Kohki Fujikawa

Both Isoforms of Human UDP-glucose:glycoprotein Glucosyltransferase are Enzymatically Active

Being recognized as an important constituent of the glycoprotein folding cycle, uridine diphosphate-glucose:glycoprotein glucosyltransferase (UGGT) has been a subject of intense study. Up to now, it is two isoforms, UGGT1 and 2 have been identified, which share ∼ 50% amino acid identity. UGGT1 is a well-documented enzyme which functions as a folding sensor in the endoplasmic reticulum, by the virtue of its ability to transfer a glucose residue to non-glucosylated high-mannose-type glycans of immature glycoproteins exhibiting non-native conformation. On the other hand, direct evidence to support the glucosyltransferase activity of UGGT2 has been lacking, leaving it unclear as to whether it has any function in the glycoprotein folding process. This study aimed to reveal the property of human UGGT2 by using synthetic substrates such as fluorescently labeled glycans and N-glycosylated proteins. The analysis, for the first time, revealed the glucosyltransferase activity of UGGT2, whose specificity was shown to be quite similar to UGGT1, in terms of both glycan specificity and preferential recognition of proteins having non-native conformations. Finally, Sep15 was found to form the heterodimeric complex with both isoforms of UGGT and markedly enhanced its glucosyltransferase activity.

The First Total Synthesis of Ganglioside GalNAc‐GD1a, a Target Molecule for Autoantibodies in Guillain–Barré Syndrome

The first synthesis of ganglioside GalNAc-GD1a, featuring efficient glycan assembly and a cyclic glucosyl ceramide as a versatile unit for ganglioside synthesis is described. Although ganglioside GalNAc-GD1a was first found as a brain ganglioside, IgG autoantibodies to GalNAc-GD1a were subsequently found to be closely related to a human peripheral-nerve disorder, Guillain-Barré syndrome, which is the commonest cause of acute flaccid paralysis worldwide. In this study, the characteristic hexasaccharide part carrying two sialic acid residues was synthesized efficiently by use of a readily accessible GM2-core unit as a common unit. The potentially difficult coupling of the oligosaccharide and ceramide moieties was carried out by using a cyclic glucosyl ceramide as a coupling partner for the hexasaccharide part, thereby successfully providing the framework of the target compound. Global deprotection delivered the homogenous ganglioside GalNAc-GD1a. An enzyme-linked immunosorbent assay showed that sera from patients with Guillain-Barré syndrome reacted both with natural and with synthetic GalNAc-GD1a.

Parallel quantification of lectin–glycan interaction using ultrafiltration

Using ultrafiltration membrane, a simple method for screening protein-ligand interaction was developed. The procedure comprises three steps: mixing ligand with protein, ultrafiltration of the solution, and quantification of unbound ligands by HPLC. By conducting analysis with variable protein concentrations, affinity constants were easily obtained. Multiple ligands can be analyzed simultaneously as a mixture, when concentration of ligands was controlled. Feasibility of this method for lectin-glycan interaction analysis was examined using fluorescently labeled high-mannose-type glycans and recombinant intracellular lectins or endo-α-mannosidase mutants. Estimated Ka values of malectin and VIP36 were in good agreement indeed with those evaluated by conventional methods such as isothermal titration calorimetry (ITC) or frontal affinity chromatography (FAC). Finally, several mutants of endo-α-mannosidase were produced and their affinities to monoglucosylated glycans were evaluated.

Construction of a high-mannose-type glycan library by a renewed top-down chemo-enzymatic approach.

A comprehensive method for the construction of a high-mannose-type glycan library by systematic chemo-enzymatic trimming of a single Man9-based precursor was developed. It consists of the chemical synthesis of a non-natural tridecasaccharide precursor, the orthogonal demasking of the non-reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high-mannose-type glycans in their mono- or non-glucosylated forms. It employed glucose, isopropylidene, and N-acetylglucosamine groups for blocking the A-, B-, and C-arms, respectively. After systematic trimming of the precursor, thirty-seven high-mannose-type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N-acetylglucosaminyltransferase-I toward M7-M3 glycans, clarifying the substrate specificity in the context of high-mannose-type glycans.

Synthesis of a GM3 ganglioside analogue carrying a phytoceramide moiety by intramolecular glycosylation as a key step.

A novel analogue of ganglioside GM3, in which sphingosine was replaced with a phytosphingosine moiety, was synthesized by intramolecular glycosylation as a key step. Glucose, a reducing terminal of the saccharide, and phytoceramide were first tethered by succinic acid and the derivative used for the subsequent glycosidic bond formation. The obtained glycosyl phytoceramide was further glycosylated with the sialyl galactose residue to afford a fully protected GM3 derivative, which was converted into the desired, final compound by using conventional deprotection procedures.

Glycan specificity of a testis-specific lectin chaperone calmegin and effects of hydrophobic interactions.

BACKGROUND Testis-specific chaperone calmegin is required for the generation of normal spermatozoa. Calmegin is known to be a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin. Although functional similarity between calnexin and calmegin has been predicted, detailed information concerned with substrate recognition by calmegin, such as glycan specificity, chaperone function and binding affinity, are obscure. METHODS In this study, biochemical properties of calmegin and calnexin were compared using synthetic glycans and glycosylated or non-glycosylated proteins as substrates. RESULTS Whereas their amino acid sequences are quite similar to each other, a certain difference in secondary structures was indicated by circular dichroism (CD) spectrum. While both of them inhibited protein heat-aggregation to a similar extent, calnexin exhibited a higher ability to facilitate protein folding. Similarly to calnexin, calmegin preferentially recognizes monoglucosylated glycans such as Glc1Man9GlcNAc2 (G1M9). While the surface hydrophobicity of calmegin was higher than that of calnexin, calnexin showed stronger binding to substrate. We reasoned that lectin activity, in addition to hydrophobic interaction, contributes to this strong affinity between calnexin and substrate. CONCLUSIONS Although their similarity in carbohydrate binding specificities is high, there seems to be some differences in the mode of substrate recognition between calmegin and calnexin. GENERAL SIGNIFICANCE Properties of calmegin as a lectin-chaperone were revealed in comparison with calnexin.

Extending the Glucosyl Ceramide Cassette Approach: Application in the Total Synthesis of Ganglioside GalNAc-GM1b

The development of a novel cyclic glucosyl ceramide cassette acceptor for efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB) groups were selected as protecting groups at C2 and C3 of the glucose residue with the aim of improving the functionality of the cassette acceptor. The choice of the PMB group resulted in a loss of β-selectivity, which was corrected by using an appropriate tether to control the spatial arrangement and the nitrile solvent effect. To investigate the effect of linker structure on the β-selectivity of intramolecular glycosylation, several linkers for tethering the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl, dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing the cassette form. The newly designed glucosyl ceramide cassette acceptor was then applied in the total synthesis of ganglioside GalNAc-GM1b.

Endoplasmic Reticulum (ER)-Targeted, Galectin-Mediated Retrograde Transport by Using a HaloTag Carrier Protein

Investigations into metabolic processes within the cell have often relied on genetic methods such as forced expression and knockout or knockdown techniques. An alternative approach would be introducing a molecule into the desired location inside the cell. To translocate compounds from outside cells into the endoplasmic reticulum (ER), we constructed a delivery carrier protein. This comprised N‐terminal galectin‐1 for cell‐surface binding (G1), a protease cleavable sequence (ps), a HaloTag domain for attaching exogenous compounds (Halo), and a C‐terminal KDEL sequence for ER retention. Fluorescently labeled G1‐ps‐Halo‐KDEL passed through the Golgi apparatus and reached the ER. By using Man9GlcNAc2‐BODIPY as a cargo compound, the carrier protein was also delivered into the ER with concomitant processing of mannose to Man5,6, by the ER‐resident α1,2‐mannosidase. G1‐ps‐Halo‐KDEL might serve as a new type of delivery carrier protein to direct compounds into the ER.

Influence of high-mannose glycan whose glucose moiety is substituted with 5-thioglucose on calnexin/calreticulin cycle

Our study first revealed that UDP-5-thioglucose functions as a glycosyl donor of UDP-glucose: glycoprotein glucosyltransferase to produce 5-thio-glucosylated Man9 (5S-G1M9). Subsequently, we observed that only calreticulin can interact with 5S-G1M9. Finally, the 5-thioglucose residue was resistant to hydrolysis by glucosidase II.