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Glycobiology-related Enzymes

Introduction of Glycobiology-related Enzymes

Glycomics—the science of studying the structure and function of glycans has become the third research hotspot after genomics and proteomics. Glycans bind to proteins or lipids in a covalent manner, forming a complex and diverse structure in the organism. Among them, glycosylation-modified proteins have been shown to be involved in many biological processes related to cell level and biological development, such as protein maturation and folding, cell adhesion, molecular transport and clearance, receptor binding and activation, signal transduction, and endocytosis (Figure 1). In eukaryotes, there are two main forms of protein glycosylation modification: N-glycosylation and O-glycosylation. The biosynthesis of the sugar chains of cell surface is mainly catalyzed by the glycosyltransferases. The glycosyltransferase superfamily can be divided into multiple functional subfamilies according to the sugar donor substrate and the nature of the catalyzed glycosidic bond, such as the sialyltransferase family, the fucosyltransferase family, and the GlcNAc transferases family. Each subfamily forms a unique glycosidic bond by catalyzing each specific substrate. Abnormal glycosylation modification, i.e., the abnormal expression of glycosyltransferases and abnormalities in their sugar chain structure are common features of malignant tumor development and metastasis. In addition, glycosidase is a type of enzyme that hydrolyzes glycosic bonds, and plays an important role in the hydrolysis and synthesis of carbohydrates and glycoconjugates in organisms. Glycosidases are found in almost all organisms. They are a class of enzymes that hydrolyze glycosidic bonds in various sugar-containing compounds (including monoglycosides, oligosaccharides, polysaccharides, saponins, glycoproteins, etc.) to form monosaccharides, oligosaccharides or glycoconjugates by endo- or exo-cleavage. Glycosidases play an important role in oligosaccharide synthesis, synthesis of alkyl glycosides and aryl glycosides, glycosylation of amino acids and polypeptides, and glycosylation of antibiotics.

Glycans Permeate Cellular Biology.

Figure 1. Glycans Permeate Cellular Biology.

Classification of Glycobiology-related Enzymes

Fucosyltransferases (FUTs) are a class of enzymes that can catalyze the transfer of fucosyl groups from GDP-fucose donors to their corresponding receptors to form fucosyl-modified glycoproteins or glycolipids (Figure 2). Hydrophobic cluster analysis (HCA) found that fucosyltransferases have five conserved peptide motifs. Fucosyltransferases include the following four classes: protein O-fucosyltransferases (POFUT1 and POFUT2) that can recognize the protein serine, threonine side chain hydroxyl groups; α-1, 2-fucosyltransferases (FUTI and FUTII) that can recognize the galactose residues on the type II sugar chain Galβ1, 4GlcNAc structure; α-1, 3/ 4-fucosyltransferases (FUTIII~VII, FUTIX~XI) that can recognize N-acetylglucosamine residue on the sugar chain; α-1, 6-fucosyltransferase (FUTVIII) that can recognize the N-acetylglucosamine residue bound to asparagine. According to the position of the fucosylation modification, these modifications can be further divided into three types: core fucosylation modification (FUTVIII), terminal fucosylation modification (FUTI~VII, FUTIX~XI) and protein O-fucosylation modification (POFUTI and POFUTII). The expression of fucosyltransferases exhibits complex cellular and tissue specificity. Although most fucosyltransferases locate to the Golgi apparatus, the protein O-fucosyltransferases are found to locate to the endoplasmic reticulum.

Fucosylation sites of human a-1, 2-, a-1, 3/ 4-, a-1, 6- and O-FucTs. FucTs I–IX (or FucTs 1–9) catalyze the fucosyl transfer to different sugar residues of O-glycans and N-glycans, as indicated by the arrows.

Figure 2. Fucosylation sites of human a-1, 2-, a-1, 3/ 4-, a-1, 6- and O-FucTs. FucTs I–IX (or FucTs 1–9) catalyze the fucosyl transfer to different sugar residues of O-glycans and N-glycans, as indicated by the arrows.

Sialylation modification is one of the most important glycosylation modifications at the end of the sugar chain. The cytidine-5’-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) is the donor substrate of sialyltransferases (STs) which catalyzes sialic acid with different glycosidic bonds (α2-3, α2-6 or α2-8) to link to glycoproteins (or galactoses at the end of glycolipid, N-acetylgalactosamine or other sialic acids). The sialyltransferase in eukaryotic cells is similar to other glycosyltransferases and belongs to the type II transmembrane protein (i.e., the C-terminus of protein is in the extracellular region while the N-terminus is in the intracellular region). The domain of a typical sialyltransferase consists mainly of a stem region and a catalytic domain, wherein the catalytic domain comprises a sialic acid motif L with 48 to 49 amino acids and a sialic acid motif S with 23 amino acids. The sialyltransferase is generally localized to the Golgi apparatus, and some sialyltransferases are present in the form of soluble enzymes. There are 20 STs: ST3Gal I-VI catalyzing α2-3 sialylation; ST6Gal I-II (N-linked) and ST6GalNAc I-VI (O-linked) catalyzing α2-6 sialylation; ST8Sia I-VI catalyzing α2-8 sialylation. The process of sialyltransferase-mediated sialylation of the glycoconjugate makes the sialylated glycoconjugates tissue-, cell- and molecular-specific due to the complex and variability of the structure of sialic acid, ligation and receptor.

N-acetylglucosaminyltransferases (GnTs) are localized to the Golgi of eukaryotes. And they can catalyze the connection between the D-mannose (Man) and the GlcNAc domain of the UDP-N-acetylglucosamine, forming multiple N-glycan branching structures. GnTs belong to type II transmembrane proteins, including intracellular, transmembrane, stem and catalytic domains and there is a conserved peptide domain, DxD motif, in the catalytic domain of GnTs. GnTs can be classified into the following six types according to the catalytic starting position: GnTI (α-1, 3-mannosyl-glycoprotein β-1, 2-N-acetylglucosaminyltransferase I); GnTII (α-1, 6-mannosyl-glycoprotein β-1,2-N-acetylglucosaminyltransferase II); GnTIII (β-1, 4-N-acetylglucosaminyltransferase III); GnTIV (α-1, 3-mannosyl-glycoprotein β-1, 4-N-acetylglucosaminyltransferase IV); GnTV (α- 1, 6-mannosyl-glycoprotein β-1, 6-N-acetylglucosaminyltransferase V); GnTVI (α-1, 6-mannosyl-glycoprotein β-4-N-acetylglucosaminyltransferase VI). The most important enzymes in the current research and N-glycan synthesis are GnTIII and GnTV.

Function of Glycobiology-related Enzymes

In mammals, the fucosylation modification on the cell surface is carried out by fucosyltransferase. Human ABO blood group antigen and Lewis system are oligosaccharides synthesized by the sequential action of glycosyltransferases, in which fucosyltransferase plays a key role. For example, LeY blood group antigen is an oligosaccharide and its α-1, 3 fucosylation is mainly catalyzed by FUTIV. The fucosylation of proteins plays an important role in many physiological processes in eukaryotes, including cell adhesion during development, inflammatory response, leukocyte transport or lymphocyte homing, and fertilization. In the pathological situation such as immunization or tumor, the body often is abnormal in fucosylation. Abnormal fucosylation is the most important abnormal glycosylation modification in hepatocellular carcinoma, and fucosylated alpha-fetoprotein (AFP-L3) was used as a tumor marker for hepatocellular carcinoma in the United States in 2005. The expression and activity of core fucosyltransferase FUTVIII is abnormal in tumors such as lung cancer, pancreatic cancer, ovarian cancer, liver cancer, and colon cancer. Mathieu et al found that the overexpression of FUTI in hepatoma cell HepG2 can selectively prevent the interaction of sLex with endothelial E-selectin, thereby inhibiting the adhesion and rolling of HepG2 to activated endothelial cells. The expression level of POFUTI is significantly increased in oral cancer, liver cancer, gastric cancer and breast cancer, and it is closely related to the primary tumor size of oral cancer, phenotype of invasive gastric cancer, lymph node metastasis of breast cancer.

In view of the negative charge of sialic acid itself, strong hydrophilicity and the localization of the end of the sugar chain, sialic acid can directly or indirectly participate in various life activities of cells. The sialylation of cell surface glycoconjugates not only participates in the regulation of membrane protein conformation, but also participates in the transmission of normal signals such as growth, migration, and apoptosis between cells. In addition, the biological functions of sialylation also include structural and physical functions such as regulation of glomerular filtration membrane charge, plasticity of nerve cells, and charge repulsion of erythrocyte membranes. And sialic acid at the end of the cell surface sugar chain can block the corresponding antigen site recognition to protect the glycoconjugate from being recognized and degraded by the immune system. Sialic acid can also interact with ligands such as inorganic ions, hormones, lectins, and antibodies to mediate inflammation and immune responses by regulating cell adhesion. The abnormal sialylation on the cell surface is also involved in the development of tumor cells, such as proliferation, migration, invasion and immune escape. Tumor-specific metastasis is closely related to the adhesion between tumor cells and specific vascular endothelial cells (Figure 3). Multiple adhesion molecules such as E-selectin and its ligands sLeX and sLeA play important roles in tumor metastasis, while α-2, 3-sialyltransferases are the main enzymes that catalyze the formation of sLeX and sLeA. It has been reported that ST6GalI mediates the adhesion and migration of ovarian cancer cells to Collagen 1 by modulating the α2-6 sialylation of integrin β1. ST8SiaI and GD3 are highly expressed in neuroectodermal-derived malignancies such as melanoma, glioblastoma, neuroblastoma, and estrogen receptor (ER)-negative breast cancer, and they are also involved in tumor cell proliferation, migration, adhesion, and angiogenesis.

Aberrant sialylation favors tumor growth and progression.

Figure 3. Aberrant sialylation favors tumor growth and progression.

N-acetylglucosaminyltransferase is involved in cell adhesion, signal transduction and other life activities due to its species diversity and high specificity. It mediates the N-acetylglucosaminylation of glycans, which involves in various physiological and pathological processes of cells. Mgat1 (a gene encoding GnTI)-dependent N-glycans are essential for the development of vertebrates and invertebrate metazoans. N-acetylglucosaminylation on the cell surface may have a role in stabilizing the structure of the protease. For example, GnTV is involved in post-translational modification of matrix protease, and the stability of matrix protease is increased by adding β1, 6 branching structures to protect it from degrading by trypsin. In addition, GnTV can modify receptor glycoproteins on cell membranes. For example, the high expression of GnTV will increase the downstream signal transduction by increasing the binding of EGF to its receptor by increasing the β1, 6 branching structures of the EGFR. It has been reported that the increase of GlcNAcβl, 6 branching structures caused by the increase of GnTV expression can promote tumor development and metastasis. GnTIII can alter the related signaling pathways by affecting the sugar chain modification of NGFs or EGFs, further affecting the biological behaviors of tumor cells such as migration, invasion and apoptosis. Overexpression of GnTV in gastric cancer cells induces the mislocalization of E-cadherin from the cell membrane to the cytoplasm.


  1. Tu Z, et al. ChemInform abstract: Development of fucosyltransferase and fucosidase inhibitors. Chemical Society Reviews. 2013, 42(10):4459-4475.
  2. Büll C, et al. Sialic acids sweeten a tumor's life. Cancer Research. 2014, 74(12):3199-3204.
  3. Hart G W, Copeland R J. Glycomics Hits the Big Time. Cell. 2010, 143(5):672-676.

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