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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.

No MeSH data available.


Schematic representation of RG-I region of the complex multi-component pectin structure (adapted from Somerville et al. [6]). Side chain galactans are composed of β-(1 → 4)-linked galactosyl residues which form the backbone structure which can be further decorated with β-(1,6)-linked galactosyl residues to introduce branching points or α-(1 → 3)- and α-(1 → 5)-linked Araf residues.
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fig1: Schematic representation of RG-I region of the complex multi-component pectin structure (adapted from Somerville et al. [6]). Side chain galactans are composed of β-(1 → 4)-linked galactosyl residues which form the backbone structure which can be further decorated with β-(1,6)-linked galactosyl residues to introduce branching points or α-(1 → 3)- and α-(1 → 5)-linked Araf residues.

Mentions: Pectins are structurally and functionally amongst the most complex polysaccharides [1] and they have versatile functions in plant morphology, growth, development, cell adhesion and plant defence. Pectins and their derivatives also find many applications [2], especially in the food industry as gelling and stabilizing agents. Current research also suggests potential biomedical use for pectins, since it has been shown that they can stimulate the human immune response, lower cholesterol and serum glucose levels, as well as have beneficial effects in cancer [3]. The ability to manipulate pectin structure at the whole plant level therefore has implications for both fundamental science and application. While many advances have been made in the past decade, detailed understanding of the enzymes required for pectin biosynthesis is still at best patchy [4]. With this in mind, we have considered the development of fluorescent glycan probes suitable for the analysis of plant cell wall glycosyltransferase activities [5]. In the current study we report on galactoside-based probes as acceptor substrates for biosynthesis studies on pectic galactan (Fig. 1). This galactan is a linear polymer composed of β-(1 → 4)-linked galactopyranose residues attached via its reducing end to the rhamnopyranose residue of rhamnogalacturonan-I (RG-I) which is one of the pectin backbone polysaccharides.


The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β -galactosyltransferase activities
Schematic representation of RG-I region of the complex multi-component pectin structure (adapted from Somerville et al. [6]). Side chain galactans are composed of β-(1 → 4)-linked galactosyl residues which form the backbone structure which can be further decorated with β-(1,6)-linked galactosyl residues to introduce branching points or α-(1 → 3)- and α-(1 → 5)-linked Araf residues.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig1: Schematic representation of RG-I region of the complex multi-component pectin structure (adapted from Somerville et al. [6]). Side chain galactans are composed of β-(1 → 4)-linked galactosyl residues which form the backbone structure which can be further decorated with β-(1,6)-linked galactosyl residues to introduce branching points or α-(1 → 3)- and α-(1 → 5)-linked Araf residues.
Mentions: Pectins are structurally and functionally amongst the most complex polysaccharides [1] and they have versatile functions in plant morphology, growth, development, cell adhesion and plant defence. Pectins and their derivatives also find many applications [2], especially in the food industry as gelling and stabilizing agents. Current research also suggests potential biomedical use for pectins, since it has been shown that they can stimulate the human immune response, lower cholesterol and serum glucose levels, as well as have beneficial effects in cancer [3]. The ability to manipulate pectin structure at the whole plant level therefore has implications for both fundamental science and application. While many advances have been made in the past decade, detailed understanding of the enzymes required for pectin biosynthesis is still at best patchy [4]. With this in mind, we have considered the development of fluorescent glycan probes suitable for the analysis of plant cell wall glycosyltransferase activities [5]. In the current study we report on galactoside-based probes as acceptor substrates for biosynthesis studies on pectic galactan (Fig. 1). This galactan is a linear polymer composed of β-(1 → 4)-linked galactopyranose residues attached via its reducing end to the rhamnopyranose residue of rhamnogalacturonan-I (RG-I) which is one of the pectin backbone polysaccharides.

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.