The past half a century has witnessed a tremendous progress in structural determination of glycans in glycoconjugates. From the establishment of GlcNAc-Asn linkage in glycoproteins, a common core structure in N-glycans was soon elucidated. Subsequent meticulous structural studies utilizing chromatographic separation of labeled oligosaccharides accompanied by various chemical and enzymatic methods led to hundreds of established structures. Advancement in instrumentation (e.g., high performance liquid chromatography and nuclear magnetic resonance) was indispensable in the process, and now mass spectrometry of different modes has become essential, especially for high-throughput elucidation of structures. As more and more structures become known, the importance of database also has increased. All these progress contribute to expanded realm of glycomics and proteoglycomics.
Many of the most valuable recombinant DNA products, including monoclonal antibodies, are secreted glycoproteins, which contain N-glycans. The biochemical nature of these attached glycans depends on the characteristics of the host cell used, the protein synthesized, and the culture environment. The capacity to produce functional glycoproteins with desired glycosylation patterns depends on the presence of proper cellular enzymatic machinery required for the processing of N-glycans. It is well recongnized that expression of therapeutic proteins using insect cells in combination with the baculovirus-expression system has potential advantages in terms of the ease of large scale, low cost production of heterologous glycoproteins. However, the glycoforms expressed in several commonly used insect cell lines are markedly different from those expressed in human cells. As N-glycans often affect the in vivo biological activity, pharmacokinetics, and several other properties of glycoproteins, the glycoforms produced in this system have become a major concern in the last few years. Accordingly, considerable effort has been invested in the past decade in order to gain a better understanding of the processing of N-glycans in insect cells that generate different glycoforms, and also to overcome the bottlenecks to producing glycoproteins with humanized N-glycans that are more suitable for therapeutic use.
Herein the importance of the 2-dimensional mapping (2-DM) method in medical research area is introduced. This is the most powerful method for the analysis of glycan isomers. Glycans are synthesized by specific enzymes, and the analysis of isomers can lead to the identification of further directly related enzymes. Therefore, structural analysis using 2-DM connects readily to gene or protein expression analysis. Further, glycan purification during 2-DM analysis is also useful for sample preparation for mass spectroscopic analyses. This review describes the application of 2-DM to three research areas, namely, the α-mannosidase II alternative pathway, drug resistance in cancer cells, and the diagnosis of bladder cancer.
The multidimensional HPLC mapping method developed by Dr. Noriko Takahashi and coworkers has been widely recognized as a useful tool for the structural analysis of neutral and sialyl N-glycans. The utility of this method would be further extended if it could be used to analyze anionic oligosaccharides, including sulfated or glucuronylated glycans, since they play important roles in cell-cell communications. Hence, we developed HPLC maps of sulfated and glucuronylated oligosaccharides produced by recombinant sulfotransferases and glycosyltransferases. Here, we describe the HPLC map of anionic glycans and demonstrate its utility by applying this map for the characterization of sulfotransferase N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) and glycosylation profiling of influenza virus.
More than twenty recombinant antibody molecules are now licensed for treatment of a variety of cancers and chronic diseases. In addition there are, literally, hundreds in development and early phase clinical trials. Initially, the attraction of antibodies was their specificity for target antigens; however, it is now appreciated that the immune complexes formed trigger downstream biological activities that are critical to clinical success. It has been demonstrated that, although accounting for only 2-3% of antibody mass, glycosylation of the IgG-Fc is essential to the activation of such biologic mechanisms (effector functions). Additionally the precise structure of the attached oligosaccharide can determine protein conformation and stability and hence downstream biologic efficacy. Cellular engineering has been embraced to generate cell lines that synthesise selected homogeneous antibody glycoforms that may be optimal for a given disease indication. The advantage of being able to formulate therapeutic antibody preparations at high concentration might be aided by additional glycosylation within the IgG-Fab region. Novel production vehicles are also being developed that offer the possibility of lower production costs, with consequent lower cost of goods.
Thirty years ago, I discovered a new enzyme, glycoamidase A that cleaves carbohydrate moiety from glycopeptides without affecting peptide structures. This brought me to determination that I should commit comprehensive analysis of N-linked oligosaccharide structures in glycoproteins by using this enzyme. Now, the HPLC mapping method developed by myself is a powerful tool to identify the structures of N-glycans. Here I described my 30 years in the dream of the structural analyses of the N-linked oligosaccharides from the discovery of glycoamidase A to the development of the GALAXY database.