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The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities

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ABSTRACT

Aminopyrene trisulfonate (APTS)-labelled disaccharides are demonstrated to serve as readily accessible acceptor substrates for galactosyltransferase activities present in Arabidopsis microsome preparations. The reductive amination procedure used to install the fluorophore results in loss of the ring structure of the reducing terminal sugar unit, such that a single intact sugar ring is present, attached via an alditol tether to the aminopyrene fluorophore. The configuration of the alditol portion of the labelled acceptor, as well as the position of alditol galactosylation, substantially influence the ability of compounds to serve as Arabidopsis galactosyltransferase acceptor substrates. The APTS label exhibits an unexpected reaction-promoting effect that is not evident for structurally similar sulfonated aromatic fluorophores ANDS and ANTS. When APTS-labelled β-(1 → 4)-Gal3 was employed as an acceptor substrate with Arabidopsis microsomes, glycan extension generated β-(1 → 4)-galactan chains running to beyond 60 galactose residues. These studies demonstrate the potential of even very short glycan-APTS probes for assessing plant galactosyltransferase activities and the suitability CE-LIF for CAZyme profiling.

No MeSH data available.


APTS-labelled β-Gal-containing acceptors derived from disaccharides Gal-β-(1 → 4)-Gal, Gal-β-(1 → 4)-Glc and Gal-β-(1 → 6)-Gal. Acronyms gal and glc denote galactitol and glucitol, respectively.
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fig2: APTS-labelled β-Gal-containing acceptors derived from disaccharides Gal-β-(1 → 4)-Gal, Gal-β-(1 → 4)-Glc and Gal-β-(1 → 6)-Gal. Acronyms gal and glc denote galactitol and glucitol, respectively.

Mentions: For batch to batch consistency, we opted to employ microsomal preparations from cultured Arabidopsis cells as a source of galactosyltransferase activities. To optimise conditions for microsomal GalT-catalysed reactions, a CE-LIF-based quantitative analysis was established for which basic principles were adapted from published research [16]. The assay followed the conversion of Gal-β-(1 → 4)-Gal-β-(1 → 4)-gal-APTS (Gal2-gal-APTS) into Gal-β-(1 → 4)-Gal-β-(1 → 4)-Gal-β-(1 → 4)-gal-APTS (Gal3-gal-APTS, see legend to Fig. 2) under the action of UDP-Gal and GalT in the presence of Triton X-100 at 20 °C. In order to facilitate simple end-point kinetic analyses, reactions were allowed to proceed to a maximum of 15% yield of Gal3-gal-APTS (ca. 6 h) when formation of longer oligomers was negligible (<2%). Thus it was established that GalT is active over a broad range of pH and Mn2+ concentrations, with maximal activity at pH 6 in the presence of 15–20 mM Mn2+ (SI Fig. S1A, S1B). Notably, the GalT was found to tolerate a broad range of detergent/protein ratios and detergent concentrations (SI Fig. S1C). Throughout the following series of experiments, 0.5% Triton X-100 was used at a detergent/protein ratio of 5:1. For optimal GalT activity the detergent needed to be peroxide-free and used fresh.


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
APTS-labelled β-Gal-containing acceptors derived from disaccharides Gal-β-(1 → 4)-Gal, Gal-β-(1 → 4)-Glc and Gal-β-(1 → 6)-Gal. Acronyms gal and glc denote galactitol and glucitol, respectively.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036537&req=5

fig2: APTS-labelled β-Gal-containing acceptors derived from disaccharides Gal-β-(1 → 4)-Gal, Gal-β-(1 → 4)-Glc and Gal-β-(1 → 6)-Gal. Acronyms gal and glc denote galactitol and glucitol, respectively.
Mentions: For batch to batch consistency, we opted to employ microsomal preparations from cultured Arabidopsis cells as a source of galactosyltransferase activities. To optimise conditions for microsomal GalT-catalysed reactions, a CE-LIF-based quantitative analysis was established for which basic principles were adapted from published research [16]. The assay followed the conversion of Gal-β-(1 → 4)-Gal-β-(1 → 4)-gal-APTS (Gal2-gal-APTS) into Gal-β-(1 → 4)-Gal-β-(1 → 4)-Gal-β-(1 → 4)-gal-APTS (Gal3-gal-APTS, see legend to Fig. 2) under the action of UDP-Gal and GalT in the presence of Triton X-100 at 20 °C. In order to facilitate simple end-point kinetic analyses, reactions were allowed to proceed to a maximum of 15% yield of Gal3-gal-APTS (ca. 6 h) when formation of longer oligomers was negligible (<2%). Thus it was established that GalT is active over a broad range of pH and Mn2+ concentrations, with maximal activity at pH 6 in the presence of 15–20 mM Mn2+ (SI Fig. S1A, S1B). Notably, the GalT was found to tolerate a broad range of detergent/protein ratios and detergent concentrations (SI Fig. S1C). Throughout the following series of experiments, 0.5% Triton X-100 was used at a detergent/protein ratio of 5:1. For optimal GalT activity the detergent needed to be peroxide-free and used fresh.

View Article: PubMed Central - PubMed

ABSTRACT

Aminopyrene trisulfonate (APTS)-labelled disaccharides are demonstrated to serve as readily accessible acceptor substrates for galactosyltransferase activities present in Arabidopsis microsome preparations. The reductive amination procedure used to install the fluorophore results in loss of the ring structure of the reducing terminal sugar unit, such that a single intact sugar ring is present, attached via an alditol tether to the aminopyrene fluorophore. The configuration of the alditol portion of the labelled acceptor, as well as the position of alditol galactosylation, substantially influence the ability of compounds to serve as Arabidopsis galactosyltransferase acceptor substrates. The APTS label exhibits an unexpected reaction-promoting effect that is not evident for structurally similar sulfonated aromatic fluorophores ANDS and ANTS. When APTS-labelled &beta;-(1&nbsp;&rarr;&nbsp;4)-Gal3 was employed as an acceptor substrate with Arabidopsis microsomes, glycan extension generated &beta;-(1&nbsp;&rarr;&nbsp;4)-galactan chains running to beyond 60 galactose residues. These studies demonstrate the potential of even very short glycan-APTS probes for assessing plant galactosyltransferase activities and the suitability CE-LIF for CAZyme profiling.

No MeSH data available.