A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors

Abstract

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy

“…N -glycans can be prepared by fewer steps starting from a N -glycan precursor obtained from natural sources such as a glycopeptide from egg yolk 21–25 , an invertase glycoprotein from wild-type S. cerevisiae 45 , a lipid-linked oligosaccharide from glyco-engineered E. coli 45 , or bovine fetuin 46 . After deglycosylation of these natural products, glycan cores are obtained that can be modified by a series of glycosidase and glycosyl transferase catalyzed transformations to give more complex structures.…”

Recent Progress in Chemo-Enzymatic Methods for the Synthesis of N-Glycans

“…For example, different human symmetrically N-glycans (e.g. GlcNAc 2 Man 3 GlcNAc 2 ) were assembled from the bacteriaderived core pentasaccharide precursor with subsequent extension by different GnTs (Hamilton et al, 2017). In particular, isolated SGP from egg yolk, which can be trimmed by sialidases and β1, 4-galactosidase to give various complex type N-glycans with symmetrical branches have been widely used as the substrate (Huang et al, 2009;Li et al, 2016b;Wu et al, 2016).…”

“…These GlcNAc moieties could be converted to N-acetyllactosamines (LacNAc) by GalTs, leading to further structural diversity (Cummings, 2009). In the laboratory, the commercially available or expressed recombinant GnTs (e.g., GnT I, GnT II, and GnT IV) could be applied to the preparation of hybrid-and complex type N-glycan libraries including asymmetric multi-antennary complex type N-glycans by structurally remodeling microbial oligosaccharides, which could be used to generate the N-glycan microarrays for highthroughput screening of glycan-binding proteins (Hamilton et al, 2017).…”

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

“…Reactions generally proceed under mild, aqueous conditions without the need for toxic and harsh organic reagents. Using bio- and/or chemoenzymatic synthesis tools, several natural and engineered glycan libraries have recently been constructed including asymmetric multi-antennary N -glycans (Wang Z. et al, 2013 ), glycosphingolipid glycans (Yu et al, 2016 ), authentic human type N -glycans (Li L. et al, 2015 ; Hamilton et al, 2017 ), O -mannosyl glycans (Meng et al, 2018 ; Wang S. et al, 2018 ), human milk oligosaccharides (HMOs) (Xiao et al, 2016 ; Prudden et al, 2017 ), and tumor-associated antigens (Li P. J. et al, 2019 ; 'T Hart et al, 2019 ). Similar strategies have been adopted for cell-free enzymatic synthesis of glycolipid libraries including those from bacterial (Glover K. et al, 2005 ), animal (Stubs et al, 2010 ), and human origins (Li S. T. et al, 2019 ).…”