Expression System for Human Glycosyltransferases and Its Application

Abstract

Glycosylation of proteins and lipids is the most complex of their various co translational and post translational modifications. Glycan modification contributes to the folding and conformational stability of many proteins, 1) and modified glycoproteins and glycolipids on the cell surface are known to act as ligands for host pathogen interactions, 2) cell adhesion and cell signaling.3) These phenomena are influenced by changes in glycan structures. Since it is known that glycans on proteins and lipids are synthesized by sequential reactions by glycosyltransferases, the alteration of the glycan structure is dependent on the expression of glycan synthesis related genes encoding glycosyltransferases, sulfotransferases, glycosidases, sugar nucleotide synthetases, and sugar nucleotide transporters. Glycan structures are defined by not only the expression level of the genes but also several other factors, such as substrate specificity, competition of more than two glycosyltransferases for the same substrate, localization of glycosyltransferases, and concentration of donor substrate as sugar nucleotides in the Golgi apparatus. Therefore, the expression of glycosyltransferases and the determination of their enzymatic characterizations in detail are significant for the elucidation of the biosynthetic machinery of glycans.In addition to basic study of glycosyltransferases, recombinant enzymes are available for the synthesis of glycans and glycopeptides and for the modification of glycoproteins in vitro. In recent years, mass spectrometry, 4,5) high performance liquid chromatography (HPLC), 6,7) and lectin microarray systems 8,9) have been developed for the structural analysis of glycans. A glycan library composed of a variety of structure defined glycans is indispensable for quantitative analyses using these technologies. Since human glycosyltransferases have a strict specificity toward donor and acceptor substrates, it is reasonable to employ in vitro synthesis of glycans by glycosyltransferases for producing glycan standards. Glycan array technology, and the ease with which the glycans can be customized for display, may enhance our ability to detect unknown lectins in mammals and to probe pathogen specific glycan interactions. 10,11) Application of glycosyltransferases in other areas of interest includes the modification of glycans attached to glycoproteins, which has also become an important topic in recent years. Because biologics and biosimilar drugs such as therapeutic antibodies and cytokines are the largest class of new drug candidates being developed by pharmaceutical companies, there are strong concerns that the heterogeneity of the glycan structure may sometimes affect their in vivo activity. Therefore, the development of technology to produce glycoproteins with homogeneous glycans is essential.In our research center, the Glycogene (GG) Project was started in 2001, in which as many novel genes as possible, which were the candidates for glycogenes including glycosyltransferases and sugar nucleotide transporters, ...