Identification of the binding roles of terminal and internal glycan epitopes using enzymatically synthesized N-glycans containing tandem epitopes

Abstract

Glycans play diverse roles in a wide range of biological processes. Research on glycan-binding events is essential for learning their biological and pathological functions. However, the functions of terminal and internal glycan epitopes exhibited during binding with glycan-binding proteins (GBPs) and/or viruses need to be further identified. Therefore, a focused library of 36 biantennary asparagine (Asn)-linked glycans with some presenting tandem glycan epitopes was synthesized via a combined Core Isolation/Enzymatic Extension (CIEE) and one-pot multienzyme (OPME) synthetic strategy. These N-glycans include those containing a terminal sialyl N-acetyllactosamine (LacNAc), sialyl Lewis x (sLex) and Siaα2–8-Siaα2–3/6-R structures with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) sialic acid form, LacNAc, Lewis x (Lex), α-Gal, and Galα1–3-Lex; and tandem epitopes including α-Gal, Lex, Galα1–3-Lex, LacNAc, and sialyl LacNAc, presented with an internal sialyl LacNAc or 1–2 repeats of an internal LacNAc or Lex component. They were synthesized in milligram-scale, purified to over 98% purity, and used to prepare a glycan microarray. Binding studies using selected plant lectins, antibodies, and viruses demonstrated, for the first time, that when interpreting the binding between glycans and GBPs/viruses, not only the structure of the terminal glycan epitopes, but also the internal epitopes and/or modifications of terminal epitopes needs to be taken into account.

An enzymatic strategy to asymmetrically branched N-glycans

“…8 And due to the diversity and complexity, as well as low abundance of most structures, it is extremely challenging to isolate adequate amounts of N -glycans with specific structures from natural sources. On the other hand, despite numerous efforts on chemical synthesis of N -glycans, 9 only a few groups had recently developed strategies for synthesizing libraries of structurally defined N -glycans, including Boons’s general strategy for chemoenzymatic synthesis of asymmetric N -glycans, 6, 10 Wong’s modular synthesis of HIV N -glycans, 11 and our core synthesis/enzymatic extension (CSEE) strategy to prepare N -glycan isomers. 12 Nevertheless, all strategies require expertise and proficient skills for chemical synthesis of key asymmetric N -glycan core or modular structures for further enzymatic diversification.…”

“…10b Herein, we introduce a novel enzymatic strategy to generate asymmetric N -glycans excluding any chemical synthesis. Briefly, two symmetric N -glycan structures, N001 and N301 (Fig.…”

Converting Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) to a regioselective α2–6-sialyltransferase by saturation mutagenesis and regioselective screening

“…10 Both DSLNnT and DSLNT were effective in protecting neonatal rats from necrotizing enterocolitis in an animal model and could be potentially developed into therapeutic agents. 9, 10 This property of Pd2,6ST also allowed facile synthesis of multisialylated poly- N -acetyllactosamine (poly-LacNAc) extensions on O- and N-linked glycans for microarray studies, 12, 22 as well as the synthesis of glycans with α2–3-sialylation at the terminal Gal and α2–6-sialylation at the internal Gal. 22, 23 However, it generated complications for synthesizing the terminal disialyl tetrasaccharide sequence of gangliosides GD1α, GT1aα, GQ1bα.…”

“…9, 10 This property of Pd2,6ST also allowed facile synthesis of multisialylated poly- N -acetyllactosamine (poly-LacNAc) extensions on O- and N-linked glycans for microarray studies, 12, 22 as well as the synthesis of glycans with α2–3-sialylation at the terminal Gal and α2–6-sialylation at the internal Gal. 22, 23 However, it generated complications for synthesizing the terminal disialyl tetrasaccharide sequence of gangliosides GD1α, GT1aα, GQ1bα. 21 In addition, it would not be suitable for obtaining mono-α2–6-sialylated longer glycans such as α2–6-linked sialyl LNT or sialyl LNnT structures in the milk and on the cell surfaces of human and animals.…”

Facile Chemoenzymatic Synthesis of O‐Mannosyl Glycans

“…[16] These GTs are highly active and recognize minimal motifs (e.g., GlcNAc for NmLgtB, LN for Pd26ST and Hp3FT, LN or Le X for PmST1-M144D) on oligosaccharide acceptors, and have been previously applied for preparing complex glycans. [17] …”