Characterization of the 1,3- -glucan synthase of Aspergillus fumigatus

Beauvais, Anne; Drake, Richard; Ng, Ken; Diaquin, Michel; Latgé, Jean‐Paul

doi:10.1099/00221287-139-12-3071

Cited by 61 publications

(50 citation statements)

References 26 publications

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“…The K m for A. nidulans preparations of 2 to 2.5 mM UDPG closely resembles the value of 1.9 mM reported for A. fumigatus preparations by Beauvais et al (8) and is about five times higher than the value of 400 M determined by Beaulieu et al (7). The V max of 6 to 10 nmol/min/mg for A. nidulans is very similar to the value determined for A. fumigatus.…”

Section: Discussionsupporting

confidence: 65%

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Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

et al. 1996

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Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-␤-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of ϳ200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched ϳ300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.

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Section: Discussionsupporting

confidence: 65%

“…Two groups have reported extraction of crude GS from A. fumigatus by different detergents but have not reported further purification (7,8). The only filamentous fungus from which highly purified GS has been obtained is Neurospora crassa (3,4).…”

mentioning

confidence: 99%

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

et al. 1996

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“…A microsomal membrane pellet was recovered after 1 h of centrifugation at 36,500 ϫ g at 4°C of the membrane extract as described previously (2) and was resuspended in EB (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2 M NaF, 1 M sucrose). This extract was stored under liquid nitrogen and named the microsomal membrane fraction (MMF).…”

Section: ј-Aagaattcaagactgagttcttccc-3јmentioning

confidence: 99%

Glucan Synthase Complex of Aspergillus fumigatus

Bruneau²,

et al. 2001

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The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for ␤(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized ␤(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H ؉ -ATPase, and a 160-kDa protein which was labeled by an anti-␤(1-3) glucan antibody and was homologous to ABC bacterial ␤(1-2) glucan transporters.The fungal cell wall, which is specific and essential to fungal life, is mainly constituted of polysaccharides. Among all polysaccharides identified to date in the cell wall, ␤(1-3) glucans are the most prevalent, and they are present in all yeast and filamentous fungi investigated to date (14). Although ␤(1-3) glucan biosynthesis has been the subject of intensive research efforts for the last 30 years, the ␤(1-3) glucan biosynthetic pathway is not fully understood. It has been known since the early studies of Cabib and coworkers (22,31,35,36) that ␤(1-3) glucans are synthesized from UDP glucose by a membrane protein complex, ␤(1-3) glucan synthase (EC 2.4.1.34; UDP-glucose/liter 1,3-␤-D-glucan-3-␤-D-glucosyltransferase). Synthesis occurs on the cytoplasmic side of the plasma membrane, and ␤(1-3) glucan chains are extruded towards the periplasmic space (15,35). The glucan synthase complex has been characterized at the molecular level almost exclusively in the yeast Saccharomyces cerevisiae (5,7,12,19,29) and has been shown to be composed of two proteins: (i) the putative catalytic subunit Fksp, a large-molecular-size (Ͼ200 kDa) polypeptide with 16 transmembrane domains (12,29,30), and (ii) the regulatory subunit Rho1p, a small-molecular-size GTPase, which stimulates ␤(1-3) glucan synthase activity in its prenylated form (1,11,17,18,24,28,33).If the ␤(1-3) glucan synthase has been extensively analyzed in yeast, then this enzymatic complex has been poorly studied in filamentous fungi. Only one FKS gene had been cloned and sequenced to date in Aspergillus nidulans (23), and neither has a regulatory partner been identified nor has the cellular localization of the glucan synthase complex been investigated.This study was centered on the char...

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“…The unsaturated long-chain FAs were also tested at lower concentrations, from 0.33 to 33 mg l-l, using the following GS assay. A solubilized GS extract (P30) was isolated from Aspergilltls fumigatus as described by Beauvais et al (1993). For Trichopbyton rubrum and T. mentagropbytes, the high-speed membrane extract (before solubilization) was used as a source of GS.…”

Section: Test Of Inhibitory Activity On Gsmentioning

confidence: 99%

Unsaturated fatty acids are the active molecules of a glucan-synthase-inhibitory fraction isolated from entomophthoralean protoplasts

MacKichan

Thomsen²,

Kerwin

et al. 1995

Microbiology

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A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, glucan synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of glucan synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 "C and were shown to be non-competitive and non-specif ic inhibitors of glucan synthase.

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Characterization of the 1,3- -glucan synthase of Aspergillus fumigatus

Cited by 61 publications

References 26 publications

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

Glucan Synthase Complex of Aspergillus fumigatus

Unsaturated fatty acids are the active molecules of a glucan-synthase-inhibitory fraction isolated from entomophthoralean protoplasts

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