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

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 (sLe(x)) and Sia alpha 2-8-Sia alpha 2-3/6-R structures with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) sialic acid form, LacNAc, Lewis x (Le(x)), alpha-Gal, and Gal alpha 1-3-Le(x); and tandem epitopes including alpha-Gal, Le(x), Gal alpha 1-3-Le(x), LacNAc, and sialyl LacNAc, presented with an internal sialyl LacNAc or 1-2 repeats of an internal LacNAc or Le(x) 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.