CDNA Cloning and Chromosomal Mapping of Human N-Acetylglucosaminyltransferase-V

doi:10.1006/bbrc.1994.1045

Biochemical and Biophysical Research Communications

Volume 198, Issue 1, 15 January 1994, Pages 318-327

https://doi.org/10.1006/bbrc.1994.1045 Get rights and content

Abstract

Human N-acetylglucosaminyltransferase V (GnT-V, EC 2.4.1.155) cDNA was isolated from a human fetal liver cDNA library. Oligonucleotide primers for polymerase chain reaction were designed according to the amino acid sequence of human GnT-V. Screening for the cDNA was carried out by plaque hybridization using PCR products of about 500 bp. Human GnT-V has 741 amino acids and six putative N-glycosylation sites. The homology to rat GnT-V is 88% at the nucleotide level and is 97 % at the amino acid level, and there is one amino acid insertion. Using the cDNA clones as probe, five overlapping genomic clones have been isolated from a human phagemid DNA library. The GnT-V gene has been mapped to chromosome 2q21 using flourescence in situ hybridization.

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Cited by (102)

Glycans and cancer: Role of N-Glycans in cancer biomarker, progression and metastasis, and therapeutics
2015, Advances in Cancer Research
Glycosylation is catalyzed by various glycosyltransferase enzymes which are mostly located in the Golgi apparatus in cells. These enzymes glycosylate various complex carbohydrates such as glycoproteins, glycolipids, and proteoglycans. The enzyme activity of glycosyltransferases and their gene expression are altered in various pathophysiological situations including cancer. Furthermore, the activity of glycosyltransferases is controlled by various factors such as the levels of nucleotide sugars, acceptor substrates, nucleotide sugar transporters, chaperons, and endogenous lectin in cancer cells. The glycosylation results in various functional changes of glycoproteins including cell surface receptors and adhesion molecules such as E-cadherin and integrins. These changes confer the unique characteristic phenotypes associated with cancer cells. Therefore, glycans play key roles in cancer progression and treatment. This review focuses on glycan structures, their biosynthetic glycosyltransferases, and their genes in relation to their biological significance and involvement in cancer, especially cancer biomarkers, epithelial–mesenchymal transition, cancer progression and metastasis, and therapeutics.
Major N-glycan branching structures which are directly related to cancer are β1,6-GlcNAc branching, bisecting GlcNAc, and core fucose. These structures are enzymatic products of glycosyltransferases, GnT-V, GnT-III, and Fut8, respectively. The genes encoding these enzymes are designated as MGAT5 (Mgat5), MGAT3 (Mgat3), and FUT8 (Fut8) in humans (mice in parenthesis), respectively. GnT-V is highly associated with cancer metastasis, whereas GnT-III is associated with cancer suppression. Fut8 is involved in expression of cancer biomarker as well as in the treatment of cancer. In addition to these enzymes, GnT-IV and GnT-IX (GnT-Vb) will be also discussed in relation to cancer.
Predominant expression of N-acetylglucosaminyltransferase V (GnT-V) in neural stem/progenitor cells
2015, Stem Cell Research
Citation Excerpt :
N-acetylglucosaminyltransferase (GnT)-III, which is encoded by the Mgat3 gene, transfers a GlcNAc to β-mannose via β1,4-linkage, thus leading to the formation of bisecting GlcNAc. A β1,6-branch is formed by GnT-V, which is encoded by the Mgat5 gene, thus resulting in conversion to triantennary oligosaccharide (Saito et al., 1994; Shoreibah et al., 1993). Another glucosaminyltransferase that synthesizes β1,6-branched complex-type N-glycans in vitro is GnT-Vb (IX), a paralog of GnT-V with 53% amino acid similarity (Inamori et al., 2003; Kaneko et al., 2003).
Neural stem/progenitor cells (NPCs) express a variety of asparagine-linked oligosaccharide chains, called N-glycans, on the cell surface, and mainly produce hybrid-type and complex-type N-glycans. However, the expression profiles and roles of N-acetylglucosaminyltransferase-V (GnT-V), an enzyme that forms β1,6-branched N-glycans, in NPCs remain unknown. In this study, cultured NPCs were prepared from adult or embryo cortex, and were maintained as either proliferating NPCs or differentiated cells in vitro. Analysis using reverse-transcriptase polymerase chain reaction, Western blot and lectin blot revealed that GnT-V and its reaction products were distinctly expressed in proliferating NPCs; moreover expression of GnT-V and its reaction products were markedly diminished in differentiated cells. In brain slices, many GnT-V-positive neurogenic cells were detected throughout the cerebral cortex on embryonic day 13, while only a few doublecortin (Dcx)- and GnT-V-double positive NPCs were detected around the subventricular zone of the lateral ventricle in the adult brain. However, in the mice in which motor function was spontaneously recovered after cryoinjury to the motor cortex, many Dcx- and GnT-V-double positive NPCs were found to have accumulated around the brain lesion of the adult cerebral cortex compared with the mice in which the function did not recover. These results indicate that GnT-V expression is under rigorous control during NPC differentiation. Furthermore, expression of GnT-V and its reaction products in NPCs may be necessary for the functional recovery after brain injury, and could be used as a marker for visualization of NPCs.
Overexpression of N-acetylglucosaminyltransferases III and v in human melanoma cells. Implications for MCAM N-glycosylation
2014, Biochimie
An important role in cancer pathogenesis is attributed to N-glycans with “bisecting” N-acetylglucosamine and beta1-6 branches but the exact mechanisms still remain to be elucidated. Two structures are formed by Golgi beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (EC = 2.4.1.144, GnT-III) and alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A (EC = 2.4.1.155, GnT-V) respectively. The enzymes are encoded by MGAT3 and MGAT5 genes. The aim of this study was to establish two human melanoma cell lines with induced overexpression of GnT-III or GnT-V and to perform a preliminary functional characterization. WM-266-4 cells were stably transfected with human MGAT3 or MGAT5 cDNAs. GnT-III and GnT-V activities were assayed with a novel HPLC method based on labeling of N-glycan acceptor with 2-aminobenzamide (adapted from Taniguchi et al., 1989). Higher expression and activities of glycosyltransferases were detected. Increased amounts of “bisected” and beta1-6 branched N-glycans were present on melanoma cell adhesion molecule (known as MCAM/MUC18). However, cells did not display significant differences in viability and capabilities to migrate through an endothelial layer. To the best of our knowledge, the result of our study is the first to demonstrate that “bisected” N-glycans can be carried by MCAM. Moreover, increased modification of this protein by the two glycosyltransferases in WM-266-4-GnT-III cells was the consequence of the overexpression of only one enzyme. The obtained model can be useful for studying mechanisms of N-glycans branching and better understanding of their role in cancer progression. The proposed modification of the glycosyltransferase activity assay has shown to be a good alternative for 2-aminopyridine based HPLC systems.
E-cadherin and adherens-junctions stability in gastric carcinoma: Functional implications of glycosyltransferases involving N-glycan branching biosynthesis, N-acetylglucosaminyltransferases III and v
2013, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
Overall, we decoded the functions of the bisecting GlcNAc N-glycans (product of GnT-III) and β1,6 GlcNAc branching N-glycans (product of GnT-V), in the modulation and regulation of E-cadherin in human gastric cancer. MKN45 gastric cell line stably transfected with GnT-III or GnT-V cDNAs and with the empty vector (mock cells) were used [24,27,28]. These cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (1000 μg/ml), under the selection of G418 (500 μg/ml) in 5% CO2.
E-cadherin is a cell–cell adhesion molecule and the dysfunction of which is a common feature of more than 70% of all invasive carcinomas, including gastric cancer. Mechanisms behind the loss of E-cadherin function in gastric carcinomas include mutations and silencing at either the DNA or RNA level. Nevertheless, in a high percentage of gastric carcinoma cases displaying E-cadherin dysfunction, the mechanism responsible for E-cadherin dysregulation is unknown. We have previously demonstrated the existence of a bi-directional cross-talk between E-cadherin and two major N-glycan processing enzymes, N-acetylglucosaminyltransferase-III or -V (GnT-III or GnT-V).
In the present study, we have characterized the functional implications of the N-glycans catalyzed by GnT-III and GnT-V on the regulation of E-cadherin biological functions and in the molecular assembly and stability of adherens-junctions in a gastric cancer model. The results were validated in human gastric carcinoma samples.
We demonstrated that GnT-III induced a stabilizing effect on E-cadherin at the cell membrane by inducing a delay in the turnover rate of the protein, contributing for the formation of stable and functional adherens-junctions, and further preventing clathrin-dependent E-cadherin endocytosis. Conversely, GnT-V promotes the destabilization of E-cadherin, leading to its mislocalization and unstable adherens-junctions with impairment of cell–cell adhesion.
This supports the role of GnT-III on E-cadherin-mediated tumor suppression, and GnT-V on E-cadherin-mediated tumor invasion.
These results contribute to fill the gap of knowledge of those human carcinoma cases harboring E-cadherin dysfunction, opening new insights into the molecular mechanisms underlying E-cadherin regulation in gastric cancer with potential translational clinical applications.
Enhanced epithelial-mesenchymal transition-like phenotype in N-acetylglucosaminyltransferase V transgenic mouse skin promotes wound healing
2011, Journal of Biological Chemistry
Citation Excerpt :
In particular, N-acetylglucosaminyltransferase V (GnT-V)3 plays an important role in carcinogenesis and tumor metastasis (2). To characterize the detailed molecular mechanisms underlying GnT-V-related tumor metastasis, we and other groups succeeded in purifying and cloning GnT-V (3–5). In addition, we developed a sugar remodeling system of cancer cells and demonstrated the biological function of GnT-V in tumor metastasis through biochemical analysis of its target glycoproteins (6).
N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the β1,6 branching of N-acetylglucosamine on N-glycans. GnT-V expression is elevated during malignant transformation in various types of cancer. However, the mechanism by which GnT-V promotes cancer progression is unclear. To characterize the biological significance of GnT-V, we established GnT-V transgenic (Tg) mice, in which GnT-V is regulated by a β-actin promoter. No spontaneous cancer was detected in any organs of the GnT-V Tg mice. However, GnT-V expression was up-regulated in GnT-V Tg mouse skin, and cultured keratinocytes derived from these mice showed enhanced migration, which was associated with changes in E-cadherin localization and epithelial-mesenchymal transition (EMT). Further, EMT-associated factors snail, twist, and N-cadherin were up-regulated, and cutaneous wound healing was accelerated in vivo. We further investigated the detailed mechanisms of EMT by assessing EGF signaling and found up-regulated EGF receptor signaling in GnT-V Tg mouse keratinocytes. These findings indicate that GnT-V overexpression promotes EMT and keratinocyte migration in part through enhanced EGF receptor signaling.
Cancer glycomics
2010, Handbook of Glycomics
New technologies are continuously evolving to identify specific glycan, glycopeptide, and glycoprotein differences found in cancer patient sera when compared to non-diseased, control sera. This chapter emphasizes recent studies that have focused on using analytical technologies to characterize altered glycan and glycoconjugate expression in cancer cells, tissues, and fluids such as serum from patients with cancer. Particular emphasis is placed on those that seek to exploit these aberrant glycan changes to develop potential tumor markers that could be used to direct chemotherapeutic agents or serve as diagnostic or prognostic markers of cancer or cancer risk. With the possibility of utilizing actual tumor-derived glycans and glycopeptides immobilized in arrays on slides, the odds of detecting antibodies to cancer-specific glycopeptides appear to significantly increase. There is new promise in the generation of robust immune responses to glycopeptides. Applying glycomic technologies for the identification of cancer-specific glycopeptides could, therefore, lead to the generation of efficacious anti-cancer vaccines. A formidable challenge that impacts all glycomics marker discoveries is to develop reliable clinical assay systems for detection of specific cancer glycoprotein glycoform such that large numbers of samples can be assayed during clinical trials. The results show that complicated sample preparation and time-consuming analytical procedures must be reduced and adapted so that analyses can be performed in a specialized clinical laboratory.

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Regular ArticleCDNA Cloning and Chromosomal Mapping of Human N-Acetylglucosaminyltransferase-V

Abstract

Regular Article
CDNA Cloning and Chromosomal Mapping of Human N-Acetylglucosaminyltransferase-V