Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins
GLYCOBIOLOGY(2021)
Abstract
Paucimannosidic glycans are restricted to the core structure [Man(1-3)GlcNAc(2)Fuc(0-1)] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man(2-3)GlcNAc(2)Fuc(1)] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man(3)GlcNAc(2) with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man(3)GlcNAc(2) and its alpha 1,6 core-fucosylated variant, Man(3)GlcNAc(2)Fuc(1). Unexpectedly and in contrast to earlier findings, Man(5)GlcNAc(2)-type structures bind equally well and a large tolerance was observed for substitutions on the alpha 1,6 arm. It was confirmed that any substitution on the single alpha 1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the alpha 1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted alpha 1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.
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Key words
core fucose,IgM,Mannitou,N-glycan,paucimannosidic epitopes
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