| The cell wall structure of mushroom was investigated by comparing the cell wall components including polysaccharides and proteins among three different developmental stages including mycelium, sclerotium and fruiting body of a medicinal mushroom Pleurotus tuber-regium (PTR). A differential solvent fractionation scheme using hot water (HW), cold alkali (CA) and hot alkali (HA) solutions was used to extract cell wall fractions according to the principle of removing the cell wall materials from the outer layer to the inner layer with mild-to-strong extraction conditions. The crude mushroom cell wall fractions thus obtained were purified by use of membrane ultrafiltration followed by liquid column chromatography to give cell wall polysaccharides (CWPSs) that were characterized for their monosaccharide composition, linkage pattern, molecular mass, as well as structural features by spectroscopic (IR, NMR) and microscopic (TEM, AFM) techniques. Mushroom cell wall proteins (CWPs) were also obtained from the different developmental stages of PTR and analyzed by proteomic and glycomic methods.;A four-fraction sandwich model for the structure of PTR cell wall was proposed according to the present findings. In the first outer fraction, proteins and glycoproteins are found together with CWPSs which can be extracted using boiling water. They are associated via non-covalent bondings with other cell wall components. The second fraction is mainly consisted of cold alkali-soluble polysaccharides constituted with mainly glucose and small amount of other sugars including mannose, galactose, xylose and rhamnose. The third fraction is mainly consisted of hot alkali-soluble polysaccharides that have more glucose units than the second fraction. This polysaccharide fraction requires more severe alkaline treatment for its isolation due to a stronger linkage to the innermost part of the mushroom cell walls. The fourth and innermost fraction that is attached to the cell membrane is composed of an alkali-insoluble glucan-chitin complex.;From the results of proteomic analysis, 103, 48 and 91 proteins were obtained from PTR cell wall of mycelium, sclerotium and fruiting body, respectively. More than 60% of these PTR proteins were found to function as translation, ribosomal structure and biogenesis. Results from glycomic analysis revealed that all N-glycans released from the glycoproteins of the hot water soluble fraction of sclerotial cell wall (SHW80) were found to share a common pentasaccharide core region of Man3GlcNAc2. O-glycans from the glycoproteins of SHW80 were composed of GlcNAc, fucose, galactose, GalNAc and sialic acid only but had a more complicated structure than N-glycans.;A few purified CWPSs that are the main components of mushroom cell wall were characterized for their fine chemical structures. The main component of the structural skeleton of mCW is a hot alkali soluble fraction MHA-I, which has a molecular mass of 4.502~104 g/mol, while its R.M.S. radius was calculated to be 33.2 nm.Differing from mCW, more glycoproteins with lower molecular mass (< 25 kDa) was detected in the first fraction of sCW, which were associated with glucan via non-covalent linkage in this fraction. The main component of the structural skeleton of sCW consists of glucans (SHW50-I, SCA-I, SHA-IF1 and SHA-IF2) that all had a v molecular mass over 106 g/mol, and their radii were as small as MHA-I. The major branching points of these glucans were at O-3 and/or O-6 positions of the Glcp main chain and DB were from 63% to 70%. The four-fraction structure of fCW was similar to that of mCW, but the sugar unit and linkage type of its hot alkali-soluble fraction FHA-I was similar to that of glucans from sCW. The results from AFM and TEM further confirmed the structural conformation of these mushroom cell wall hyper-branched polysaccharides. Hence, it is proven that the structural skeletons of mushroom cell wall among the three different developmental stages are mainly composed of alkali-soluble glycans, which are mainly consisted of hyper-branched polysaccharides. (Abstract shortened by UMI.). |
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