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Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall.
Eukaryot Cell. 2009 Nov; 8(11):1626-36.EC

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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Authors+Show Affiliations

Cabib E
Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Department of Health andHuman Services, Bethesda, Maryland 20892, USA. enricoc@bdg10.niddk.nih.gov

MeSH

Cell WallChemistry Techniques, AnalyticalChitinGlucansGlycoside HydrolasesProtein BindingSaccharomyces cerevisiaeSaccharomyces cerevisiae Proteins

Pub Type(s)

Evaluation Study
Journal Article
Research Support, N.I.H., Intramural

Language

eng

PubMed ID

19734368

Citation

Cabib, Enrico. "Two Novel Techniques for Determination of Polysaccharide Cross-links Show That Crh1p and Crh2p Attach Chitin to Both Beta(1-6)- and Beta(1-3)glucan in the Saccharomyces Cerevisiae Cell Wall." Eukaryotic Cell, vol. 8, no. 11, 2009, pp. 1626-36.
Cabib E. Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall. Eukaryot Cell. 2009;8(11):1626-36.
Cabib, E. (2009). Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall. Eukaryotic Cell, 8(11), 1626-36. https://doi.org/10.1128/EC.00228-09
Cabib E. Two Novel Techniques for Determination of Polysaccharide Cross-links Show That Crh1p and Crh2p Attach Chitin to Both Beta(1-6)- and Beta(1-3)glucan in the Saccharomyces Cerevisiae Cell Wall. Eukaryot Cell. 2009;8(11):1626-36. PubMed PMID: 19734368.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall. A1 - Cabib,Enrico, Y1 - 2009/09/04/ PY - 2009/9/8/entrez PY - 2009/9/8/pubmed PY - 2010/1/15/medline SP - 1626 EP - 36 JF - Eukaryotic cell JO - Eukaryot Cell VL - 8 IS - 11 N2 - 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. SN - 1535-9786 UR - https://www.unboundmedicine.com/medline/citation/19734368/Two_novel_techniques_for_determination_of_polysaccharide_cross_links_show_that_Crh1p_and_Crh2p_attach_chitin_to_both_beta_1_6___and_beta_1_3_glucan_in_the_Saccharomyces_cerevisiae_cell_wall_ L2 - https://journals.asm.org/doi/10.1128/EC.00228-09?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -