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Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Gene Section Mini Review GCNT3 (glucosaminyl (N-acetyl) transferase 3, mucin type) Prakash Radhakrishnan, Pi-Wan Cheng Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA Published in Atlas Database: November 2007 Online updated version: http://AtlasGeneticsOncology.org/Genes/GCNT3ID44105ch15q21.html DOI: 10.4267/2042/38541 This work is licensed under a Creative Commons Attribution-Non-commercial-No Derivative Works 2.0 France Licence. © 2008 Atlas of Genetics and Cytogenetics in Oncology and Haematology Identity DNA/RNA Hugo: GCNT3 Other names: C2GnT-M; hC2GnT-M; C2GnT2; C2/C4gnT; GnT-M; mucus type C2GnT Location: 15q21.3 Note: GCNT3/C2GnT-M is a single pass type II membrane protein belonging to glycosyltransferase 14 family. Note: Human GCNT3/C2GnT-M is located on chromosome 15 in the region of q21.3, oriented from centromere to telomere. Description Human GCNT3/C2GnT-M gene is approximatively 8.26 kb in size and located in chromosome 15q21.3 at the position of 57,691,415 - 57,699,501. Schematic representation of Human GCNT3/C2GnT-M gene and transcripts. There are three different sized transcripts. (TIS, Transcription Initiation Site designated as +1; E, Exon; I, Intron; UTR, Untranslated region; ATG, start codon; ORF, Open reading frame). Atlas Genet Cytogenet Oncol Haematol. 2008;12(4) 276 GCNT3 (glucosaminyl (N-acetyl) transferase 3, mucin type) Radhakrishnan P, Cheng PW Recently, the GCNT3/C2GnT-M promoter (-417/+187) containing two basal cis-regulatory region (-291/-182 and -62/-43) was identified. The Th2 cytokine and retinoic acid responsive cis-regulatory elements reside in -417/+187 region. Expression Human GCNT3/C2GnT-M gene is expressed in mucussecretory tissues in the following decreasing order of expression: Colon; testis; stomach; small intestine; adrenal gland; kidney; trachea; thyroid gland; Uterus; Ovary; Pancreas; fetal liver; Prostate. Unlike bovine GCNT3/C2GnT-M gene, the type of transcript expressed by hC2GnT-M gene is not tissue specific among the mucus secretory tissues. Expression of GCNT3/C2GnT-M gene is down regulated in colon and colorectal tumors and various colorectal cancer cells. GCNT3/C2GnT-M expression is regulated by various external agent(s). It is inhibited by EGF and enhanced by Th2 cytokines, retinoic acids and sodium butyrate. Transcription Human GCNT3/C2GnT-M contains three different sized transcripts: 2.3-2.5, 3.6-3.8 and 6.8-7.0 kb. The transcript 1 (approximatively 2.3-2.5kb) is made of 3 exons, exon 1 (69-198 bp), exon 2 (333-401 bp), and exon 3 (1864 bp). Exon 3 contains 59 bp of 5' UTR, 1314 bp of ORF and 491 bp of 3'UTR. It does not contain any introns. Whereas, the intermediate sized transcript (3.6-3.8kb) contains 1.3kb of intron 2 and the large sized transcript (6.8-7.0 kb) contains 4.5kb of intron 1 in addition to all three exons. Exon 1 is heterogeneous in size, which ranges from 69 to 198 bp depending on tissues and cells. Exon 1 is present in all transcripts and has same 3' end but different 5' ends. A 333 bp Exon 2 is identified in most of the mucus secreting tissues and airway epithelial cells while a 401 bp of exon 2 is only detected in A549 cells. Localisation Golgi membrane. Function GCNT3/C2GnT-M is responsible for the synthesis of all three branch structures, including core 2, core 4, and I antigen found in the glycans of secreted mucins. These three branch structures are generated by the transfer of GlcNAc from UDP-GlcNAc to core 1, core 3, and I antigen, respectively as shown below. 1. UDP-GlcNAc + Galbeta1-3GalNAca1-S/T gives Galbeta1-3(GlcNAcbeta1-6) GalNAca1-S/T + UDP. 2. UDP-GlcNAc + GlcNAcbeta1-3GalNAc1a-S/T gives GlcNAcbeta1-3(GlcNAcbeta1-6) GalNAca1-S/T + UDP. 3. UDP-GlcNAc + GlcNAcbeta1-3Galbeta1-R gives GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-R + UDP(R: sugars). The primary function of secreted mucins is to protect mucus secretory epithelium by retention of water and maintenance of the rheological properties of the mucus, and adherence to airborne and ingested pathogens to facilitate their removal from these tissues. The first two properties depend primarily on the carbohydrate content and this property depends on the heterogeneity of carbohydrate structure. Secreted mucins contain very high carbohydrate content, i.e. 70-90% by weight, and very heterogeneous carbohydrate structure, e.g. up to 100 different oligosaccharides in mucins isolated from a single donor. The three b6GlcNAc branch structures found in the secreted mucins are responsible for the increase of carbohydrate content and structural complexity. Decrease of GCNT3/C2GnT-M in the secretory epithelium can result in dehydration of the mucus and compromise of bacterial clearance. Protein Note: Human GCNT3/C2GnT-M (EC 2.4.1.102) has 438 amino acids and molecular weight of 50,863 Da. The predicted GCNT3/C2GnT-M structure shows a short Nterminal cytoplasmic tail (CT), a transmembrane domain (TM), a stem region and a long catalytic domain at the C-terminal region. Description GCNT3/C2GnT-M is a type II membrane protein located in the Golgi apparatus. It contains a nine-amino acid peptide tail at the N-terminus located in the cytoplasm, which is followed by a transmembrane domain consisted of 18 amino acids, a stem region, and a catalytic domain located in the Golgi lumen. The protein contains 13 cysteines, including 4 at the Nterminal region, which are conserved among GCNT3/C2GnT-M across species, and 9 at the Cterminal region, which are conserved among all mucin glycan b6GlcNAc branching enzymes. Structural information obtained from bovine GCNT3/C2GnT-M shows that among the 9 conserved cysteines, the second cysteine is unconjugated and the other 8 cysteines form 4 cystine bonds between first and ninth, third and seventh, fourth and fifth, and sixth and eighth. The disulfide bonds formed from the nine conserved cysteines are different between GCNT3/C2GnT-M and C2GnT-L. GCNT3/C2GnT-M contains two potential N-glycosyltaion sites at N-69 and N-289. Atlas Genet Cytogenet Oncol Haematol. 2008;12(4) Homology Human GCNT3/C2GnT-M shows a very high level of similarity to other non-human GCNT3/C2GnT-M: bovine (83%), rat (78%) and mouse (77%). Further, it shows moderate level of (48% and 38%) similarity to human C2GnT-L and C2GnT-T, respectively. 277 GCNT3 (glucosaminyl (N-acetyl) transferase 3, mucin type) Radhakrishnan P, Cheng PW Implicated in References Colorectal cancer Schwientek T, Nomoto M, Levery SB, Merkx G, van Kessel AG, Bennett EP,Hollingsworth MA, Clausen H. Control of Oglycan branch formation. Molecular cloning of human cDNA encoding a novel beta1,6-N-acetylGlucosaminyl transferase forming core 2 and core 4. J Biol Chem 1999;274(8):45044512. Note: GCNT3/C2GnT-M enzyme is down regulated in colon and colorectal tumors and most cancerous cells derived from mucus-secretory tissues. Re-expression of GCNT3/C2GnT-M suppresses tumor growth in the xenografts of nude mice. Disease Colorectal cancer is the 3rd most common form of cancer and the 2nd leading cause of cancer-related death among men and women in the Western world. It causes 655,000 deaths worldwide per year. The survival rate of colorectal cancer is not much higher than 50% even if the disease is diagnosed at an early stage. Colorectal cancer is mostly formed from adenomatous polyps. These polyps can be detected and removed during colonoscopy, which would decrease cancer death by greater than 80%. Metastasis of cancer cells through bowel wall of the colon to lymph nodes is very common. If metastasis is detected, 5 year survival rate is less than 10%. Prognosis Recent reports suggest that deficiency or down regulation of human GCNT3/C2GnT-M expression is associated with development of colitis and colorectal cancer. This enzyme may be used as a prognostic marker for colorectal cancer. Oncogenesis GCNT3/C2GnT-M expression is down regulated in colorectal cancers. Down regulation of GCNT3/C2GnT-M would lead to the production of secreted mucins with lower carbohydrate content and less heterogeneous carbohydrate, which would compromise the protective function of these mucins. As a result, bacteria can not be cleared effectively, which causes irritation of the epithelium and chronic inflammation, and eventually cancer. Its re-expression suppresses tumor cell spreading, adhesion, motility, and invasion. It also inhibits cell growth and colonyforming ability, and induces apoptotic cell death. In addition, expression of C2GnT-M suppresses tumor growth in the xenografts of nude mice. The results suggest that GCNT3/C2GnT-M is important in protecting the normal functional architecture of colon epithelial cells. Atlas Genet Cytogenet Oncol Haematol. 2008;12(4) Yeh JC, Ong E, Fukuda M. Molecular cloning and expression of a novel beta-1,6-N-acetyl glucosaminyltransferase that forms core 2, core 4, and I branches. J Biol Chem 1999;274(5):3215-3221. Beum PV, Cheng PW. Biosynthesis and function of beta 1,6 branched mucin-type glycans. Adv Exp Med Biol 2001;491:279-312. (Review). Beum PV, Bastola DR, Cheng PW. Mucin biosynthesis: epidermal growth factor downregulates core 2 enzymes in a human airway adenocarcinoma cell line. Am J Respir Cell Mol Biol 2003;29(1):48-56. Singh J, Khan GA, Kinarsky L, Cheng H, Wilken J, Choi KH, Bedows E, Sherman S,Cheng PW. Identification of disulfide bonds among the nine core 2 Nacetylglucosaminyltransferase-M cysteines conserved in the mucin beta6-N-acetylglucosaminyltransferase family. J Biol Chem 2004;279(37):38969-38977. Beum PV, Basma H, Bastola DR, Cheng PW. Mucin biosynthesis: upregulation of core 2 beta 1,6 N-acetyl glucosaminyltransferase by retinoic acid and Th2 cytokines in a human airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol 2005;288(1):L116-L124. Ishibashi Y, Inouye Y, Okano T, Taniguchi A. Regulation of sialyl-Lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line. Glycoconj J 2005;22(1-2):53-62. Huang MC, Chen HY, Huang HC, Huang J, Liang JT, Shen TL, Lin NY, Ho CC, Cho IM, Hsu SM. C2GnT-M is downregulated in colorectal cancer and its re-expression causes growth inhibition of colon cancer cells. Oncogene 2006;25(23):32673276. Hashimoto M, Tan S, Mori N, Cheng H, Cheng PW. Mucin biosynthesis: molecular cloning and expression of mouse mucus-type core 2 beta1,6 N-acetylglucosaminyl transferase. Glycobiology 2007;17(9):994-1006. Radhakrishnan P, Beum PV, Tan S, Cheng PW. Butyrate induces sLex synthesis by stimulation of selective glycosyltransferases genes. Biochem Biophys Res Commun 2007;359(3):457-462. Tan S, Cheng PW. Mucin biosynthesis: identification of the cisregulatory elements of human C2GnT-M gene. Am J Respir Cell Mol Biol 2007;36(6):737-745. This article should be referenced as such: Radhakrishnan P, Cheng PW. GCNT3 (glucosaminyl (Nacetyl) transferase 3, mucin type). Atlas Genet Cytogenet Oncol Haematol.2008;12(4):276-278. 278