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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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CE-LIF analyses of GalT assays with APTS-labelled β-(1 → 4)-galacto-oligosaccharides. Arrows show peak positions of acceptor substrates and values in parenthesis indicate percentage of their turnover in the corresponding assay. The galactosyltransferase reaction was performed for 4 h at 15 °C with 50 μg of A. thaliana 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, 50 μM APTS-labelled acceptors and 0.5 mM UDP-Gal.
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fig5: CE-LIF analyses of GalT assays with APTS-labelled β-(1 → 4)-galacto-oligosaccharides. Arrows show peak positions of acceptor substrates and values in parenthesis indicate percentage of their turnover in the corresponding assay. The galactosyltransferase reaction was performed for 4 h at 15 °C with 50 μg of A. thaliana 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, 50 μM APTS-labelled acceptors and 0.5 mM UDP-Gal.

Mentions: In the first series of experiments with APTS-labelled galacto-oligosacharides, GalT assays were carried out with the A. thaliana enzyme preparation at 15 °C for 4 h. Under these conditions, formation of higher oligomers was readily monitored with the help of CE-LIF analysis (Fig. 5). Quantification of acceptor turnover was possible based on CE-LIF and the data are shown above corresponding electropherograms. The turnover increased with increasing acceptor galactan chain length, from a small but detectable extent in the case of Gal-β-(1 → 4)-gal-APTS (Gal-gal-APTS) to nearly complete consumption of the labelled acceptor substrate in case of Gal5-gal-APTS. Similar GalT assays with parent unlabelled galacto-oligosaccharides, followed by APTS labelling, revealed a small amount of polymerisation products only for β-(1 → 4)-galacto-pentaose and –hexaose, whereas chain elongation for β-(1 → 4)-galacto-biose, -triose, and –tetraose was undetectable by CE-LIF analysis (Fig. S6 in SI).


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
CE-LIF analyses of GalT assays with APTS-labelled β-(1 → 4)-galacto-oligosaccharides. Arrows show peak positions of acceptor substrates and values in parenthesis indicate percentage of their turnover in the corresponding assay. The galactosyltransferase reaction was performed for 4 h at 15 °C with 50 μg of A. thaliana 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, 50 μM APTS-labelled acceptors and 0.5 mM UDP-Gal.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig5: CE-LIF analyses of GalT assays with APTS-labelled β-(1 → 4)-galacto-oligosaccharides. Arrows show peak positions of acceptor substrates and values in parenthesis indicate percentage of their turnover in the corresponding assay. The galactosyltransferase reaction was performed for 4 h at 15 °C with 50 μg of A. thaliana 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, 50 μM APTS-labelled acceptors and 0.5 mM UDP-Gal.
Mentions: In the first series of experiments with APTS-labelled galacto-oligosacharides, GalT assays were carried out with the A. thaliana enzyme preparation at 15 °C for 4 h. Under these conditions, formation of higher oligomers was readily monitored with the help of CE-LIF analysis (Fig. 5). Quantification of acceptor turnover was possible based on CE-LIF and the data are shown above corresponding electropherograms. The turnover increased with increasing acceptor galactan chain length, from a small but detectable extent in the case of Gal-β-(1 → 4)-gal-APTS (Gal-gal-APTS) to nearly complete consumption of the labelled acceptor substrate in case of Gal5-gal-APTS. Similar GalT assays with parent unlabelled galacto-oligosaccharides, followed by APTS labelling, revealed a small amount of polymerisation products only for β-(1 → 4)-galacto-pentaose and –hexaose, whereas chain elongation for β-(1 → 4)-galacto-biose, -triose, and –tetraose was undetectable by CE-LIF analysis (Fig. S6 in SI).

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