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T Fontaine

Publications and source records attributed to T Fontaine.

At least 19 recordsLinked to original sources
PubMed2002

Molecular characterization of a cell wall-associated beta(1-3)endoglucanase of Aspergillus fumigatus.

A 74 kDa beta(1-3)endoglucanase of Aspergillus fumigatus was recently isolated from a cell wall autolysate and biochemically characterized. In this study, we report the cloning and the disruption of the ENGL1 gene encoding this beta(1-3)endoglucanase. ENGL1 contains an open reading frame of 2181 bp encoding a polypeptide of 727 amino acids. Sequence analysis showed that ENGL1 is the first characterized member of a new family of beta(1-3)glucanases. Disruption of ENGL1, however, did not lead to a phenotype distinct from the parental strain, indicating that this cell wall-associated beta(1-3)endoglucanase does not play an essential role in constitutive cell growth.

Amino Acid Sequence↗
PubMed2001

Biochemical characterization and surfactant properties of horse allergens.

A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri-antennary tri-sialylated N-linked glycan. Linkages of terminal N-acetylneuraminic acid to galactose were: alpha-(2-->6) in Equ c 4, and both alpha-(2-->3) and alpha-(2-->6) in Equ c 1. Oligosaccharide portions of Equ c 1 or Equ c 4 were barely involved in IgE-immunoreactivity. Partial N-terminal sequence of Equ c 4 shares a significant sequence homology with the rat submandibular gland protein A. No matching was found for two internal peptides of Equ c 5. Surfactant properties of horse allergens as well as other proteins were investigated. In contrast to Equ c 2 and Equ c 3, solutions of Equ c 1, Equ c 4 and Equ c 5 significantly lowered the surface tension. Relationship between a property such as this, involving oriented hydrophobic patches of a molecule and allergenicity, is addressed.

Allergens↗
PubMed2000

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall.

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.

Aspergillus fumigatus↗
PubMed2000

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation.

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Aldehyde-Ketone Transferases↗
PubMed2000

Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall.

A novel 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-beta-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1, 3-beta-glucan molecule forming a 1,3-beta linkage, resulting in the elongation of 1,3-beta-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-beta-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-beta-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.

Amino Acid Sequence↗
PubMed2000

Identification of the catalytic residues of the first family of beta(1-3)glucanosyltransferases identified in fungi.

A new family of glycosylphosphatidylinositol-anchored beta(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis. Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72. We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases.

Amino Acid Sequence↗
PubMed1999

Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa.

A cAMP-activatable Ca2+-dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus.

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.

Aspergillus fumigatus↗
PubMed1997

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose.

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.

Amino Acid Sequence↗
PubMed1997

Differential patterns of activity displayed by two exo-beta-1,3-glucanases associated with the Aspergillus fumigatus cell wall.

Two exo-beta-1,3-glucanases (herein designated exoG-I and exoG-II) were isolated from the cell wall autolysate of the filamentous fungus Aspergillus fumigatus and purified by ion-exchange, hydrophobic-interaction, and gel filtration chromatographies. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 82 kDa for the monomeric exoG-I and 230 kDa for the dimeric exoG-II. exoG-I and exoG-II were glycosylated, and N glycans accounted, respectively, for 2 and 44 kDa. Their pH optimum is 5.0. Their optimum temperatures are 55 degrees C for exoG-I and 65 degrees C for exoG-II. By a sensitive colorimetric method and high-performance anion-exchange chromatography for product analysis, two patterns of exo-beta-1,3-glucanase activities were found. The 230-kDa exoG-II enzyme acts on p-nitrophenyl-beta-D-glucoside, beta-1,6-glucan, and beta-1,3-glucan. This activity, which retains the anomeric configuration of glucose released, presented a multichain pattern of attack of the glucan chains and a decrease in the maximum initial velocity (Vm) with the increasing size of the substrate. In contrast, the 82-kDa exoG-I, which inverts the anomeric configuration of the glucose released, hydrolyzed exclusively the beta-1,3-glucan chain with a minimal substrate size of 4 glucose residues. This enzyme presented a repetitive-attack pattern, characterized by an increase in Vm with an increase in substrate size and by a degradation of the glucan chain until it reached laminaritetraose, the limit substrate size. The 82-kDa exoG-I and 230-kDa exoG-II enzymes correspond to a beta-1,3-glucan-glucohydrolase (EC 3.2.1.58) and to a beta-D-glucoside-glucohydrolase (EC 3.2.1.21), respectively. The occurrence and functions of these two classes of exo-beta-1,3-glucanases in other fungal species are discussed.

Aspergillus fumigatus↗
PubMed1996

A novel beta-(1-3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus.

Cell wall transferases utilizing beta-(1-3)-glucan chains as substrates may play important roles in cell wall assembly and rearrangement, as beta-(1-3)-glucan is a major structural component of the cell wall of many fungi. A novel beta-(1-3)-glucanosyltransferase was purified to apparent homogenei ty from an autolysate of the cell wall of Aspergillus fumigatus. The enzyme had a molecular mass of 49 kDa and contained approximately 5 kDa of N-linked carbohydrate. The enzyme catalyzed an initial endo-type splitting of a beta-(1-3)-glucan molecule, followed by linkage of the newly generated reducing end to the nonreducing end of another beta-(1-3)-glucan molecule. Laminarioligosaccharides of size G10 and greater were donor substrates for the transferase. Laminarioligosaccharides of size G5 and greater formed acceptors. The enzyme was able to reuse initial transferase products as donors and acceptors in extended incubations, resulting in the formation of increasingly larger transferase products until they became insoluble. The major initial products from an incubation of the transferase with borohydride-reduced G11 (rG11) were rG6 and rG16. 1H NMR analysis of the rG16 transferase product showed it was a laminarioligosaccharide, indicating that the enzyme forms a beta-(1-3)-linkage during transfer. The enzyme may have a key function in vivo by allowing the integration of newly synthesized glucan into the wall and promoting cell wall expansion during cell growth.

Aspergillus fumigatus↗
PubMed1995

Differentiation of capsular polysaccharides from Acetobacter diazotrophicus strains isolated from sugarcane.

Capsular polysaccharides (CPSs) from six representative strains of Acetobacter diazotrophicus were isolated and fractionated by gel filtration and anion-exchange chromatography. Purified CPSs obtained in the non-adsorbed fraction of a DEAE-Sephadex A-25 column were qualitatively and quantitatively analyzed for sugar composition. Uronic acid and amino sugars were not detected in all purified CPSs. Basically the CPSs of A. diazotrophicus are composed of rhamnose, mannose, galactose and glucose. The presence of fucose was only observed in the CPS of strains PR2 and PAL3. Based on these results, the six strains of A. diazotrophicus could be divided into four groups according to the sugar content of their capsules: (i) fucose-containing capsules (PR2 and PAL3, localized in roots), (ii) mannose-rich capsule (PAL5, localized in root), (iii) capsules with a high ratio of hexose to rhamnose (PR4 and PR20, localized in stems) and (iv) capsules with a low ratio of hexose to rhamnose (PR14, localized in rhizosphere). For all CPSs, sodium dodecy sulfate-polyacrylamide gel electrophoresis showed diffuse bands of slow mobility in silver-stained gels. The different CPS migration patterns could not be correlated with sugar composition. The purified CPS of strain PAL3 was found to be immunogenic and immunochemically similar to the CPS of strain PR2. The serological specificity to CPS of strains PAL3 and PR2 correlated well with the presence of focuse, indicating that this deoxyhexose is immunodominant.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetobacter↗
PubMed1994

Toward an understanding of the biogenesis of the starch granule. Determination of granule-bound and soluble starch synthase functions in amylopectin synthesis.

Plant starch synthesis can be distinguished from those of bacterial, fungal, and animal glycogen by the presence of multiple elongation (starch synthases) and branching enzymes. This complexity has precluded genetic assignment of functions to the various soluble starch synthases in the building of amylopectin. In Chlamydomonas, we have recently shown that defects in the major soluble starch synthase lead to a specific decrease in the amount of a subset of amylopectin chains whose length ranges between 8 and 40 glucose residues (Fontaine, T., D'Hulst, C., Maddelein, M.-L., Routier, F., Marianne-Pepin, T., Decq, A., Wieruszeski, J. M., Delrue, B., Van Den Koornhuyse, N., Bossu, J.-P., Fournet, B., and Ball, S. G. (1993) J. Biol. Chem. 268, 16223-16230). We now demonstrate that granule-bound starch synthase, the enzyme that was thought to be solely responsible for amylose synthesis, is involved in amylopectin synthesis. Disruption of the Chlamydomonas granule-bound starch synthase structural gene establishes that synthesis of long chains by this enzyme can become an absolute requirement for amylopectin synthesis in particular mutant backgrounds. In the sole presence of soluble starch synthase I, Chlamydomonas directs the synthesis of a major water-soluble polysaccharide fraction and minute amounts of a new type of highly branched granular material, whose structure is intermediate between those of glycogen and amylopectin. These results lead us to propose that the nature of the elongation enzyme conditions the synthesis of distinct size classes of glucans in all starch fractions.

Amylopectin↗
PubMed1993

Toward an understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas soluble starch synthase II controls the synthesis of intermediate size glucans of amylopectin.

Low starch mutants of Chlamydomonas reinhardtii were isolated after x-ray mutagenesis of wild-type strain 137C. The mutants accumulated 20-40% of the normal amount and displayed a 2-fold decrease of the total glycogen-primed soluble starch synthase activity. Three different mutant alleles of the st-3 gene were isolated that were characterized by similar defects and displayed a net increase in amylose content. Amylose-primed synthesis of glucan in native gels revealed a complete wipe out of one of the soluble starch synthases. Zymograms and kinetic analyses performed both in the mutant and in partially purified wild type extracts reveal at least two distinct activities that are partly analogous to higher plant soluble starch synthases I and II (SSI and II). The st-3 mutants were defective for SSII. Methylation and debranching of the purified amylopectin fraction clearly show a decrease in the number of intermediate size glucans (dp8 to 50) and an absolute and relative increase of very short glucans (dp2 to 7). These results suggest that a soluble starch synthase may be necessary for the synthesis or maintenance of intermediate size glucans that are the main component of the branched clusters of amylopectin.

Amylopectin↗
PubMed1993

Isolation and characterisation of hemicelluloses from sunflower hulls.

The hemicelluloses extracted from sunflower hulls by repeated alternating oxidative and alkaline treatments were purified by precipitation with Cetavlon and then ion-exchange chromatography on DEAE-trisacryl. The resulting fractions were examined by hydrolysis, methylation, GLC-MS, and NMR spectroscopy. The hemicelluloses are of the glucuronoxylan type with the following structure -->4)-beta-D-Xylp-(1-->4)-[4-O-Me-alpha-D-GlcpA-(1-->2)]-bet a-D-Xylp-(1-->. The polysaccharides differed in the amount of branching; the ratio of the main fraction 4-O-MeGlcA:Xyl was 1:8-9.

Carbohydrate Conformation↗
PubMed1992

Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.

Alleles↗