Biomedical subjects

T Hennet

Publications and source records attributed to T Hennet.

At least 19 recordsLinked to original sources

PubMed2000

The beta 1,3-galactosyltransferase beta 3GalT-V is a stage-specific embryonic antigen-3 (SSEA-3) synthase.

We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.

Animals↗

PubMed2000

Secretion and purification of recombinant beta1-4 galactosyltransferase from insect cells using pFmel-protA, a novel transposition-based baculovirus transfer vector.

The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.

Amino Acid Sequence↗

PubMed2000

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.

Alleles↗

PubMed2000

Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie.

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures. We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features. We detected hypoglycosylation on serum transferrin and cerebrospinal fluid beta-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients. Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings. Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity. Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients. Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.

Amino Acid Sequence↗

PubMed2000

Clinical and biochemical characteristics of congenital disorder of glycosylation type Ic, the first recognized endoplasmic reticulum defect in N-glycan synthesis.

We report on 8 patients with a recently described novel subtype of congenital disorder of glycosylation type Ic (CDG-Ic). Their clinical presentation was mainly neurological with developmental retardation, muscular hypotonia, and epilepsy. Several symptoms commonly seen in CDG-Ia such as inverted nipples, abnormal fat distribution, and cerebellar hypoplasia were not observed. The clinical course is milder overall, with a better neurological outcome, than in CDG-Ia. The isoelectric focusing pattern of serum transferrin in CDG-Ia and CDG-Ic is indistinguishable. Interestingly, beta-trace protein in cerebrospinal fluid derived from immunoblot analysis of the brain showed a less pronounced hypoglycosylation pattern in CDG-Ic patients than in CDG-Ia patients. Analysis of lipid-linked oligosaccharides revealed an accumulation of Man9GlcNAc2 intermediates due to dolichol pyrophosphate-Man9GlcNAc2 alpha-1,3 glucosyltransferase deficiency. All patients were homozygous for an A333V mutation.

Amino Acid Substitution↗

PubMed1999

The remodeling of glycoconjugates in mice.

A role for glycoconjugates in mediating cellular interactions is well established. To further understand the formation, function and regulation of various glycoconjugates in vivo, gene targeting approaches have been applied to glycosyltransferase and glycosidase enzymes involved in different biosynthetic pathways. The growing number of gene targeted mice generated have brought confirmations of the importance of both core and terminal glycosylation enzymes in normal development and physiology. Of particular interest has been the degree of cell and tissue specificity of phenotypes generated by systemic null mutations as well as the number of enzymes belonging to multigene families having overlapping activities.

Animals↗

PubMed1999

A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic.

Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation. CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders. The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2. Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients. Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients. The mutation cosegregated with the disease in a Mendelian recessive manner. Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S. cerevisiae alg5 and alg6 deficiency. By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype. The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.

Amino Acid Sequence↗

PubMed1999

A beta-1,3-N-acetylglucosaminyltransferase with poly-N-acetyllactosamine synthase activity is structurally related to beta-1,3-galactosyltransferases.

Human and mouse cDNAs encoding a new beta-1, 3-N-acetylglucosaminyltransferase (beta3GnT) have been isolated from fetal and newborn brain libraries. The human and mouse cDNAs included ORFs coding for predicted type II transmembrane polypeptides of 329 and 325 aa, respectively. The human and mouse beta3GnT homologues shared 90% similarity. The beta3GnT gene was widely expressed in human and mouse tissues, although differences in the transcript levels were visible, thus indicating possible tissue-specific regulation mechanisms. The beta3GnT enzyme showed a marked preference for Gal(beta1-4)Glc(NAc)-based acceptors, whereas no activity was detected on type 1 Gal(beta1-3)GlcNAc and O-glycan core 1 Gal(beta1-3)GalNAc acceptors. The new beta3GnT enzyme was capable of both initiating and elongating poly-N-acetyllactosamine chains, which demonstrated its identity with the poly-N-acetyllactosamine synthase enzyme (E.C. 2.4.1.149), showed no similarity with the i antigen beta3GnT enzyme described recently, and, strikingly, included several amino acid motifs in its protein that have been recently identified in beta-1,3-galactosyltransferase enzymes. The comparison between the new UDP-GlcNAc:betaGal beta3GnT and the three UDP-Gal:betaGlcNAc beta-1,3-galactosyltransferases-I, -II, and -III reveals glycosyltransferases that share conserved sequence motifs though exhibiting inverted donor and acceptor specificities. This suggests that the conserved amino acid motifs likely represent residues required for the catalysis of the glycosidic (beta1-3) linkage.

Amino Acid Sequence↗

PubMed1999

Molecular cloning of a human UDP-galactose:GlcNAcbeta1,3GalNAc beta1, 3 galactosyltransferase gene encoding an O-linked core3-elongation enzyme.

Using the full-length amino-acid sequences of the human beta1,3 galactosyltransferase (beta3GalT)-I, -II and III enzymes as query, we have identified an additional member of the beta3GalT gene family within a sequenced region of the human chromosome 21 as found in GenBank. The novel human beta3GalT-V gene included an open reading frame of 933 bp encoding a protein of 310 amino acids with a short N-terminal cytoplasmic tail, a single predicted transmembrane domain and a large lumenal catalytic domain. The human beta3GalT-V protein showed 34%, 27%, 31% and 23% sequence identity with the human beta3GalT-I, -II, -III and -IV enzymes, respectively. The expression of beta3GalT-V as a recombinant protein in Sf9 insect cells confirmed the galactosyltransferase activity catalyzed by this enzyme. Similarly to beta3GalT-I, -II and -III, the beta3GalT-V enzyme used beta-linked GlcNAc as an acceptor, but unlike the former enzymes beta3GalT-V exhibited a marked preference for the O-linked core3 GlcNAcbeta1,3GalNAc substrate. The beta3GalT-V gene was mainly expressed in human small intestine and to a lesser extent in pancreas and testis. Although beta3GalT-V transcripts were not detected in normal colon tissue, based on Northern analysis, beta3GalT-V mRNA was found in the adenocarcinoma cell line Colo 205.

Amino Acid Sequence↗

PubMed1998

Immune regulation by the ST6Gal sialyltransferase.

The ST6Gal sialyltransferase controls production of the Siaalpha2-6Galbeta1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.

Animals↗

PubMed1998

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

Amino Acid Sequence↗

PubMed1998

[Transgenic mice in basic research].

What are transgenic mice and what do we learn from them? In this review, we focus on the generation of "classical" transgenic and "knock-out" mice. The establishment of transgenic and gene-targeted mice provides an unique tool to study the function(s) of a given gene in the context of a whole organism. Based on selected examples, we demonstrate the potential of this transgenic technology to understand the interactions between cells, organs and organ systems in genetically engineered mice.

Alzheimer Disease↗

PubMed1996

TNF alpha alters mitochondrial membrane potential in L929 but not in TNF alpha-resistant L929.12 cells: relationship with the expression of stress proteins, annexin 1 and superoxide dismutase activity.

Tumour necrosis factor alpha (TNF alpha) cytotoxicity is mediated, at least in part, by oxidative stress and phospholipase A2 activation. The first post-receptor events to be observed in TNF alpha-sensitive lines are the generation of superoxide anion (O2-) within the mitochondria and the activation of phospholipase A2. Using the lipophilic dye JC-1 to determine mitochondrial membrane potential, we showed that TNF alpha induces time-dependent alterations in mitochondrial membrane potential in L929 cells but not in the TNF alpha-resistant L929. 12 subclone. Heat shock (HS) proteins (HSP) and superoxide dismutase (SOD) have been shown to protect cells from TNF alpha cytotoxicity, while glucose regulated proteins (GRP) and annexins might also be involved in cellular protection. We thus compared the expression of HSP, grp78 and annexin 1 as well as SOD activity in TNF alpha sensitive and resistant lines. We found no difference in the expression of HSP, grp78 or annexin 1, but an increase in the constitutive activity of SOD in the L929.12 cells as compared to L929. Furthermore, SOD was inducible by TNF alpha in L929 cells, but not in L929.12 cells. These data suggest that in TNF alpha-resistant lines, mitochondrial damage by TNF alpha is prevented by an increase in SOD rather than in overexpression of stress proteins or annexins.

Animals↗

PubMed1995

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

Alleles↗

PubMed1993

Expression of BCL-2 protein enhances the survival of mouse fibrosarcoid cells in tumor necrosis factor-mediated cytotoxicity.

Tumor necrosis factor (TNF) kills some types of tumor cells in vitro and participates in tumor elimination in vivo. TNF has been shown to kill cells by altering their mitochondria structurally and functionally. The oncogene BCL-2 codes for a protein located in the inner membrane of mitochondria which is able to inhibit the commitment to cell death in various cell types. We have therefore investigated whether TNF-mediated killing of the cell line L929 could be modulated by expression of the protein BCL-2. We report here that L929 cells transfected with a BCL-2 expression vector have an increased survival compared to wild type cells after TNF challenge. The protective effect is greatest at moderate TNF concentrations and is still significant at concentrations that killed 100% of wild type cells. The action of BCL-2 is selective inasmuch as cells are not protected against other cytotoxic agents blocking various mitochondrial functions. We show that cells expressing BCL-2 have a higher mitochondrial membrane potential (delta psi) than wild type cells. The increase in delta psi could be linked with the enhanced survival of cells after TNF challenge. Indeed, we found that treatment of wild type L929 cells with the ionophore nigericin, which increases delta psi, protects them even at high TNF concentrations.

Animals↗

PubMed1993

Tumour necrosis factor-alpha induces superoxide anion generation in mitochondria of L929 cells.

Within a few minutes after addition to L929 cells, tumour necrosis factor-alpha (TNF alpha) induced an increase in lucigenin-enhanced chemiluminescence that could be inhibited by superoxide dismutase. The generation of superoxide anion (O2.-) was sensitive to treatment with rotenone, antimycin A and cyanide, indicating that the signal originated from mitochondria. The mechanism of production of O2.- was shown to be independent of ATP synthesis, as uncoupling of this event from mitochondrial electron transport did not alter the generation of O2.- induced by TNF alpha. Chemiluminescence was further dependent on the presence of extracellular calcium, suggesting a role for this cation as a second messenger. This hypothesis was supported by the finding that inhibition of mitochondrial calcium uptake by Ruthenium Red exerted a protective effect on TNF alpha-treated L929 cells. Increased O2.- generation was followed by a marked decrease in mitochondrial dehydrogenase activity and cellular ATP levels, while cell membrane permeability was moderately increased. A role for mitochondrial O2.- generation in TNF alpha cytotoxicity was further supported by the finding that resistant L929 cells had decreased ability to produce O2.- in response to TNF alpha. In addition, we detected a decreased activity of the mitochondrial enzyme succinate dehydrogenase in these cells, suggesting that this component of the respiratory chain might be an important contributor to the TNF alpha-induced generation of O2.-.

Adenosine Triphosphate↗

PubMed1992

A kinetic study of immune mediators in the lungs of mice infected with influenza A virus.

We investigated a broad spectrum of immunoactive mediators in a mouse model of influenza. ICR mice (4-5 wk old) that were infected with a 10 LD50 dose of influenza A/PR8/34 virus died after 6 days without evidence of bacterial superinfection. Maximal virus titers were reached by day 2 postinfection, whereas the multifocal pneumonia with mononuclear cell infiltration reached its maximum at the end of infection. We measured the cytokines IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha, granulocyte (G)/macrophage (M)-CSF, G-CSF, M-CSF, and the lipid mediators leukotriene B4 and platelet-activating factor in the cellfree bronchoalveolar lavage fluid of mice during infection. We found an early increase of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF, IFN-gamma, and leukotriene B4. Levels of these factors peaked between 36 h and day 3 postinfection, with the exception of IL-6 that remained at elevated levels throughout infection. G-CSF and M-CSF increased slowly and reached a maximum by day 5 postinfection. We were unable to detect IL-2, IL-3, or IL-4. PAF remained at the same level throughout infection. Our results suggest that lung-resident cells, and possibly the alveolar macrophages, participate actively in the onset of the inflammatory response against the invading virus. The inability to detect the T cell products IL-2, IL-3, and IL-4 was unexpected considering the role of T cells in the elimination of the virus in infected mice. Our observation confirms thus earlier findings about the inability of specific T cell clones to elicit an unspecific antiviral effect.

Animals↗

PubMed1992

Alterations in antioxidant defences in lung and liver of mice infected with influenza A virus.

We investigated the possible involvement of oxidative mechanisms in the pathogenesis of influenza A/PR8/34 virus infection in mice. As a biochemical marker of oxidative stress, we determined the endogenous concentrations of the antioxidants glutathione and vitamins C and E in their reduced and oxidized forms in the lungs, liver and blood plasma of control and infected animals. Following intranasal infection with 8 to 10 LD50, influenza virus was detected in the lungs, but not in the plasma, liver or other organs. Infection resulted in a decrease in the total concentration of glutathione and vitamins C and E, whereas no relevant change in the ratio of oxidized to total concentration of antioxidants was observed. Changes in the concentration of hepatic antioxidants were significant in the early stages of the infection. The results suggest that hepatic alterations may be caused indirectly by mechanisms related to the host response to virus infection. The observed general decrease in the antioxidant buffering capacity may reduce the ability of tissues to protect against potential oxidative stress. Such stress can occur during bacterial superinfections, which are common in influenza, thereby rendering the host more susceptible to the pathogenic effects of such agents. In addition, reactive oxygen species produced in the lung may inactivate protease inhibitors, resulting in increased protease activity. Using an in vitro system consisting of alpha 1-antiprotease, trypsin and HOCl as the oxidant, we have shown that the infectivity of influenza viruses can be increased up to 10,000-fold by proteolytic cleavage of haemagglutinin, leading to activation of the fusogenic properties of this protein.

Animals↗