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

Copper (II) has been implicated in the pathology of Alzheimer's 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 Alzheimer's disease.

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

In the brains of Alzheimer's 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 Alzheimer's disease.

Application of ultra-high magnetic field for saccharide molecules: 1H NMR spectra of 6-O-alpha-D-glucopyranosyl-cyclomaltoheptaose and -cyclomaltohexaose.

1H NMR spectra of G1-alpha-CD and G1-beta-CD were recorded using a spectrometer equipped with a 21.6 T magnet. An ultra-high magnetic field was effective for detecting 1H NMR signals with a small difference in chemical shifts. Introducing a glucosyl group onto CDs as a branch caused deformation of equilibrated 1H signals of cyclodextrin. Particularly, 1H signals in branched glucose were shifted greatly.

Molecular cloning and characterization of beta1,4-N-acetylgalactosaminyltransferases IV synthesizing N,N'-diacetyllactosediamine.

A sequence highly homologous to beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3) was found in a database of human expressed sequence tags. The full-length open reading frame of the gene, beta4GalNAc-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 beta4GalNAc-T3. The recombinant enzyme transferred N-acetylgalactosamine to N-acetylglucosamine-beta-benzyl with a beta1,4-linkage to form N,N'-diacetyllactosediamine as did beta4GalNAc-T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to beta4GalNAc-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.

1H NMR spectra of branched-chain cyclomaltohexaoses (alpha-cyclodextrins).

The 1H NMR spectra of seven branched alpha-cyclodextrins (alpha-CDs) were observed and analyzed in detail. They were compared with spectra of alpha-CD and amylose. Although these branched alpha-CDs consist only of alpha-D-glucose with the same alpha-(1-->4) O-glucosyl binding, aside from one exception, differences in chemical shifts of corresponding signals were significantly large. Especially, differences in the chemical shift in anomeric protons were considerably large. Subtle differences in glucosyl binding directly influences chemical shifts of these protons because anomeric protons are located adjacent to the glucosyl binding sites.

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

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and beta-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 beta-cyclodextrin as an entrance of the inclusion and the small rim of beta-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of beta-cyclodextrin.

Molecular cloning and characterization of a novel UDP-GlcNAc:GalNAc-peptide beta1,3-N-acetylglucosaminyltransferase (beta 3Gn-T6), an enzyme synthesizing the core 3 structure of O-glycans.

The core 3 structure of the O-glycan, GlcNAcbeta1-3GalNAcalpha1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide beta1,3-N- acetylglucosaminyltransferase (beta3Gn-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 beta3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it beta3Gn-T6. The putative amino acid sequence of beta3Gn-T6 retains the beta3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of beta3Gn-T6 expressed in insect cells showed beta3Gn-T activity toward GalNAcalpha-p-nitrophenyl and GalNAcalpha1-serine/threonine. The beta1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. beta3Gn-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 beta3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that beta3Gn-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.

A novel human beta1,3-N-acetylgalactosaminyltransferase that synthesizes a unique carbohydrate structure, GalNAcbeta1-3GlcNAc.

We found, using a BLAST search, a novel human gene (GenBank trade mark accession number BC029564) that possesses beta3-glycosyltransferase motifs. The full-length open reading frame consists of 500 amino acids and encodes a typical type II membrane protein. This enzyme has a domain containing beta1,3-glycosyltransferase motifs, which are widely conserved in the beta1,3-galactosyltransferase and beta1,3-N-acetylglucosaminyltransferase families. The putative catalytic domain was expressed in human embryonic kidney 293T cells as a soluble protein. Its N-acetylgalactosaminyltransferase activity was observed when N-acetylglucosamine (GlcNAc) beta1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a beta1,3-linkage by NMR spectroscopic analysis, and was therefore named beta1,3-N-acetylgalactosaminyltransferase-II (beta3GalNAc-T2). The acceptor substrate specificity of beta3GalNAc-T2 was examined using various oligosaccharide substrates. Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for beta3GalNAc-T2, followed by GlcNAcbeta1-4GlcNAcbeta1-O-benzyl, and GlcNAcbeta1-6GalNAcalpha1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the beta3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, mbeta3GalNAc-T2, was also found in a data base of mouse expressed sequence tags. In situ hybridization analysis with mouse testis showed that the transcripts are expressed in germ line cells. beta3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and beta-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAcbeta1-3GlcNAcbeta1-R structure has not been reported in humans or other mammals, we have discovered a novel human glycosyltransferase producing this structure on N- and O-glycans.

Molecular cloning and characterization of a novel human beta 1,4-N-acetylgalactosaminyltransferase, beta 4GalNAc-T3, responsible for the synthesis of N,N'-diacetyllactosediamine, galNAc beta 1-4GlcNAc.

We found a novel human glycosyltransferase gene carrying a hypothetical beta1,4-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 beta-benzyl. The product was deduced to be GalNAc beta 1-4GlcNAc beta-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme beta1,4-N-acetylgalactosaminyltransferase-III (beta 4GalNAc-T3). beta 4GalNAc-T3 effectively synthesized N,N'-diacetylgalactosediamine, GalNAc beta 1-4GlcNAc, 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 beta 4GalNAc-T3 to synthesize N,N'-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and beta1,4-galactosidase was utilized as an acceptor protein, beta 4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that beta 4GalNAc-T3 could transfer GalNAc residues, producing N,N'-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.