Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001).

Fucose-deficient hematopoietic stem cells have decreased self-renewal and aberrant marrow niche occupancy.

BACKGROUND: Modification of Notch receptors by O-linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study was to assess the self-renewal and stem cell niche features of fucose-deficient HSCs. STUDY DESIGN AND METHODS: Homeostasis and maintenance of HSCs derived from FX(-/-) mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two-photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose-deficient and wild-type (WT) HSCs. RESULTS: Marrow progenitors from FX(-/-) mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose-deficient HSCs from FX(-/-) mice displayed decreased self-renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by two-photon intravital imaging, the fucose-deficient HSCs were found localized farther from the endosteum of the calvarium marrow than the WT HSCs. CONCLUSIONS: The current reported aberrant niche occupancy by HSCs from FX(-/-) mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O-fucosylation, suggests that fucosylation-modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.

Secretory cell hyperplasia and defects in Notch activity in a mouse model of leukocyte adhesion deficiency type II.

BACKGROUND & AIMS: Leukocyte adhesion deficiency II (LAD II) is a rare condition caused by defective protein fucosylation, causing decreased leukocyte rolling, psychomotor retardation, and poor growth. The ligand-binding activity of Notch, a gastrointestinal signaling protein, depends on O-fucosylation. We investigated Notch signaling and intestinal epithelial architecture in a mouse model of LAD II. METHODS: Mice lacking 3,5-epimerase/4-reductase (FX) or FX(-/-) bone marrow chimeras (with either wild-type or FX(-/-) bone marrow) were maintained on a fucose-free diet. Intestinal secretory epithelial cells were quantified by histology and immunohistochemistry. Reverse transcription-polymerase chain reaction and immunoblot analyses were used to detect Notch-regulated genes in isolated crypt epithelium. Intestinal leukocyte-endothelial interaction was quantified by intravital microscopy. The intestinal epithelium of 2-week-old FX(-/-) mice was transfected with an adenoviral vector expressing a constitutively active form of Notch. RESULTS: FX(-/-) mice rapidly exhibited secretory epithelial cell hyperplasia, reduced cell proliferation, and altered epithelial gene expression patterns consistent with reduced Notch signaling. These effects were reversed when mice were given dietary fucose or by adenoviral transfection of the intestinal epithelium with the Notch intracellular domain. CONCLUSIONS: In a mouse model of LAD II, secretory cell hyperplasia occurs in the small intestine and colon; these effects depend on Notch signaling. Defects in Notch signaling might therefore be involved in the pathogenesis of this rare pediatric condition.

Resistance to Bacillus thuringiensis toxin in Caenorhabditis elegans from loss of fucose.

A mutation in the Caenorhabditis elegans bre-1 gene was isolated in a screen for Bacillus thuringiensis toxin-resistant (bre) mutants to the Cry5B crystal toxin made by B. thuringiensis. bre-1 mutant animals are different from the four other cloned bre mutants in that their level of resistance is noticeably lower. bre-1 animals also display a significantly reduced brood size at 25 degrees C. Here we cloned the bre-1 gene and characterized the bre-1 mutant phenotype. bre-1 encodes a protein with significant homology to a GDP-mannose 4,6-dehydratase, which catalyzes the first step in the biosynthesis of GDP-fucose from GDP-mannose. Injection of GDP-fucose but not fucose into C. elegans intestinal cells rescues bre-1 mutant phenotypes. Thus, C. elegans lacks a functional fucose salvage pathway. Furthermore, we demonstrate that bre-1 mutant animals are defective in production of fucosylated glycolipids and that bre-1 mutant animals make quantitatively reduced levels of glycolipid receptors for Cry5B. We finally show that bre-1 mutant animals, although viable, show a lack of fucosylated N- and O-glycans, based on mass spectrometric evidence. Thus, C. elegans can survive with little fucose and can develop resistance to crystal toxin by loss of a monosaccharide biosynthetic pathway.

Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.

The core fucosylation (alpha1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated alpha1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8(-/-) mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8(-/-) mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-beta1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8(-/-) mice have a marked dysregulation of TGF-beta1 receptor activation and signaling, as assessed by TGF-beta1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-beta1 receptor defects found in Fut8(-/-) cells can be rescued by reintroducing Fut8 into Fut8(-/-) cells. Furthermore, exogenous TGF-beta1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8(-/-) lung. We propose that the lack of core fucosylation of TGF-beta1 receptors is crucial for a developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.

Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose.

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).

Alpha 1,6-linked fucose affects the expression and stability of polysialic acid-carrying glycoproteins in Chinese hamster ovary cells.

To determine the effect of alpha1,6-linked fucose modification of N-glycans on the expression of polysialic acids (PSAs), the expression of PSAs in a fucose-lacking mutant of Chinese hamster ovary (CHO) cells, Lec13, was compared with that in CHO K1 cells. PSA synthase activity in these cells and the antennary structures of N-glycans associated with the neural adhesion molecule (NCAM), which is a major PSA-carrying glycoprotein, did not differ between the two types of cells. Metabolic labeling of cells with [3H]glucosamine for 48 h followed by immunoprecipitation with anti-PSA monoclonal antibodies revealed that the amount of labeled PSA-carrying glycoproteins obtained from Lec13 cells was 10-times less than that from K1 cells, although the incorporation of [3H]glucosamine into total extracts and NCAM was almost the same. In contrast, when cells were pulse labeled with [35S]methionine followed by a 1 h chase, there was not such a great difference in PSA-carrying protein synthesis between K1 and Lec13 cells. However, during a prolonged chase period, PSA-carrying proteins rapidly decreased in Lec13 cells, whereas those in K1 cells did not change. The degradation of PSA-carrying glycoproteins in Lec13 cells was partly prevented when the cells were grown in fucose-containing medium. Therefore, fucose modification of core N-glycans may affect the efficient expression of PSAs through the intracellular stability of PSA-carrying glycoproteins.