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

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 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.

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Related in: MedlinePlus

GalT assay with A. thaliana microsomes and Gal2-gal-APTS, Gal2-gal-ANDS and Gal2-gal-ANTS analysed by FACE as described in section 2.6. The reactions were performed overnight at 20 °C with 0.25 or 0.5 mM fluorescently-labelled galactotriose, 0.5 mM UDP-Gal and 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 and 20 mM MnCl2.
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fig7: GalT assay with A. thaliana microsomes and Gal2-gal-APTS, Gal2-gal-ANDS and Gal2-gal-ANTS analysed by FACE as described in section 2.6. The reactions were performed overnight at 20 °C with 0.25 or 0.5 mM fluorescently-labelled galactotriose, 0.5 mM UDP-Gal and 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 and 20 mM MnCl2.

Mentions: The difference in GalT activities detected with APTS-labelled and unlabelled acceptors clearly indicates the positive effect of APTS on the ability of galactose-containing oligosaccharides to serve as substrates in β-galactan chain extension reactions. To gain insight into the influence of the chemical structure of APTS on APTS-labelled substrates, experiments were also performed with two other fluorescent labels, ANTS and ANDS, which are also used for fluorophore-assisted carbohydrate electrophoresis (FACE) [29], [35]. Galactotriose was fluorescently-labelled with ANDS and ANTS to give compounds similar to Gal2-gal-APTS. All three fluorescent derivatives were applied as substrates in parallel GalT assays, which were analysed by FACE (Fig. 7). With Gal2-gal-APTS, at least seven product bands were evident, which can be assigned to oligosaccharide products with DP 4–10. In contrast, only minor quantities of products arising from the addition of one or two galactose residues were detectable when either Gal2-gal-ANDS or Gal2-gal-ANTS were used.


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
GalT assay with A. thaliana microsomes and Gal2-gal-APTS, Gal2-gal-ANDS and Gal2-gal-ANTS analysed by FACE as described in section 2.6. The reactions were performed overnight at 20 °C with 0.25 or 0.5 mM fluorescently-labelled galactotriose, 0.5 mM UDP-Gal and 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 and 20 mM MnCl2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036537&req=5

fig7: GalT assay with A. thaliana microsomes and Gal2-gal-APTS, Gal2-gal-ANDS and Gal2-gal-ANTS analysed by FACE as described in section 2.6. The reactions were performed overnight at 20 °C with 0.25 or 0.5 mM fluorescently-labelled galactotriose, 0.5 mM UDP-Gal and 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 and 20 mM MnCl2.
Mentions: The difference in GalT activities detected with APTS-labelled and unlabelled acceptors clearly indicates the positive effect of APTS on the ability of galactose-containing oligosaccharides to serve as substrates in β-galactan chain extension reactions. To gain insight into the influence of the chemical structure of APTS on APTS-labelled substrates, experiments were also performed with two other fluorescent labels, ANTS and ANDS, which are also used for fluorophore-assisted carbohydrate electrophoresis (FACE) [29], [35]. Galactotriose was fluorescently-labelled with ANDS and ANTS to give compounds similar to Gal2-gal-APTS. All three fluorescent derivatives were applied as substrates in parallel GalT assays, which were analysed by FACE (Fig. 7). With Gal2-gal-APTS, at least seven product bands were evident, which can be assigned to oligosaccharide products with DP 4–10. In contrast, only minor quantities of products arising from the addition of one or two galactose residues were detectable when either Gal2-gal-ANDS or Gal2-gal-ANTS were used.

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 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.


Related in: MedlinePlus