In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo-α-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α-glycosyl-fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1- 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc Man GlcNAc ).

Glycoprotein oligosaccharides function as tags for protein quality control in the endoplasmic reticulum (ER). Since most of proteins are glycosylated and function only after they are properly folded, glycoprotein glycan profiles in the ER might be useful to analyze various cellular status including diseases. Here, we examined whether ER glycan-processing profiles in diabetic rats and osteoporotic mice as models might have different cellular status from those of normal controls. Direct analysis of glycoprotein-processing profiles in the ER is often hampered by glycoforms that are retro-translocated to the ER from other cellular compartments. Moreover, when we focus on the mixture of glycoproteins as the processing substrates, the glycan-processing efficiencies are influenced by the aglycon states including their polypeptide folding. To overcome this problem, we reconstructed glycan profiles using ER extracts as an enzymatic source and synthetic glycoprotein mimetic having homogeneous aglycon as a substrate, resulted in disease-specific glycan profiles. To understand such differences, we also analyzed the activity, and expression level, of each glycan-related enzyme. These glycan profiles are expected to be useful indexes for operational status of the ER glycoprotein quality control, and may also give information to classify some diseases.

N-Linked glycans play important roles in various cellular and immunological events. Endo-␤-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently ␣-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucosespecific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies. N-Linked glycans are oligosaccharides attached to Asn residues of proteins and have key functionalities in various cellular and immunological systems (1, 2). N-Glycans are categorized into three major types, high-mannose, complex, and hybrid types, and there are bi-, tri-, and tetra-antennary glycans with respect to the number of branches (3). Mammalian N-glycans are frequently ␣-1,6-fucosylated at the Asn-linked GlcNAc of the core N,NЈ-diacetylchitobiose unit. A high-throughput analysis of the IgG glycome in three isolated human populations revealed that between 91 and 97.7% of N-glycans are core-fucosylated (4). The ␣-1,6-fucosylation plays important roles in the functionalities of epidermal growth factor receptors, cell adhesion molecules (5), and antibody-dependent cellular toxicity (6), and altered core fucosylation levels have been observed in certain types of diseases (7-9). Endo-␤-N-acetylglucosaminidases (ENGases, 2 EC 3.2.1.96) hydrolytically cleave the ␤-1,4-glycosidic bonds within the core N,NЈ-diacetylchitobiose unit to release N-glycan, leaving a GlcNAc, with or without the core fucose, linked to the Asn residue of proteins (Fig. 1A) (10). The ability of ENGases to chemoenzymatically synthesize homogeneousl...

We developed a new 1,2-cis-α-glucosylation reaction, mediated by enhancing the α-favoured transition state via an electron-donating form the TBS group.