Abstract
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.
Highlights
The fungal cell wall is a physically rigid layer that protects the fungal cell from its environment, mediates cell-cell interaction, and is responsible for the shape of the cell
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
The covalent bond between the two polysaccharides has been characterized in Saccharomyces cerevisiae by Kollar et al [6], who showed that chitin is linked to the nonreducing end of a -1,3-glucan chain
Summary
A. fumigatus CBS 144 – 89 was grown in a 15-liter fermenter in a liquid medium containing a 2% glucose and 1% mycopeptone (Biokar Diagnostics) as described previously [17]. The insoluble pellet residue was resuspended in 80 ml of 50 mM Tris-HCl, pH 8.0 containing 5 mM sodium azide and incubated at 37 °C for 5 days with 4 ml of recombinant chitinase A (0.5 mg of protein/ml) from Serratia marcescens produced in Escherichia coli and purified as described previously [18]. All fractions were desalted by gel filtration on a Sephadex G15 column (35 ϫ 2.5 cm; Amersham Pharmacia Biotech) eluted with 20 mM acetic acid at 2 ml/min and freeze dried. Transgalactosylation of terminal nonreducing GlcNAc residues was performed using the following procedure: samples containing 5 mg of carbohydrate were incubated in 600 l of 50 mM Tris-HCl, pH 7.5, containing 1 mM MnCl2, and 5 mM sodium azide, with 120 l of UDPGal (10 mg/200 l) and 30 l of galactosyl transferase (1 milliunit/ml; Roche Molecular Biochemicals) at 37 °C during 3 days. Gas chromatograph was equipped with a CP-Sil 5CB/MS capillary column (25 m ϫ 0.32 mm, Chrompack) gas vector, and helium was at the flow rate of 2 ml/min; the column temperature was 100 –240 °C at 5 °C/min
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