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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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GalT assay with Gal2-gal-APTS. (A) CE-LIF analysis of reaction products after 17 h incubation; (B) Zoomed in view of electropherogram A. Estimated DPs (total number of Gal and gal residues) of products are shown above selected peaks. Peaks coloured red are likely to represent galactose-containing oligosaccharides with β-(1 → 3)- or β-(1 → 6)-linkages. The galactosyltransferase reaction was performed overnight at room temperature with 50 μg of Arabidopsis microsomal protein in 0.5% Triton X-100 (detergent/protein ratio 5:1), 25 mM MES-KOH buffer pH 6.5 with 15 mM MnCl2, 100 μM Gal2-gal-APTS and 1 mM UDP-Gal.
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fig6: GalT assay with Gal2-gal-APTS. (A) CE-LIF analysis of reaction products after 17 h incubation; (B) Zoomed in view of electropherogram A. Estimated DPs (total number of Gal and gal residues) of products are shown above selected peaks. Peaks coloured red are likely to represent galactose-containing oligosaccharides with β-(1 → 3)- or β-(1 → 6)-linkages. The galactosyltransferase reaction was performed overnight at room temperature with 50 μg of Arabidopsis microsomal protein in 0.5% Triton X-100 (detergent/protein ratio 5:1), 25 mM MES-KOH buffer pH 6.5 with 15 mM MnCl2, 100 μM Gal2-gal-APTS and 1 mM UDP-Gal.

Mentions: Finally, we examined the effect of prolonged action of Arabidopsis microsomes onto Gal3-gal-APTS. When the reaction was carried out overnight at 20 °C a wide range of products with DP reaching more than 60 was identified by CE-LIF (Fig. 6). Product peaks were regularly spaced, except at low retention time (<10 min) where additional set of signals were present (Fig. 6B, coloured red). The latter are likely to originate from products with β-(1 → 3) or β-(1 → 6)-linked galactose residues.


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
GalT assay with Gal2-gal-APTS. (A) CE-LIF analysis of reaction products after 17 h incubation; (B) Zoomed in view of electropherogram A. Estimated DPs (total number of Gal and gal residues) of products are shown above selected peaks. Peaks coloured red are likely to represent galactose-containing oligosaccharides with β-(1 → 3)- or β-(1 → 6)-linkages. The galactosyltransferase reaction was performed overnight at room temperature with 50 μg of Arabidopsis microsomal protein in 0.5% Triton X-100 (detergent/protein ratio 5:1), 25 mM MES-KOH buffer pH 6.5 with 15 mM MnCl2, 100 μM Gal2-gal-APTS and 1 mM UDP-Gal.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig6: GalT assay with Gal2-gal-APTS. (A) CE-LIF analysis of reaction products after 17 h incubation; (B) Zoomed in view of electropherogram A. Estimated DPs (total number of Gal and gal residues) of products are shown above selected peaks. Peaks coloured red are likely to represent galactose-containing oligosaccharides with β-(1 → 3)- or β-(1 → 6)-linkages. The galactosyltransferase reaction was performed overnight at room temperature with 50 μg of Arabidopsis microsomal protein in 0.5% Triton X-100 (detergent/protein ratio 5:1), 25 mM MES-KOH buffer pH 6.5 with 15 mM MnCl2, 100 μM Gal2-gal-APTS and 1 mM UDP-Gal.
Mentions: Finally, we examined the effect of prolonged action of Arabidopsis microsomes onto Gal3-gal-APTS. When the reaction was carried out overnight at 20 °C a wide range of products with DP reaching more than 60 was identified by CE-LIF (Fig. 6). Product peaks were regularly spaced, except at low retention time (<10 min) where additional set of signals were present (Fig. 6B, coloured red). The latter are likely to originate from products with β-(1 → 3) or β-(1 → 6)-linked galactose residues.

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.


Related in: MedlinePlus