AI Article Synopsis
- Researchers used various techniques to analyze yeast cell walls and found that two transglycosylases, Crh1p and Crh2p/Utr2p, are essential for attaching chitin to beta(1-6)glucan.
- New methods were developed to study polysaccharide cross-links in cell walls, including the use of curdlan and in situ deacetylation of chitin to extract chitosan.
- A reexamination revealed that prior results were impacted by contamination in the enzyme preparation, confirming that Crh1p and Crh2p facilitate the transfer of chitin to both beta(1-3) and beta(1-6)glucans in the yeast cell wall.
Article Abstract
Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with beta(1-3)- or beta(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to beta(1-6)glucan. With this technique, crh1Delta crh2Delta mutants still appeared to contain a substantial percentage of chitin linked to beta(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a beta(1-3)glucan, for beta(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1Delta crh2Delta strains is free. Reexamination of the previously used procedure revealed that the beta(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both beta(1-3)- and beta(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772413 | PMC |
| http://dx.doi.org/10.1128/EC.00228-09 | DOI Listing |
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