Akihiko Koizumi

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.

Top‐Down Chemoenzymatic Approach to a High‐Mannose‐Type Glycan Library: Synthesis of a Common Precursor and Its Enzymatic Trimming

From the stacks: A novel method for construction of a high-mannose-type glycan library by systematic enzymatic trimming of a single synthetic Man9-based precursor was developed. Efficient chemical synthesis of the tetradecasaccharide common precursor and orthogonal enzymatic trimming to obtain all M(8-9) and G(1)M(8-9) derivatives was demonstrated. G = glucose, M = mannose.

Synthetic studies on the carbohydrate moiety of the antigen from the parasite Echinococcus multilocularis

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Gal beta 1-->3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (A), tetrasaccharide Gal beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (D), and pentasaccharides Gal beta 1-->3(Gal beta 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (E) and Gal beta1-->3(Gal alpha 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (F) (R = 2-(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-azido-4-O-benzyl-2-deoxy-alpha-d-galactopyranoside served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear trisaccharide Gal beta 1-->4Gal beta 1-->3GalNAc alpha 1-OR (B) and branched tetrasaccharide Gal beta 1-->4Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (C) were synthesized by stepwise condensation.

The surface carbohydrates of the Echinococcus granulosus larva interact selectively with the rodent Kupffer cell receptor

The larvae of the cestodes belonging to the genus Echinococcus dwell primarily in mammalian liver. They are protected by the laminated layer (LL), an acellular mucin-based structure. The glycans decorating these mucins constitute the overwhelming majority of molecules exposed by these larvae to their hosts. However, their decoding by host innate immunity has not been studied. Out of 36 mammalian innate receptors with carbohydrate-binding domains, expressed as Fc fusions, only the mouse Kupffer cell receptor (KCR; CLEC4F) bound significantly to the Echinococcus granulosus LL mucins. The receptor also bound the Echinococcus multilocularis LL. Out of several synthetic glycans representing Echinococcus LL structures, the KCR bound strongly in particular to those ending in Galα1-4Galβ1-3 or Galα1-4Galβ1-4GlcNAc, both characteristic LL carbohydrate motifs. LL carbohydrates may be optimized to interact with the KCR, expressed only in liver macrophages, cells known to contribute to the tolerogenic antigen presentation that is characteristic of this organ.

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.

Synthesis of the carbohydrate moiety from the parasite Echinococcus multilocularis and their antigenicity against human sera

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galβ1-3GalNAc core of the Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galβ1-3(GlcNAcβ1-6)GalNAcα1-R (G), tetrasaccharide Galβ1-3(Galβ1-4GlcNAcβ1-6)GalNAcα1-R (J) and pentasaccharide Galβ1-3(Galα1-4Galβ1-4GlcNAcβ1-6)GalNAcα1-R (K) (R=biotinylated probe) were synthesized by block synthesis by the use of 5-(methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. Moreover, linear trisaccharide Galα1-4Galβ1-3GalNAcα1-R (H) and branched tetrasaccharide Galα1-4Galβ1-3(GlcNAcβ1-6)GalNAcα1-R (I) were synthesized by stepwise condensation. We examined the antigenicity of these five oligosaccharides by an enzyme linked immunosorbent assay (ELISA). Our results demonstrate that biotinylated oligosaccharides H, I and K show good serodiagnostic potential to detect infections caused by the parasite E. multilocularis. Among them the linear sequence Galα1-4Galβ1-3GalNAcα1-R in oligosaccharide (H) appears to show the highest sensitivity (95%). Moreover, our study clarified the dominant carbohydrate epitope of Em2 antigen.

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.

Further structural characterization of the Echinococcus granulosus laminated layer carbohydrates: the blood-antigen P1-motif gives rise to branches at different points of the O-glycan chains.

The glycobiology of the cestodes, a class of parasitic flatworms, is still largely unexplored. An important cestode species is Echinococcus granulosus, the tissue-dwelling larval stage of which causes hydatid disease. The E. granulosus larva is protected from the host by a massive mucin-based extracellular matrix termed laminated layer (LL). We previously reported ( Díaz et al. 2009. Biochemistry 48:11678-11691) the molecular structure of the most abundant LL O-glycans, comprising up to six monosaccharide residues. These are based on Cores 1 and 2, in cases elongated by a chain of Galpβ1-3 residues, which can be capped by Galpα1-4. In addition, the Core 2 GlcNAcp residue can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Larger glycans also detected contained additional HexNAc residues that could not be explained by the structural repertoire described above. In this work, we elucidate, by mass spectrometry (MS) and nuclear magnetic resonance (NMR), six additional glycans from the E. granulosus LL between six and eight residues in size. Their structures are related to those already described but in cases bear GlcNAcpβ1-6 or Galpα1-4Galpβ1-4GlcNAcpβ1-6 as ramifications on the core Galpβ1-3 residue. We also obtained evidence that noncore Galpβ1-3 residues can be similarly ramified. Thus, the new motif together with the previous information may explain all the glycan compositions detected in the LL by MS. In addition, we show that the anti-Echinococcus monoclonal antibody E492 (Parasite Immunol 21:141, 1999) recognizes Galpα1-4Galpβ1-4GlcNAcp (the blood P(1)-antigen motif). This explains the antibodys reactivity with a range of Echinococcus tissues, as the P(1)-motif is also carried on non-LL N-glycans and glycolipids from this genus.

Synthesis, Antigenicity Against Human Sera and Structure-Activity Relationships of Carbohydrate Moieties from Toxocara larvae and Their Analogues

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galβ1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meβ1-3GalNAcα1-OR (A), Fucα1-2Gal4Meβ1-3GalNAcα1-OR (B), Fuc2Meα1-2Galβ1-3GalNAcα1-OR (C), Fucα1-2Galβ1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meβ1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.

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.