Molecular cloning and functional expression of Lewis type α1,3/α1,4-fucosyltransferase cDNAs from Mangifera indica L.

In higher plants, complex type N-glycans contain characteristic carbohydrate moieties that are not found in mammals. In particular, the attachment of the Lewis a (Le) epitope is currently the only known outer chain elongation that is present in plant N-glycans. Such a modification is of great interest in terms of the biological function of complex type N-glycans in plant species.

1α,25-Dihydroxyvitamin D3 enhances γ-glutamyl transpeptidase activity in LLC-PK1 porcine kidney epithelial cells.

Mammalian γ-glutamyl transpeptidase (GGT) is expressed most highly in the kidney and serves to recover the constituent amino acids of glutathione in the proximal tubules. Serum GGT is used as a marker for obstructive jaundice and alcoholic liver disease and it has been reported that urinary GGT is a potential marker for bone resorption. The present study investigated the effect of derivatives of vitamin D3 on GGT activity in LLC-PK1 porcine renal tubular epithelial cells in order to analyze the biochemical basis of bone metabolism-associated alterations in GGT activity in the kidney.

MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface.

MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression.

Measurement of peroxiredoxin-4 serum levels in rat tissue and its use as a potential marker for hepatic disease.

Peroxiredoxin (Prx)-4, a secretable endoplasmic reticulum (ER)-resident isoform of the mammalian Prx family, functions as a thioredoxin-dependent peroxidase. It is acknowledged that Prx-4 plays a role in the detoxification of hydrogen peroxide, and potentially other peroxides, which may be generated during the oxidative folding of proteins and oxidative stress in the ER. The present study was undertaken in order to specifically quantify the tissue levels of Prx-4.

Different consequences of reactions with hydrogen peroxide and t-butyl hydroperoxide in the hyperoxidative inactivation of rat peroxiredoxin-4.

Eukaryotic typical 2-Cys type peroxiredoxin (Prx) is inactivated by hyperoxidation of the peroxidatic cysteine to a sulphinic acid in a catalytic cycle-dependent manner. This inactivation process has been well documented for cytosolic isoforms of Prx. However, such a hyperoxidative inactivation has not fully been investigated in Prx-4, a secretable endoplasmic reticulum-resident isoform, in spite of being a typical 2-Cys type, and details of this process are reported herein.

N-Glycosylation engineering of lepidopteran insect cells by the introduction of the beta1,4-N-acetylglucosaminyltransferase III gene.

The baculovirus-insect cell expression system is in widespread use for expressing post-translationally modified proteins. As a result, it is potentially applicable for the production of glycoproteins for therapeutic and diagnostic purposes. For practical use, however, remodeling of the biosynthetic pathway of host-cell N-glycosylation is required because insect cells produce paucimannosidic glycoforms, which are different from the typical mammalian glycoform, due to trimming of the non-reducing terminal beta1,2-GlcNAc residue of the core structure by a specific beta-N-acetylglucosaminidase.

Fucosylation of chitooligosaccharides by human alpha1,6-fucosyltransferase requires a nonreducing terminal chitotriose unit as a minimal structure.

FUT8, a eukaryotic alpha1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-beta-l-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally similar to that of NodZ, a bacterial alpha1,6-fucosyltransferase, which acts on a chitooligosaccharide in the synthesis of Nod factor. While the substrate specificities for the nucleotide sugar and the N-glycan have been determined, it is not known whether FUT8 is able to fucosylate other sugar chains such as chitooligosaccharides.

Expression of N-terminally truncated forms of rat peroxiredoxin-4 in insect cells.

Peroxiredoxins (Prxs), a family of thioredoxin-dependent peroxidases, are highly conserved in many organisms and function in detoxifying reactive oxygen species as well as other cellular processes. Six members of the Prx family are known in mammals, i.e.

Bidirectional N-acetylglucosamine transfer mediated by beta-1,4-N-acetylglucosaminyltransferase III.

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of the bisecting GlcNAc and plays a regulatory role in the biosynthesis of the N-linked oligosaccharide. In this study, we examined whether the glycosyl transfer catalyzed by GnT-III is reversible, and, in addition, investigated the equilibrium of the GnT-III-catalyzed reaction. Incubation of the agalactosyl-bisected biantennary oligosaccharide with GnT-III in the presence of the sufficiently high concentration of uridine diphosphate (UDP) resulted in conversion of the bisected oligosaccharide into the nonbisected one.

Identification of Pex5pM, and retarded maturation of 3-ketoacyl-CoA thiolase and acyl-CoA oxidase in CHO cells expressing mutant Pex5p isoforms.

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem.

Altered antigenic disposition of peroxisomal urate oxidase in PEX5-defective Chinese hamster ovary cells.

Since Chinese hamster ovary (CHO) cells never express urate oxidase (UO), we tried to establish cell lines stably producing UO in order to elucidate the peroxisomal import process. The enzyme is a peroxisome targeting signal 1 (PTS1) protein harboring SKL motif at the carboxy-terminus [Biochem. Biophys.

Different accumulations of 3-ketoacyl-CoA thiolase precursor in peroxisomes of Chinese hamster ovary cells harboring a dysfunction in the PEX2 protein.

The peroxisomal localization of 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase) was characterized in five Chinese hamster ovary (CHO) mutant cell lines each harboring a dysfunction in the PEX2 protein. PT54 (Pex2pN100) cells carry a nonsense mutation that results in the PEX2 protein truncated at amino acid position 100. SK24 (Pex2pC258Y) cells carry a missense mutation resulting in the amino acid substitution of a cysteine residue by a tyrosine residue at amino acid position 258 of the PEX2 protein.