Overcoming the problem of making libraries of complex sugars
July 25, 2013 § Leave a comment
Much like the way trunks, branches and twigs on trees rise from the ground, long chains of sugars rise from the surfaces of proteins and lipids. These intricately arranged chains, known as glycans, play critical roles in many physiological and pathological processes, but their complexity makes them difficult to analyze in the laboratory.
In a paper just out in Science, researchers describe a synthesis strategy to make large libraries of an important class of glycans, the N-glycans. This strategy can be a useful tool to understand how N-glycans, which are the most complex and diverse of the glycans, often with asymmetric branches with unique structures at their ends, affect many biomolecular interactions, such as in immune responses and pathogen invasion of cells.
“Despite their biological importance, there aren’t methods available to systematically and efficiently produce asymmetrically branched N-glycans needed to populate diverse glycan libraries and investigate the specificities and biology of glycan-binding proteins,” says Geert-Jan Boons at the University of Georgia, lead author on the Science paper.
Efforts to make N-glycans in the lab have focused almost exclusively on the preparation of simpler symmetrical structures. This stems from the difficulties of controlling diversification at the various sites of branching, especially when several different complex terminal structures need to be appended, says Boons. Furthermore, he says, “Isolation of well-defined complex glycans from natural sources is very difficult.”
So Boons and colleagues came up with a way to make libraries of complex, well-defined N-glycans using a combination of chemical and enzymatic reactions. The investigators used a core structure that is common to all eukaryotic N-glycans. This core structure was modified at key branching positions by chemical glycosylation. The ends produced by chemical means were then extended by glycosyltransferases, enzymes that selectively tweaked the ends of these glycans. Boons says the combination approach made it possible “to prepare the most complex N-glycans ever reported.”
To demonstrate the usefulness of their N-glycan libraries, the investigators used them to make a microarray to analyze, among other things, the binding of influenza-virus hemagglutinins to N-glycans. “We demonstrated that complex architectures of glycans determine binding of flu heamagglutinins,” says Boons.
One of the things the investigators now are working on is synthesizing libraries of N-glycans from healthy and diseased cells. “We hope to uncover how changes in cell-surface glycosylation during disease will affect protein binding and cellular physiology,” says Boons.