Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in alpha/beta anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal. Glycosaminoglycans (GAGs) are highly anionic functional polysaccharides with subtle structural variations that are very difficult to differentiate. Here the authors demonstrate proof-of-concept single-molecule detection by nanopore, taking a first step towards the ultimate goal of GAG sequencing.

Automated summary

This study demonstrates the label-free detection and analysis of glycosaminoglycan molecules using biological nanopores, reaching a high resolution in deciphering structural information. It is an important step towards the ultimate goal of glycosaminoglycan sequencing.