G lycans include oligosaccharides (short carbohydrate chains) and large complex molecules, i.e. complex carbohydrates such as glycoproteins, glycolipids, and proteoglycans. Glycans are mostly found on the cell surface and extracellular matrix (ECM), and also in various organellae such as Golgi, ER, lysosome, cytosol, and nuclei. As compared to research on DNA, RNA, and proteins, studies on glycans are technically difficult and research in this field has been not emphasized for a long period; the same is true for glycomics as compared to proteomic research. In order to characterize the structures of glycans, glycobiology including glycomics is essential for understanding of the structures and functions of proteins.Among the various post-translational modification reactions involving proteins, glycosylation is the most common; nearly 50% of all proteins are thought to be glycosylated.(1) Glycosylation reactions are catalyzed by the actions of glycosyltransferases, sugar chains being added to various complex carbohydrates. In the last couple of years most glycosyltransferases (over 180 glycosyltransferase genes) have been identified, based on the genome sequence data bases and bioinformatics approach. (4-8) Cell surface carbohydrates are involved in a variety of interactions between a cell and its extracellular environment, since they are located on the outermost layer of the cell; carbohydrates are the first molecules to be encountered and recognized by other cells, antibodies, invading viruses, and bacteria. Many secreted molecules such as hormones and toxins have also been reported to bind to carbohydrate receptors on the cell surface. In addition, most receptors on the cell surface are N-glycosylated, including epithelial growth factor receptor (EGFR), integrins, and transforming growth factor β receptor (TGFβR). Modified oligosaccharides affect protein folding and stability, and have the ability to interfere with carbohydrate-carbohydrate, carbohydrate-protein, and glycoprotein-glycoprotein interactions, and as a result, regulate many physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion, and host-pathogen interactions, cell trafficking, and transmembrane signaling. Therefore, it is not surprising that aberrant glycosylation patterns can serve as markers for certain disease states including cancer metastasis, development, and differentiation. (9) In this review, we mainly focus on the modification of N-glycans of receptors on the cell surface to further address the important roles of N-glycans in cancer science. Important functions of N-acetylglucosaminyltransferasesGnT-V. N-Acetylglucosaminyltransferase V (GnT-V) (10)(11)(12) has been thought to have a close relationship with cancer metastasis. (13,14) GnT-V catalyzes the formation of β1,6 GlcNAc branching structures, which play important roles in tumor metastasis (Fig. 1).(15) GnT-V deficient mice were generated to assess the functions of GnT-V products in normal development and cancer progression.(16) T...

The deposition of amyloid ␤-peptide (A␤) in the brain is closely associated with the development of Alzheimer's disease. A␤ is generated from the amyloid precursor protein (APP) by sequential action of ␤-secretase (BACE1) and ␥-secretase. Although BACE1 is distributed among various other tissues, its physiological substrates other than APP have yet to be identified. ST6Gal I is a sialyltransferase that produces a sialyl␣2,6galactose residue, and the enzyme is secreted out of the cell after proteolytic cleavage. We report here that BACE1 is involved in the proteolytic cleavage of ST6Gal I, on the basis of the following observations. ST6Gal I was colocalized with BACE1 in the Golgi apparatus by immunofluorescence microscopy, suggesting that BACE1 acts on ST6Gal I within the same intracellular compartment. When BACE1 was overexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I markedly increased. When APP SW (Swedish familial Alzheimer's disease mutation), a preferable substrate for BACE1, was coexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I significantly decreased, suggesting that that the ␤-cleavage of overexpressed APP SW competes with ST6Gal I processing. In addition, BACE1-Fc (Fc, the hinge and constant region of IgG) chimera cleaved protein A-ST6Gal I fusion protein in vitro. Thus, we conclude that BACE1 is responsible for the cleavage and secretion of ST6Gal I.

The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics.

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.