High throughput screening of starch structures using carbohydrate microarrays (original) (raw)

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. Starch is the principal storage polymer in the majority of plants. It accumulates as a complex granular structure made of the glucan homopolymers amylose and amylopectin, organised into large, distinctly shaped insoluble granules. Amylose typically makes up 25-30% of the starch granule, it is an essentially linear molecule that possesses an α-1,4 linkage backbone structure but can be sparsely branched by α-1,6 linkages. Amylose is mostly amorphous in the starch granule. Amylopectin is a highly branched molecule, typically comprises 70-75% of the starch granule, is more than 100-fold larger than amylose, contains clustered α-1,6 linkages which promotes crystalline lamellae to be formed in the granule 1. High throughput (HTP) screening of polysaccharide structures is becoming increasingly important, especially in the field of plant breeding to permit fast evaluation of, for example, mutant collections. Since the discovery that starch binds tri-and polyiodide and forms a strong complex with amylose, this method was used as "amylose indicator", to quantify amylose content in starch. Amylose in the presence of the iodide ligand, changes conformation to left-handed single helix V-amylose which cavity provides space for iodide and results in a bright blue complex 2,3. Starches without amylose make brown-red complex, as iodine binds only weakly to the short helical segments in amylopectin molecules. Spectrophotometric assays were developed based on this complexation 4,5 but most importantly, this technique provides an important HTP screening opportunity and permitted the identification of low amylose potato lines by screening of thousands of lines in a mutant collection 6. This method is also valuable for identifying starches that resists amylolytic degradation, so called resistant starch or RS. These starches are typically characterised by having high amylose but also includes highly branched starches 7-9. However, while iodide complexation can indicate information about amylose-amylopectin ratio and is useful for HTP screening of amylose, this method is not quantitative and does not yield any detailed information about the underlying starch structures. More exact structural information in polysaccharides may be obtained by using the very specific recognition conferred by monoclonal antibodies (mAb) and carbohydrate binding modules (CBMs). Potential starch-binding CBMs are already known, but as often with molecular probes, determining their binding specificity can be a challenging process. However, a HTP method for characterising probes was published recently 10. The method utilizes carbohydrate microarrays populated with defined oligosaccharides conjugated to BSA. One microarray