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

ABSTRACT: Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-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 approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-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.

SUBMITTER: Kelly R

PROVIDER: S-EPMC178203 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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

Kelly R R Register E E Hsu M J MJ Kurtz M M Nielsen J J

Journal of bacteriology 19960801 15

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-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- ...[more]

PMID: 8755864

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Project description:?-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two ?-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major ?-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of ?-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE ) or agsB (agsBOE ), in which the other ?-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall ?-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of ?-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in ?-1,3-glucan was almost the same (after Smith degradation: agsAOE , 41.6 ± 5.8 kDa; agsBOE , 38.3 ± 3.0 kDa). We also analyzed localization of ?-1,3-glucan in the cell wall of the two strains by fluorescent labeling with ?-1,3-glucan-binding domain-fused GFP (AGBD-GFP). ?-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with ?-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that ?-1,3-glucan covered most of ?-1,3-glucan synthesized by AgsA, although a small amount of ?-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular ?-amylase that synthesizes ?-1,4-glucooligosaccharide as a primer for ?-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of ?-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and ?-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of ?-1,3-glucan as well as the amount of ?-1,3-glucan in the cell wall of A. nidulans.

Dataset Information

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

Publications

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

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