Limits...
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.

No MeSH data available.


Related in: MedlinePlus

CE-LIF traces of the GalT assay with APTS-labelled disaccharides (peaks marked with an asterisk correspond to starting disaccharides) and UDP-Gal. A, Full scale and B, zoomed-in views. The assays were performed for 2 days at 20 °C with 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 with 15 mM MnCl2, 200 μM APTS-labelled acceptor, 2.5% glycerol and 0.5 mM UDP-Gal.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036537&req=5

fig3: CE-LIF traces of the GalT assay with APTS-labelled disaccharides (peaks marked with an asterisk correspond to starting disaccharides) and UDP-Gal. A, Full scale and B, zoomed-in views. The assays were performed for 2 days at 20 °C with 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 with 15 mM MnCl2, 200 μM APTS-labelled acceptor, 2.5% glycerol and 0.5 mM UDP-Gal.

Mentions: The resolving power of CE enabled analyses of enzyme assays with the three isomeric Gal-containing acceptors (Fig. 3), which revealed that these rather minimal models of β-galactan are all acted upon by Arabidopsis galactosyltransferases, albeit at extended time (CE-LIF data show results after 48 h incubation). The efficiencies of acceptor substrate conversion and products distributions are different in each case. If homo-oligomerisation takes place, then CE-LIF would be expected to exhibit a series of regularly separated peaks, which is clearly not the case for major products formed from any of these three acceptors investigated. The lack of regularity in the CE-LIF electropherograms is indicative of the formation of more than one linkage type during the extension of these acceptor substrates. Assays with Gal-β-(1 → 4)-gal-APTS showed relatively low turnover of the substrate, but minor amounts of polymerised products were detectable up to degree of polymerisation (DP) ∼15. In contrast, a range of seven major products with >50% overall conversion of the acceptor substrate was found when a similar reaction was performed with Gal-β-(1 → 4)-glc-APTS. Similar to this result, reactions with Gal-β-(1 → 6)-gal-APTS contained a range of different, irregularly distributed products. Overall these experiments demonstrated that even simple monosaccharide structures tethered to APTS can indeed serve as competent plant galactosyltransferase acceptor substrates. However, the configuration of alditol portion of the APTS-labelled acceptor as well as the position of alditol galactosylation substantially influence the enzymatic reaction.


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
CE-LIF traces of the GalT assay with APTS-labelled disaccharides (peaks marked with an asterisk correspond to starting disaccharides) and UDP-Gal. A, Full scale and B, zoomed-in views. The assays were performed for 2 days at 20 °C with 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 with 15 mM MnCl2, 200 μM APTS-labelled acceptor, 2.5% glycerol and 0.5 mM UDP-Gal.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig3: CE-LIF traces of the GalT assay with APTS-labelled disaccharides (peaks marked with an asterisk correspond to starting disaccharides) and UDP-Gal. A, Full scale and B, zoomed-in views. The assays were performed for 2 days at 20 °C with 100 μg of microsomal protein in 1.25% Triton X-100 (detergent/protein ratio 5:1), 25 mM Mes-KOH buffer pH 6.5 with 15 mM MnCl2, 200 μM APTS-labelled acceptor, 2.5% glycerol and 0.5 mM UDP-Gal.
Mentions: The resolving power of CE enabled analyses of enzyme assays with the three isomeric Gal-containing acceptors (Fig. 3), which revealed that these rather minimal models of β-galactan are all acted upon by Arabidopsis galactosyltransferases, albeit at extended time (CE-LIF data show results after 48 h incubation). The efficiencies of acceptor substrate conversion and products distributions are different in each case. If homo-oligomerisation takes place, then CE-LIF would be expected to exhibit a series of regularly separated peaks, which is clearly not the case for major products formed from any of these three acceptors investigated. The lack of regularity in the CE-LIF electropherograms is indicative of the formation of more than one linkage type during the extension of these acceptor substrates. Assays with Gal-β-(1 → 4)-gal-APTS showed relatively low turnover of the substrate, but minor amounts of polymerised products were detectable up to degree of polymerisation (DP) ∼15. In contrast, a range of seven major products with >50% overall conversion of the acceptor substrate was found when a similar reaction was performed with Gal-β-(1 → 4)-glc-APTS. Similar to this result, reactions with Gal-β-(1 → 6)-gal-APTS contained a range of different, irregularly distributed products. Overall these experiments demonstrated that even simple monosaccharide structures tethered to APTS can indeed serve as competent plant galactosyltransferase acceptor substrates. However, the configuration of alditol portion of the APTS-labelled acceptor as well as the position of alditol galactosylation substantially influence the enzymatic reaction.

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