High throughput screening of starch structures using carbohydrate microarrays.

Vanja Tanackovic,Maja Gro Rydahl,Mohammed Saddik Motawia,Jonatan Ulrik Fangel,Susanne Langgaard Krunic,Henriette Lodberg Pedersen,Maria Dalgaard Mikkelsen,Shahnoor Sultana Shaik,William George Tycho Willats,Andreas Blennow

doi:10.1038/srep30551

Abstract

In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches.

Highlights

Can contain hundreds of distinct samples and an unknown probe can be screened against all the defined samples on the array in a few hours
In this study we further developed carbohydrate microarray analysis by creating microarrays populated with various starch samples including oligo- and polysaccharides, and cereal grain samples transgenically modified to generate specific starch molecular structures
This paper introduces CBM20 as a specific HTP probe for starch and α-glucan recognition in carbohydrate microarray technology

Summary

Introduction

Can contain hundreds of distinct samples and an unknown probe can be screened against all the defined samples on the array in a few hours. In this study we further developed carbohydrate microarray analysis by creating microarrays populated with various starch samples including oligo- and polysaccharides, and cereal grain samples transgenically modified to generate specific starch molecular structures. These arrays were used to determine in detail the recognition profile of CBM20, a known starch-recognising protein module. Glycoamylase consists of two functional domains: N-terminal catalytic domain (residues 1–470, 55 kDa) and a C-terminal CBM 20 (residues 509–616, 12 kDa), which are connected by a heavily O-glycosylated serine/threonine-rich linker It has defined conserved residues crucial for two sites: binding site 1 contains Trp[543] and Trp[590]; while Tyr[527] and Tyr[556] are assigned to the binding site 215,17. The potentially different specificities for linear, branched and phosphorylated maltooligosaccharides were evaluated and specificity assessed in comparison with other polysaccharides using specific antibodies as probes and validated using complex transgenic plant samples

Methods

Results

Conclusion