Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins

Abstract

Paucimannosidic glycans are restricted to the core structure [Man1–3GlcNAc2Fuc0–1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] 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 recognise Man3GlcNAc2 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 Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-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 α1–3-linked mannose, and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a non-substituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

Tumor- and immune-derivedN-acetyl-β-D-hexosaminidase drive colorectal cancer and stratify patient risk

“…The core fucosylated M2F and M3F structures (Man2-3GlcNAc2Fuc1) dominated in both CRC tumor tissues and PBMCs covering 60-70% of the paucimannosidic glycans. Recapitulating findings from the glycomics experiments, an increased staining response to Mannitou, a paucimannose-reactive antibody 24,25 , was apparent for tumor tissues from CRC patients (stage II) compared to adjacent normal tissue sections from the same patients ( Figure 1E ).…”

“…To stain anti-inflammatory macrophages known to express CD163 71 , a rabbit monoclonal anti-human CD163 IgG-based antibody (1:100 dilution, 16 h, 4°C, Abcam) was applied, which was then visualized using a fluorophore-labelled goat monoclonal anti-rabbit IgG-Alexa fluor 405 (AF405) IgG (1:400 dilution, 1 h, 20°C, Abcam). As previously reported 24,25 , paucimannosidic glyco-epitopes were detected using a mouse IgM antibody ‘Mannitou’ (kindly provided by Prof Simone Diestel, Univ Bonn, Germany, 1:2 dilution of supernatant collected from cultured hybridomas, 2 h, 20°C). Mannitou was visualized using a monoclonal anti-mouse IgM produced in goat (1:400 dilution, 1 h, 20°C, Thermo) conjugated with Alexa fluor 488 (AF488).…”

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors

“…To obtain the affinity between FimH and ω1-glycoproteins, the single-cycle kinetics SPR technique was applied on a BIAcoreT200 (Cytiva). The ω1-glycoproteins had been produced in glycan-engineered plants and purified as described ( 59 ). The ω1-glycoproteins were immobilized in a single flow cell on a CM5 sensor chip, via primary amino groups using EDC/NHS activation.…”