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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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Digestion of the GalT reaction products with exo-β-(1 → 4)-galactosidase. Black trace: CE-LIF electropherogram of the products of galactan elongation after incubation of Gal-β-(1 → 4)-gal-APTS with GalT and UDP-Gal. Blue trace: CE-LIF analysis of the digestion of elongation products with exo-β-(1 → 4)-galactosidase. The galactosyltransferase reaction was performed for 5 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, 100 μM Gal-gal-APTS, 2.5% glycerol and 0.5 mM UDP-Gal. Enzymatic digestion was performed with 2 μU of exo-β-(1 → 4)-galactosidase (Streptococcus pneumoniae) for 24 h.
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fig4: Digestion of the GalT reaction products with exo-β-(1 → 4)-galactosidase. Black trace: CE-LIF electropherogram of the products of galactan elongation after incubation of Gal-β-(1 → 4)-gal-APTS with GalT and UDP-Gal. Blue trace: CE-LIF analysis of the digestion of elongation products with exo-β-(1 → 4)-galactosidase. The galactosyltransferase reaction was performed for 5 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, 100 μM Gal-gal-APTS, 2.5% glycerol and 0.5 mM UDP-Gal. Enzymatic digestion was performed with 2 μU of exo-β-(1 → 4)-galactosidase (Streptococcus pneumoniae) for 24 h.

Mentions: Detailed examination of the electropherogram originated from the reaction of Gal-β-(1 → 4)-gal-APTS revealed the presence of peaks with retention time between 7 and 10 min (Fig. 3B). When the same reaction was carried out for 5 days these products gave even more distinct evenly spaced peaks in 7–10 min region of CE-LIF trace (Fig. 4, more details in Fig. S3 in SI). Such regularity of appearances of CE signals may indicate that the products are homopolymers which originate from the action of a β-(1 → 4)-polymerising galactosyltransferase. However, clusters of poorly resolved peaks with shorter retention times (4–6.5 min) were also present suggesting possible formation of other types of inter-galactosidic linkages, most likely β-(1 → 3)- and/or β-(1 → 6)-linkages which are known to be present in plant galactans [33]. To verify identities of CE-LIF peaks the reaction mixture obtained from Gal-β-(1 → 4)-gal-APTS was treated with exo-β-(1 → 4)-galactosidase (Streptococcus pneumoniae). This digestion analysis revealed that most of the peaks in the electropherogram disappeared confirming that corresponding reaction products possessed β-(1 → 4)-linkages between galactose units and therefore synthesised by a β-1,4-galactan:β-1,4-galactosyltransferase. Several peaks remained unchanged (Fig. 4) revealing products resistant to β-(1 → 4)-galactosidase digestion. These products are likely to incorporate β-(1 → 3)- or β-(1 → 6)-linked galactose residues located at or close to the non-reducing terminus and blocking the action of the exo-β-(1 → 4)-galactosidase.


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
Digestion of the GalT reaction products with exo-β-(1 → 4)-galactosidase. Black trace: CE-LIF electropherogram of the products of galactan elongation after incubation of Gal-β-(1 → 4)-gal-APTS with GalT and UDP-Gal. Blue trace: CE-LIF analysis of the digestion of elongation products with exo-β-(1 → 4)-galactosidase. The galactosyltransferase reaction was performed for 5 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, 100 μM Gal-gal-APTS, 2.5% glycerol and 0.5 mM UDP-Gal. Enzymatic digestion was performed with 2 μU of exo-β-(1 → 4)-galactosidase (Streptococcus pneumoniae) for 24 h.
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Related In: Results  -  Collection

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fig4: Digestion of the GalT reaction products with exo-β-(1 → 4)-galactosidase. Black trace: CE-LIF electropherogram of the products of galactan elongation after incubation of Gal-β-(1 → 4)-gal-APTS with GalT and UDP-Gal. Blue trace: CE-LIF analysis of the digestion of elongation products with exo-β-(1 → 4)-galactosidase. The galactosyltransferase reaction was performed for 5 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, 100 μM Gal-gal-APTS, 2.5% glycerol and 0.5 mM UDP-Gal. Enzymatic digestion was performed with 2 μU of exo-β-(1 → 4)-galactosidase (Streptococcus pneumoniae) for 24 h.
Mentions: Detailed examination of the electropherogram originated from the reaction of Gal-β-(1 → 4)-gal-APTS revealed the presence of peaks with retention time between 7 and 10 min (Fig. 3B). When the same reaction was carried out for 5 days these products gave even more distinct evenly spaced peaks in 7–10 min region of CE-LIF trace (Fig. 4, more details in Fig. S3 in SI). Such regularity of appearances of CE signals may indicate that the products are homopolymers which originate from the action of a β-(1 → 4)-polymerising galactosyltransferase. However, clusters of poorly resolved peaks with shorter retention times (4–6.5 min) were also present suggesting possible formation of other types of inter-galactosidic linkages, most likely β-(1 → 3)- and/or β-(1 → 6)-linkages which are known to be present in plant galactans [33]. To verify identities of CE-LIF peaks the reaction mixture obtained from Gal-β-(1 → 4)-gal-APTS was treated with exo-β-(1 → 4)-galactosidase (Streptococcus pneumoniae). This digestion analysis revealed that most of the peaks in the electropherogram disappeared confirming that corresponding reaction products possessed β-(1 → 4)-linkages between galactose units and therefore synthesised by a β-1,4-galactan:β-1,4-galactosyltransferase. Several peaks remained unchanged (Fig. 4) revealing products resistant to β-(1 → 4)-galactosidase digestion. These products are likely to incorporate β-(1 → 3)- or β-(1 → 6)-linked galactose residues located at or close to the non-reducing terminus and blocking the action of the exo-β-(1 → 4)-galactosidase.

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