An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells

The structural diversity of glycans on cells – the glycome – is vast and complex to decipher. Glycan arrays have played a pivotal role in exploring the informational content of glycans. Current glycan arrays display oligosaccharides generated by chemical and chemoenzymatic synthesis or isolated from biological sources and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources, produced through considerable efforts, and they display individual saccharides without the natural context of other glycans and glycoconjugates at the cell surface. We usedmaps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic and structural basis for glycan binding epitopes in a natural context. This expandable and self-renewable cell-based glycan array reports glycosyltransferase genes required (or blocking) for interactions through logic sequential biosynthetic steps, which not only predicts essential structural features of involved glycans including the glycoconjugate context, but importantly provides instructions for enzymatic synthesis, recombinant production, and genetic strategies to dissect biological functions. The broad utility of this cell-based glycan array and its comprehensive read-out is demonstrated by dissecting glycan-binding specificities of microbial adhesins, and the discovery power is demonstrated by uncovering higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.