Crystal structure and sugar-binding ability of the C-terminal domain of N-acetylglucosaminyltransferase IV establish a new carbohydrate-binding module family.

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a ß-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind ß-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with ß-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a ß-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with ß-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of ß-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.

Multiple Stimuli-Responsive Supramolecular Gel Formed from Modified Adenosine.

Urea derivatives 1 and 2, synthesized from adenosine, were designed as low-molecular-weight gelators. Hydrophobic groups have been introduced into all or part of the hydroxy groups of the hydrophilic ribose moiety of 1 and 2 to control the solvophilicity of the molecules and their aggregates. Compound 2 selectively formed supramolecular gels in halogenated solvents such as chloroform and 1,2-dichloroethane. The supramolecular gel of 2 and chloroform was thermally stable and its gel-to-sol phase transition temperature was higher than the boiling point of chloroform. The physical properties of the supramolecular gel were investigated by determining its viscoelastic properties using a rheometer. The supramolecular gel realized multiple stimuli-responsive reversible gel-sol phase transitions. The supramolecular gel showed reversible phase transition by repeated warming-cooling cycles accompanying with the gel-sol transitions. The supramolecular gel could undergo five repeated mechano-responsive gel-sol transitions. Gel-to-sol phase transition could also be achieved by adding various anions to the supramolecular gel, such as tetrabutylammonium fluoride. Regelation was realized by adding boron trifluoride etherate to the fluoride ion containing sol. Addition of methanol to the supramolecular gel also induced gel-to-sol phase transition. Regelation was realized by adding molecular sieves 4 Å to the suspension.

Effect of Alkyl Chain Length of N-Alkyl-N'-(2-benzylphenyl)ureas on Gelation.

Urea derivatives that were substituted with a 2-benzylphenyl group and an alkyl group functioned as low molecular weight gelators for various organic solvents and ionic liquids. Urea derivatives with long alkyl chains were effective for the gelation of polar solvents. However, they were not suitable for the gelation of non-polar solvents, whereas urea derivatives with short alkyl chains were effective. Ionic liquids were similar to polar solvents in that urea derivatives with long alkyl chains were the most effective gelators. The physical properties of the formed supramolecular gels were analyzed by dynamic viscoelasticity measurements using a rheometer.

Expression and characterization of silkworm Bombyx mori ß-1,2-N-acetylglucosaminyltransferase II, a key enzyme for complex-type N-glycan biosynthesis.

N-glycans are involved in various physiological functions and their structures diverge among different phyla and kingdoms. Insect cells mainly produce high mannose-type and paucimannose-type glycans but very few mammalian-like complex-type glycans. However, many insects possess genes for proteins homologous to the enzymes involved in complex-type N-glycan synthesis in mammalian cells, and their N-glycosylation pathway is incompletely understood compared with that of mammals. Here, we cloned a candidate gene for ß-1,2-N-acetylglucosaminyltransferase II (GnTII), which is a Golgi-localized enzyme involved in a key step in the conversion to complex-type N-glycans, from silkworm Bombyx mori, and the gene was found to be expressed ubiquitously in the larval and pupal stages. In addition, recombinant B. mori GnTII was expressed as a soluble form using a silkworm-B. mori nucleopolyhedrovirus bacmid expression system. The recombinant enzyme exhibited similar pH and temperature dependency and the same substrate specificity as human GnTII, but deglycosylation with peptide:N-glycanase F did not affect its enzymatic activity. Compared with the structure of human GnTII, the amino acid residues involved in catalytic activity and substrate recognition are almost fully conserved in B. mori GnTII, which is consistent with its enzymatic properties. These results raised the possibility of mammalian-like complex-type N-glycan synthesis using the GnTII ortholog in silkworm.