Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy

Abstract

Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1 → 3)-α- d-glucan, a (1 → 3)-β- d-glucan, a (1 → 6)-β- d-glucan, and a α-galactomannan. To refine the structures of these polysaccharides, cell-wall glucans of S. pombe were extracted, fractionated, and analyzed by NMR spectroscopy. S. pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared. The alkali-insoluble fraction was treated with 0.5 M acetic acid or Zymolyase 100 T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction. 13C NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1 → 3)-α- d-glucan, a (1 → 3)-β- d-glucan, which may either be linear or slightly branched, and a highly branched (1 → 6)-β- d-glucan, in addition to α-galactomannan. The highly branched (1 → 6)-β- d-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1 → 3)-β-branched (1 → 6)-β- d-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by 13C NMR spectra in D 2O. The data collectively indicate that (1 → 3)-α- and (1 → 3)-β- d-glucans are rigid and contribute to the cell shape, while the highly branched (1 → 6)-β- d-glucan and α-galactomannan are flexible.