Characterization of the Interactions Between High Mannose Glycans and a Novel Prokaryotic Lectin Using Mass Spectrometry-Based Approaches

Importance of the Topic

Lectins are carbohydrate binding proteins with selectivity for specific glycoforms and high mannose glycans play critical roles in immunogenic response and protein stability, especially in therapeutic antibodies. Detailed knowledge of binding kinetics and affinities at the level of individual glycans improves precision of glycosylation profiling in research and quality control.

Study Objectives and Overview

This work aims to investigate the interaction between a novel prokaryotic lectin RPL Man2 and high mannose glycans ranging from Man5 to Man11. By applying mass spectrometry based approaches in solution and immobilized formats the study seeks to establish specificity constants and kinetic parameters for each glycoform.

Methodology and Instrumentation

• Catch and release native mass spectrometry was performed using native electrospray with a SYNAPT XS instrument to measure free and bound glycan populations following collision induced dissociation. Optimal cone voltage and collision voltage were determined for reliable complex survival and release.
• Affinity pull down assays employed His tag immobilization of RPL Man2 on Ni loaded polymer beads with glycan elution by methyl mannose competition. Released glycans were separated by UHPLC on an ACQUITY I Class system and detected via a Xevo G2 XS mass spectrometer.

Main Results and Discussion

• In solution catch and release MS yielded equilibrium constants from 5 micromolar for Man5 to 80 nanomolar for Man11 with tighter binding observed for higher mannose species. The Man7 isomer displayed unexpectedly stronger affinity compared to the Man8 isomers indicating structural diversity within the series.
• Immobilized kinetic assays produced consistent dissociation constants between 0.8 and 4 micromolar across all glycoforms, reflecting combined effects of association and dissociation rates. Differences between in solution and pull down measurements may arise from immobilization induced orientation effects.

Benefits and Practical Applications

The combined mass spectrometry strategies allow direct characterization of lectin glycan binding parameters at the individual glycoform level. This enhances selection of lectins for glycan profiling assays, informs the design of glycoengineering workflows and supports rigorous quality control in biotherapeutic development.

Future Trends and Opportunities

• Extension of this approach to other lectin families and broader glycan panels will expand its utility for glycomics.
• Alternative immobilization chemistries and non specific surface coupling could clarify orientation effects on binding.
• Integration with high throughput glycoproteomics and automated workflows may accelerate glycan screening and method development.

Conclusion

Mass spectrometry based catch and release and pull down assays offer complementary insights into lectin high mannose glycan interactions. The combined data demonstrate the ability to quantify affinity and kinetic parameters for individual glycoforms and lay the foundation for more precise glycan profiling methodologies.

Reference

• S Fernandez Poza A Padros R Thompson L Butler M Islam JA Mosely JH Scrivens MF Rehman Safwan Akram Tailor made recombinant prokaryotic lectins for characterisation of glycoproteins Analytica Chimica Acta 1155 2021 338 352