This review focuses on glycosides that act as metabolic decoys and inhibitors of glycosylation. These compounds resemble metabolic intermediates and act as primers of glycan biosynthesis, diverting the synthesis of sugar chains from endogenous glycoconjugates. Thus, glycosides inhibit glycoconjugate formation and provide a tool for studying glycan function in cells and whole organisms. Such primers may have future therapeutic potential through their ability to alter glycan-dependent pathologic processes in several disease states.
Sialyltransferase is one of the most demanding glycosyltransferases since chemical sialylation is usually furnished in low yield with low stereoselectivity. Recently, several bacterial sialyltransferases were shown to exhibit broader substrate specificity than that of mammalian counterparts. This suggests the potential usefulness of bacterial sialyltransferases in chemoenzymatic synthesis of natural and nonnatural sialooligosaccharides and sialoglycoconjugates.
A variety of methods for assaying glycosyltransferase activity have been developed driven by the specific interests and type of information required by researchers. These methods include the routine monitoring of activity during and after enzyme purification and in biocatalysis, detailed mechanistic and inhibition evaluations, and high throughput screening for novel glycosyltransferase activities or inhibitors. Representative assay methods for all of these applications including their advantages and limitations are summarized in this review.
Using calcitonin as a model peptide, we have systematically studied how glycosylation affects the three-dimensionalstructure and the biological activity of peptides. In the case ofthe N-glycosylation, the peptide backbone conformation did notchange, but the biological activity was shown to increase or decrease depending on the carbohydrate structure. For the O-glycosylation, both the three-dimensional structure and the biological activity were shown to change depending on the attachment site of the carbohydrate. Our results show that the carbohydrate portion of glycopeptides affects both the three-dimensional structure and the biological activity in a carbohydrate structure- and a binding site-dependent manner, and the knowledge should be useful in applying glycosylation to the development of biologically active peptides.
Fibroblast growth factor (FGF) family members cannot exert their biological activity in the absence of their interaction with heparan sulfate or heparin in the vicinity of their receptors. Cell surface proteoglycans are thought to serve such heparan sulfate sugar chains in physiological conditions. In an attempt to utilize this interaction for engineering FGFs, our aim was to construct an FGF neoglycoprotein with heparan sulfate sugar chains. A novel chimeric protein was designed in which part of syndecan-4 containing glycosaminoglycan (GAG) attachment sites was ligated to the N-terminus of FGF-1. When the cDNA encoding the protein was transfected into CHO-K1 cells, the chimeric protein was secreted into the conditioned medium with GAG modifications. One fraction of the resultant chimeric protein had acquired the ability to stimulate DNA synthesis of the target cells in the absence of exogenous or cellular heparin/heparan sulfate, indicating that the biological function of FGF-1 was successfully modified by this approach. Furthermore in artificial wound fluid which mimics inflammation, the activity of this protein was highly augmented due to the generation of small HS fragments which act as activators. These results suggest that engineering heparin-binding proteins with heparan sulfate sugar chains is highly effective.
Sugar chains are synthesized in cells by sequential reactions of glycosylation enzymes. These sugar chains do not have uniform structure, but rather constitute a mixture of various structures which correspond to the intermediates of maturation. Sugar chains on recombinant glycoproteins produced by animal cells also contain various forms. A method of producing uniform glycoproteins with specific sugar chain structure is required. We have been studying biosynthetic control of sugar chains on glycoproteins using animal cells. In the present review, we present the results of our research, focusing on the following two subjects: remodeling branch structures of complex-type sugar chains on interferon-γ (IFN-γ); control of addition of bisecting GlcNAc to immunoglobulin M (IgM). In the investigation into sugar chains on IFN-γ, we used Chinese hamster ovary cells as hosts for recombinant production of IFN-γ, and overexpressed genes for N-acetylglucosaminyltransferases IV and V, thereby changing sugar chains on IFN-γ to highly branched types. In the investigation into sugar chains on IgM, we controlled addition of bisecting GlcNAc to sugar chains on IgM by regulating intracellular activity of β1, 4-galactosyltransferase and N-acetylglucosaminyltransferase III. Our results show that regulation of the expression of glycosylation enzymes can be effectively used to control sugar chain structures.
Our principal goal was the molecular breeding of yeasts able to produce human-type sugar chains and which could be used for pharmaceutical production. This ambitious project was carried out in conjunction with two other short-and mediumterm supporting sub-projects. The short-term project lasted approximately two years and involved the development of tools for Glycobiology. The initial step involved determining the sequence of one “Mucin box”, necessary for formation of mucin-type sugar chains. Subsequently, we cloned the gene for Endoglycosidase M, an enzyme that catalyzes transglycosylation and established largescale preparation of the recombinant enzyme. We succeeded in creating a novel RNase with complex-type sugar chains instead of the original high-mannose type sugar chains generated in vitro. The theme of the medium-term project, involved the purification of the enzyme GnT-IV glycosyltransferase, which is closely associated with the activity of glycoprotein. We proceeded to clone the gene for this enzyme and discovered, to our surprise, that two types of genes exist for GnT-IV glycosyltransferase.
The yeast Saccharomyces cerevisiae is a eucaryote that is easy to use for gene engineering and in cultivation. Yeast is suitable for production of valuable secretory proteins; however, in the case of production of glycoproteins in yeast, the mannose outer chain that consists of 30-100 mannose residues is attached to the N-linked oligosaccharide of the glycoproteins, which causes problems such as immunogenecity against humans or a reduction in protein activity. We have studied the molecular breeding of a yeast that produces human compatible glycoproteins by eliminating the yeast mannose outer chain and adding of essential genes for the synthesis of mammalian type oligosaccharide. In this study, we successfully cloned and disrupted the OCH1 gene that encodes essential mannosyltransferase for the synthesis of mannose outer chain. We also succeeded in the cloning and disrupting the MNN4 and MNN6 genes that are involved in yeast specific mannosylphosphorylation. Furthermore we tried to clone glycosyltransferases, glycosidases, and sugar nucleotide transporter genes for the synthesis of mammalian type sugar chain, and tried to express these genes in yeast. In this report we introduce the results of these studies.