Accumulating evidence shows that the fates of glycoproteins in cells are determined through interactions of their carbohydrate moieties with a variety of intracellular lectins operating as molecular chaperones, cargo receptors, and ubiquitin ligases. Recent advances in developments of high-mannose type oligosaccharide libraries and structural biology of the carbohydrate-lectin interactions have revealed insights into sugar recognition by those intracellular lectins and the underlying mechanisms of quality control of glycoproteins. This article outlines the current knowledge on the molecular basis of sugar recognition by the intracellular lectins that control folding, transport, and degradation of glycoproteins in cells.
We have developed novel methods for large-scale production of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-D-mannosamine (ManNAc). For the production of Neu5Ac, we constructed two methods (chemoenzymatic synthesis and enzymation) from N-acetyl-D-glucosamine (GlcNAc) and pyruvate as substrates with N-acetylneuraminate lyase (Neu5Ac lyase) as a catalyst. In the epimerization from GlcNAc to ManNAc, one of them was carried out by the alkaline condition, and the other was by N-acyl-D-glucosamine 2-epimerase. On the other hand, we also established a novel method for the ManNAc production from Neu5Ac. It was characterized that Neu5Ac cleaved to ManNAc and pyruvate by Neu5Ac lyase on the anion exchange resin for accomplishing the complete cleavage of Neu5Ac. These methods are simple and easy, and fit for the large-scale production of Neu5Ac and ManNAc in industry, so that they may be used as raw ingredients for pharmaceuticals.
Although chemical glycosylation has certain advantages compared with enzymatic glycosylation in respect of its high flexibility and wide applicability, the reaction processes are complicated in many cases because the chemical reactions require multiple protection/de-protection steps. On the other hand, enzymatic glycosylation using glycosyltransferases is a single step process with high position- and anomer-selectivity and reaction yield. However, glycosyltransferases available for this purpose had been very limited and costly. Since then, progress has been made in the search of prokaryotic glycosyltransferases, increasing the types of enzymes that can be employed in synthesis and modification of sugar chains. Moreover, various studies have been making sugar nucleotides, which are donor substrate for the glycosyltransferases, less and less expensive. Sialic acids are present in a variety of glycoproteins and glycolipids, often at the non-reducing termini of carbohydrate chains. It has been demonstrated that sialic acids play very important roles in various biological and physiological events. Ample supply of sialosides and sialyl-glycoconjugates is indispensable to the study of their biological functions in detail. Transfer of sialic acids by sialyltransferases to appropriate substrates in the final step under a mild reaction condition can prepare these materials in quantity. Therefore, one of the most important tasks in the study of glycobiology is to provide a large amount of bacterial sialyltransferases with diverse characteristics at low prices.
Carbohydrate chains occupy truly significant positions in various fields of life sciences and biotechnology. A large number of polyclonal or monoclonal antibodies have been used as very important tools for analyzing expression of carbohydrate chains and their functions. “GlycoEpitope” is an integrated database that consists of useful information on carbohydrate antigens and their antibodies. It has been developed with the cooperation of top class researchers in the field of glycobiology and maintained by the Research Center for Glycobiotechnology in Ritsumeikan University. The GlycoEpitope Database provides a fund of information, e.g. lists of 1) glycoproteins that express carbohydrate antigens (epitopes), 2) glycolipids of which the partial structure is a carbohydrate epitope, 3) enzymes that take part in synthesis and degradation of glycoepitopes, 4) time and site of expression of carbohydrate epitopes, 5) diseases to which carbohydrate epitopes relate, and 6) suppliers where carbohydrate recognition antibodies can be obtained. This database is not limited to glycobiologists, but open to a wide range of life science researchers. Its search criteria are made flexible, so the database is very user-friendly. Here, we would like to introduce a general outline of the database and how it works.