title: Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.
abstract: Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.
authors.affiliation: Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538, Bratislava, Slovakia.
authors.forename: Kristína
authors.initials: K
authors.lastname: Kováčová
authors.affiliation: Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538, Bratislava, Slovakia. chemvfar@savba.sk.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Farkaš
mesh_terms: D002176:Candida albicans / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology; D005453:Fluorescence; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D015966:Gene Expression Regulation, Fungal; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D057166:High-Throughput Screening Assays / Q000379:methods; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D009181:Mycoses / Q000188:drug therapy; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D005656:Fungal Proteins; C066709:GAS1 protein, S cerevisiae; D008562:Membrane Glycoproteins; C091515:PHR1 protein, Candida albicans; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase; C407735:CRH1 protein, S cerevisiae; D006026:Glycoside Hydrolases; C407734:UTR2 protein, S cerevisiae
keywords: Cell walls; Fluorescence assay; High-throughput; Polysaccharide endotransglycosylases; Transglycosylation