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

Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1-->3)-alpha-D-glucan, a (1-->3)-beta-D-glucan, a (1-->6)-beta-D-glucan, and a a-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. C-13 NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1-->3)-alpha-D-glucan, a (1-->3)-beta-D-glucan, which may either be linear or slightly branched, and a highly branched (1-->6)-beta-D-glucan, in addition to alpha-galactomannan. The highly branched (1-->6)-beta-D-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1-->3)-beta-branched (1-->6)-beta-D-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by C-13 NMR spectra in D2O. The data collectively indicate that (1-->3)-alpha- and (1-->3)-beta-D-glucans are rigid and contribute to the cell shape, while the highly branched (1-->6)-beta-D-glucan and alpha-galactomannan are flexible. (C) 2004 Elsevier Ltd. All rights reserved.