Glycomics Approach Research Articles

GLYCOMICS APPROACH TO STRUCTURE-FUNCTION RELATIONSHIPS OF GLYCOSAMINOGLYCANS

Extracellular modulation of phenotype is an emerging paradigm in this current postgenomics age of molecular and cell biology. Glycosaminoglycans (GAGs) are primary components of the cell surface and the cell-extracellular matrix (ECM) interface. Advances in the technology to analyze GAGs and in whole-organism genetics have led to a dramatic increase in the known important biological role of these complex polysaccharides. Owing to their ubiquitous distribution at the cell-ECM interface, GAGs interact with numerous proteins and modulate their activity, thus impinging on fundamental biological processes such as cell growth and development. Many recent reviews have captured important aspects of GAG structure and biosynthesis, GAG-protein interactions, and GAG biology. GAG research is currently at a stage where there is a need for an integrated systems or glycomics approach, which involves an integration of all of the above concepts to define their structure-function relationships. Focusing on heparin/heparan (HSGAGs) and chondroitin/dermatan sulfate (CSGAGs), this review highlights the important aspects of GAGs and summarizes these aspects in the context of taking a glycomics approach that integrates the different technologies to define structure-function relationships of GAGs.

Profiling of Glycans in Serum for the Discovery of Potential Biomarkers for Ovarian Cancer

A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens.

Glycomics Analyses of Tear Fluid for the Diagnostic Detection of Ocular Rosacea

A Glycomics approach to detect disease is illustrated in the analyses of human tear fluid for rosacea. The diagnosis of ocular rosacea is particularly challenging in a subgroup of patients that do not present with typical facial skin findings but have ocular signs and symptoms. Indeed, up to 90% of patients with ocular rosacea may have neither obvious roseatic skin changes nor a previous diagnosis of rosacea. Tear fluid was collected from 37 subjects (21 controls and 16 patients with ocular rosacea) after conjunctival stimulation with filter (Schirmer) paper. O-linked oligosaccharides were released from tear fluid by beta-elimination and then purified using solid-phase extraction. Mass spectra were recorded on an external source HiResMALDI with a 7.0 T magnet. Mass spectra were obtained in both the positive and negative modes. However, signals were stronger in the negative mode. Tear fluid samples from rosacea patients yielded distinctive clusters of peaks that extend to higher masses. Patients with rosacea presented several oligomeric series that were not found in the controls. To discriminate the ocular rosacea cases from the normal controls, the sum of absolute intensities of 13 series corresponding to nearly 50 identified mass spectrum peaks was used. Thirty-six out of the 37 samples were correctly classified. This yields a sensitivity of 100% (95% CI 79.5-100) and specificity of 95.2% (95% CI 76.2-99.9). The high abundance of oligosaccharides in the tear fluid of patients with rosacea may lead to an objective diagnostic marker for the disease.

Identification of target proteins of N‐acetylglucosaminyl‐transferase V and fucosyltransferase 8 in human gastric tissues by glycomic approach

To understand better the mechanism underlying gastric cancer and search for potential markers for gastric cancer prognosis, the proteomic analysis of gastric cancer tissues was conducted using two-dimensional gel electrophoresis and lectin blot, followed by electrospray ionization-mass spectrometry. These approaches permitted identification of glyco- or putative glycosylated proteins which may be associated with tumorigenesis. The proteins identified include molecules involved in sugar metabolism, signal transduction, proteolysis, and stress, as well as several unknown proteins, which were aberrantly glycosylated as evidenced by the L-phytohemagglutinin blot.

Functional glycomics and evidence for gain- and loss-of-functions of target proteins for glycosyltransferases involved in <i>N</i>-glycan biosynthesis: their pivotal roles in growth and development, cancer metastasis and antibody therapy against cancer

N-Acetylglucosaminyltransferase V (GnT-V), N-acetylglucosaminyltransferase III (GnT-III) and α1-6 fucosyltransferase (Fut8) catalyze reactions that form biologically important branching N-linked sugar chains in glycoproteins. The above three branching N-glycan sugar chains, β1-6 GlcNAc branching, bisecting GlcNAc and core fucose (α1-6 fucose), play major roles in cancer invasion and metastasis, the inhibition of cancer metastasis, and antibody-dependent cellular cytotoxicity (ADCC), growth and development, respectively. A functional glycomic approach identified the gain- and loss-of-functions of glycoproteins as the result of the aberrant glycosylation. A membrane-type metal dependent serine proteinase designated matriptase which contains β1-6 GlcNAc branching became resistant to auto-digestion and proteolysis by trypsin, and resulted in a constitutively active form which might be implicated in cancer invasion and metastasis. GnT-V also acts as an angiogenic factor without the mediation of functions as a glycosyltransferase. Recently, a GnT-V homologue, GnT-IX has been identified. This gene is expressed at relatively high levels in the brain and acts on N-glycans to form a unique branched structure, as well as O-mannosyl glycans. The addition of bisecting GlcNAc to various signaling molecules or adhesion molecules suppresses cancer metastasis. Fut8 knock-out mice, due to the lack of a core fucose (α1-6 fucose) in target glycoproteins, show disorders in growth and development. The presence of a bisecting GlcNAc or the absence of a core fucose in IgG molecules enhances ADCC activity for killing tumor cells by up to 10 to 100 fold and therefore is thought to have considerable use in antibody therapy against cancer. These data clearly indicate that gain- and loss-of-functions of target proteins for these glycosyltransferases are biologically important. (Communicated by Tamio YAMAKAWA, M.J.A., Feb. 12, 2004)

A glycomic approach to proteoglycan with a two-dimensional polysaccharide chain map

Glycosaminoglycan chains were liberated from proteoglycans (bovine lung, tracheal cartilage, and cerebrum) by successive digestion with actinase and with cellulase from Aspergillus niger, which has endo-β-xylosidase activity. The glycosaminoglycan chains were fluorescence-labeled with 2-aminopyridine after digestion with Streptomyces hyaluronidase. The resulting pyridylamino-glycosaminoglycans, including heparan sulfate, chondroitin sulfate/dermatan sulfate, and heparin, were separated by high-performance liquid chromatography. Each separated fraction was analyzed by two types of high-performance liquid chromatography: gel-filtration chromatography and anion-exchange chromatography. The correlation between molecular weight and degree of sulfation could be shown on the two-dimensional polysaccharide chain map. Use of a commonly available cellulase with endo-β-xylosidase activity together with the two-dimensional polysaccharide chain map allows easy analysis of various glycosaminoglycan chains and comprehensive comparison among the structures. These techniques will become useful tools in the further development of glycotechnology and glycome analysis.

A Glycomic Approach to Hepatic Tumors in N -acetylglucosaminyltransferase III (GnT-III) Transgenic Mice Induced by Diethylnitrosamine (DEN): Identification of Haptoglobin as a Target Molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo. In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.

A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes

A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes

A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes.

The transfection of glycoprotein glycosyltransferase genes into cells leads to modification of both the structure and function of the glycoproteins and as a result, changes in glycome patterns. N-glycan branching enzymes hold some promise as a model system for the identification of glycome patterns. Both N-acetylglucosaminyltransferase III and alpha 1-6 fucosyltransferase are typical glycosyltransferases, which are involved in the branching of N-glycans. The resulting enzymatic products, bisecting N-GlcNAc and alpha 1-6 fucose residues, are no longer modified by other glycosyltransferases and it is a relatively simple task to identify their modification by means of lectins. In this review, the glycome patterns of glycosyltransferase gene transfectants and the non-transfectants were compared by two-dimensional gel electrophoresis and lectin staining, and the biological significance of the two genes are described. Analyses of glycome patterns by transfecting glycosyltransferase genes will lead to new fields of study in the area of postgenome research.

Implication of GnT-V in cancer metastasis: a glycomic approach for identification of a target protein and its unique function as an angiogenic cofactor.

It is known that sugar chains have a variety of functions and play a key role in development, differentiation, cell adhesion and cell growth in cells [1,2]. Our group has focused on the underlying mechanism of the biosynthesis of N -glycan branchings in glycoproteins, and reported on various N -glycan branching enzymes that play a pivotal role in various steps [3]. A possible link between β1-6 branched structures of N glycans and cancer metastasis has been proposed by several authors [4–6]. However before the cloning of the UDP-GlcNAc α-mannoside β1-6-N -acetylglucosaminyltransferase (GnT-V) enzyme which catalyzes the formation of β1-6 branching was carried out, it was not clear whether or not β1-6 branching is actually related to the metastatic potential of cancer cells. The enzyme was independently purified from rat kidney [7] by Pierce’s group and from human cancer cells [8] by our group, and both groups subsequently cloned their cDNAs [9,10]. Using the cloned genes several studies have presented evidence for the presence of strong association between β1-6 branching and cancer metastasis [11–13]. Knock out mice developed by Dennis’s group confirmed that the gene is highly linked to cancer development [14]. However, in the case of glycosyl-