsible for elongation of poly-lactosamine chains. This enzyme was isolated based on structural similarity with the β3GalT family. Studies showed that on a panel of invasive and noninvasive fresh transitional cell carcinomas (TCCs) showed strong down regulation of β3GnT2 in the invasive lesions, suggesting that a decline in the expression levels of some members of the glycosyltransferase (9).

The β3GnT3 and β3GnT4 enzymes were subsequently isolated based on structural similarity with the β3GalT family. β3GnT3 is a type II transmembrane protein and contains a signal anchor that is not cleaved. It prefers the substrates of lacto-N-tetraose and lacto-N-neotetraose, and is involved in the biosynthesis of poly-N-acetyllactosamine chains and the biosynthesis of the backbone structure of dimeric sialyl Lewis a. It plays dominant roles in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. The β3GnT3 was highly expressed in the non-invasive colon cancer cells. β3GnT4 is involved in the biosynthesis of poly-N-acetyllactosamine chains and prefers lacto-N-neotetraose as a substrate. It is a type II transmembrane protein and it is expressed more in bladder cancer cells (10).

β3GnT5 is responsible for lactosyltriaosylceramide synthesis, an essential component of lacto/neolacto series glycolipids (11). The expression of the HNK-1 and Lewis x antigens on the lacto/neo-lacto-series of glycolipids has shown to be developmentally and tissue-specifically regulated by β3GnT5. The overexpression of β3GnT5 in human gastric carcinoma cell lines led to increased sialyl-Lewis x expression and H. pylori adhesion (12). The β3GnT6 synthesizes the core 3 O-glycan structure and speculates that this enzyme plays an important role in the synthesis and function of mucin O-glycan in the digestive organs. In addition, the expression of β3GnT6 was markedly down regulated in gastric and colorectal carcinomas (13). Expression of β3GnT7 has been reported to be down-regulated upon malignant transformation (14). Elongation of the carbohydrate backbone of Keratan Sulfate proteoglycan is catalyzed by β3GnT7 and β1,4-galactosyltransferase 4 (15). β3GnT2-5 and β3GnT7 can transfer GlcNAc to Gal with a β1,2 linkage to synthesize a polylactosamine chain with each enzyme differing in its acceptor molecule preference. The polylactosamine and related structures plays crucial role in cell-cell interaction, cell-extracellular matrix (ECM) interaction, immune response and determining metastatic capacity (3).

The β3GnT8 enzyme extends a polylactosamine chain specifically on a tetraantennary N-glycans. β3GnT8 transfers GlcNAc to the non-reducing terminus of the Galβ1-4GlcNAc of tetra antennary N-glycan in vitro. Intriguingly, β3GnT8 is significantly upregulated in colon cancer tissues than in normal tissue (3). The co-transfection of β3GnT8 and β3GnT2 resulted in synergistic enhancement of the activity of the polylactosamine synthesis. This indicates that these two enzymes interact and complement each others function in the cell.

REFERENCES

1. Varki A, Cummings, R D, Esko J D, Freeze H H, Stanley P, Bertozzi C R, Hart G W, Etzler M E (2008). Essentials of Glycobiology, 2nd ed. Plainview (NY): Cold Spring Harbor Laboratory Press 2. Narimatsu H. Human glycogene cloning: focus on beta 3-glycosyltransferase and beta 4-glycosyltransferase families. Curr Opin Struct Biol. 2006 Oct; 16(5):567-75. Epub 2006 18. 3. Ishida H, Togayachi A, Sakai T, Iwai T, Hiruma T, Sato T, Okubo R, Inaba N, Kudo T, Gotoh M, Shoda J, Tanaka N, Narimatsu H. A novel beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer. FEBS Lett. 2005 Jan 3; 579(1):71-8. 4. Fukuda M, Hindsgaul O, Hames B D, Glover D M. (1994) in Molecular Glycobiology, eds Hames B D, Glover D M (Oxford Univ. Press, Oxford). 5. Takeya A, Hosomi O, Kogure T (1985). The presence of N-acetyllactosamine and lactose: β (1-3) N-acetylglucosaminyltransferase activity in human urine. Jpn J Med Sci Biol, 38:1-8 6. Martin Jinek, Ya-Wen Chen, Henrik Clausen, Stephen M Cohen & Elena Conti. Structural insights into the Notch-modifying glycosyltransferase Fringe. Nature Structural & Molecular Biology 13, 945 - 946 (2006) 7. Dapeng Zhou, Andre Dinter, Ricardo Gutierrez Gallego, Johannis P Kamerling, Johannes F G Vliegenthart, Eric G Berger and Thierry Hennet (1999). A β-1,3-N-acetylglucosaminyl-transferase with poly-N-acetyllactosamine synthase activity is structurally related to β-1,3-galactosyltransferases. PNAS, Vol. 96, 406-411

8. Xingfeng Baoa, Motohiro Kobayashib, Shingo Hatakeyamaa, Kiyohiko Angataa, Donald Gullbergc, Jun Nakayamab, Michiko N Fukudaa and Minoru Fukuda (2009). Tumor suppressor function of laminin-binding -dystroglycan requires a distinct β-3-N-acetylglucosaminyltransferase. PNAS, Vol. 106:29, 12109-12114

9. Irina Gromova, Pavel Gromov and Julio E. Celis (2001). A Novel Member of the Glycosyltransferase Family, β3GnT2, highly down regulated in invasive human bladder Transitional Cell Carcinomas. Molecular Carcinogenesis, Vol. 32, 61-72

10. Norihiko Shiraishi, Ayumi Natsume, Akira Togayachi, Tetsuo Endo, Tomohiro Akashima, Yoji Yamada, Nobuyuki Imai, Satoshi Nakagawa, Satoshi Koizumi, Susumu Sekine, Hisashi Narimatsu and Katsutoshi Sasaki (2001). Identification and characterization of 3 novel β 1,3-N-Acetylglucosaminyl-transferases. Structurally Related to the β1,3-Galactosyltransferase family. The Journal of Biological Chemistry, Vol. 276: 5, 3498-3507

11. A. Togayachi, T. Akashima, R. Ookubo, T. Kudo, S. Nishihara, H. Iwasaki, A. Natsume, H. Mio, J. Inokuchi and T. Irimura et al., Molecular cloning and characterization of UDP-GlcNAc: Lactosylceramide β1,3-N-acetylglucosaminyltransferase (β3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids, J Biol Chem 276 (2001), pp. 22032–22040

12. Nuno T Marcos, Ana Magalhães, Bibiana Ferreira, Maria J Oliveira, Ana S Carvalho, Nuno Mendes, Tim Gilmartin, Steven R Head, Céu Figueiredo, Leonor David, Filipe Santos-Silva, and Celso A Reis (2008). Helicobacter pylori induces β3GnT5 in human gastric cell lines, modulating expression of the SabA ligand Sialyl-Lewis X. Journal of Clinical Investigation, Vol. 118: 6

13. Toshie Iwai, Takashi Kudo, Risa Kawamoto, Tomomi Kubota, Akira Togayachi, Toru Hiruma, Tomoko Okada, Toru Kawamoto, Kyoei Morozumi, and Hisashi Narimatsu (2005). Core 3 synthase is down-regulated in colon carcinoma and profoundly suppresses the metastatic potential of carcinoma cells. PNAS, Vol. 102:12, 4572-4577

14. Kataoka K and Huh N H (2002). A novel β1,3-N-acetylglucosaminyltransferase involved in invasion of cancer cells as assayed in vitro. Biochem Biophys Res Commun, Vol. 294, 843-848

15. Norihito Hayatsu, Satoshi Ogasawara, Mika Kato Kaneko, Yukinari Kato and Hisashi Narimatsu. Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells. Biochemical and Biophysical Research Communications. Volume 368, Issue 2, 4 April 2008, Pages 217-222