Yasuko Ishizuka

Molecular Cloning and Characterization of a Novel UDP-GlcNAc:GalNAc-peptide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T6), an Enzyme Synthesizing the Core 3 Structure of O-Glycans

The core 3 structure of theO-glycan, GlcNAcβ1–3GalNAcα1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide β1,3-N- acetylglucosaminyltransferase (β3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human β3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it β3Gn-T6. The putative amino acid sequence of β3Gn-T6 retains the β3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of β3Gn-T6 expressed in insect cells showed β3Gn-T activity toward GalNAcα-p-nitrophenyl and GalNAcα1-serine/threonine. The β1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. β3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the β3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that β3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

We found a novel human glycosyltransferase gene carrying a hypothetical β1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5′-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank™ accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc β-benzyl. The product was deduced to be GalNAcβ1–4GlcNAcβ-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme β1,4-N-acetylgalactosaminyltransferase-III (β4GalNAc-T3). β4GalNAc-T3 effectively synthesized N,N′-diacetylgalactosediamine, GalNAcβ1–4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N′-diacetylgalactosediamine structures in their N-glycans, we examined the ability of β4GalNAc-T3 to synthesize N,N′-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and β1,4-galactosidase was utilized as an acceptor protein, β4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that β4GalNAc-T3 could transfer GalNAc residues, producing N,N′-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.

Molecular cloning and characterization of β1,4‐N‐acetylgalactosaminyltransferases IV synthesizing N,N′‐diacetyllactosediamine1

A sequence highly homologous to β1,4‐N‐acetylgalactosaminyltransferase III (β4GalNAc‐T3) was found in a database of human expressed sequence tags. The full‐length open reading frame of the gene, β4GalNAc‐T4 (GenBank accession number AB089939), was cloned using the 5′ rapid amplification of cDNA ends method. It encodes a typical type II transmembrane protein of 1039 amino acids having 42.6% identity with β4GalNAc‐T3. The recombinant enzyme transferred N‐acetylgalactosamine to N‐acetylglucosamine‐β‐benzyl with a β1,4‐linkage to form N,N′‐diacetyllactosediamine as did β4GalNAc‐T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to β4GalNAc‐T3 in vitro, however, the tissue distributions of the two enzymes were quite different. These results indicated that the two enzymes have similar roles in different tissues.

Copper (II) modulates in vitro aggregation of a tau peptide.

Copper (II) has been implicated in the pathology of Alzheimers disease (AD) for the impaired homeostatic mechanism found in the brains of AD patients. Here we studied the binding properties of Cu(II) with the first microtubule-binding repeat, encompassing residues 256-273 of the human tau441 sequence. Additionally, the effect of Cu(II) on the assembly of this repeat was also investigated. Our results indicate that Cu(II) can bind to this repeat with His(268) involved and has an inhibiting effect on the in vitro aggregation of this repeat. This work provides new insight into the role of Cu(II) in Alzheimers disease.

Post‐source Decay Fragment Spectra of Cyclomalto‐octaose and Branched Cyclomalto‐hexaose by Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry

gamma-Cyclodextrin, maltosyl-alpha-cyclodextrin and diglucosyl-alpha-cyclodextrin were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. All of these compounds have the same molecular weight (M.W. = 1297.15) and consist of only D-glucopyranose. From a comparison of the intensities in the post-source decay (PSD) fragment spectra of these cyclodextrin derivatives, correlation between the chemical structures and the relative intensities in the PSD fragment ions was found. The correlation is considered to be caused by the difference in the number of cleavage sites at the glycosyl binding. It was found that the intensity of the PSD ion resulting from one cleavage is higher than that resulting from two cleavages at a glycosyl bond. The results show that PSD fragment-ion spectrum method used in MALDI-TOF mass spectrometry is a very powerful technique for the structural analyses of the sugar-substituted cyclodextrins.

Analysis of Glycosidic Linkages in Saccharide Compounds by Post-source Decay Fragment Methods in Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy

Maltotriosyl- and panosyl-alpha-cyclodextrins and the nonaose of pullulan were analyzed by post-source decay (PSD) fragment methods of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. By the mass number analysis, it was found that all of the PSD fragment ions were produced by cleavages of glycosidic linkages. Comparison of the relative intensities of the ions in those compounds enabled us to distinguish two kinds of glycosidic linkages, alpha 1-4 and alpha 1-6, by MALDI-TOFMS with a new type of ion reflector: the curved field reflectron.

Synthesis and conformational properties of phosphopeptides related to the human tau protein

In the brains of Alzheimers disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to determine the effects of abnormal phosphorylation on the local structure. A series of peptides corresponding to isolated regions of tau protein have been successfully synthesized using Fmoc-based chemistry and their conformations were determined by 1H NMR spectroscopy and circular dichroism (CD) spectroscopy. Immunodominant peptides corresponding to tau-(256-273), tau-(350-367) and two phosphorylated derivatives in which a single Ser was phosphorylated at positions 262 and 356, respectively, were the main focus of the study. A direct alteration of the local structure after phosphorylation constitutes a new strategy through which control of biological activity can be enforced. In our study on Ser262 in R1 peptide and Ser356 in R4 peptide, phosphorylation modifies both the negative charge and the local conformation nearby the phosphorylation sites. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the binding domain of tau protein to alter specificity and affinity of binding to microtubule, particularly in response to the abnormal phosphorylation events associated with Alzheimers disease.

Three-dimensional structure of the inclusion complex between phloridzin and β-cyclodextrin

Abstract The inclusion of phloridzin into β-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and β-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and β-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and β-cyclodextrin was simulated with distance constraints estimated by the intensity of NOE peaks using the DADAS90 programs. Two inclusion possibilities were suggested—the large rim of β-cyclodextrin as an entrance of the inclusion and the small rim of β-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of β-cyclodextrin.

An NMR Study of the Buckminster Fullerene Complex with Cyclodextrin in Aqueous Solution

Abstract NMR measurements have shown that C60 forms inclusion complexes with γ-CD in aqueous solution at high temperatures but does not form the complexes with α-CD and β-CD. The 1H NMR spectral analyses indicate that in the complex, the C60 molecule intrudes into the hydrophobic cavity of γ-CD.

Circular dichroism spectra of the inclusion complexes of phlorizin in cyclodextrins

Two aromatic rings of a phlorizin molecule form inclusion complexes with β-CD and γ-CD. Induced circular dichroism spectra of these complexes have been measured to estimate the orientation of the two aromatic rings in the hydrophobic space of CDs. Apparent complex formation constants have been also estimated for each complex. It is concluded that phlorizin forms a stronger inclusion complex with β-CD than with γ-CD.