Abstract
Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS are N-glycosylated, but the biological functions of their glycans has remained elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structural stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. The enzyme was expressed in CHO Lec1 cells, which produce high-mannose type N-glycans similar to the TS N-glycosylation pattern in vivo. MALDI-TOF MS data revealed that up to eight putative N-glycosylation sites were glycosylated. N-glycan removal via EndoHf treatment of TconTS1 led to a decrease in substrate affinity relative to the untreated enzyme, but apparently has no impact on the conversion rate. No changes in secondary structure elements of hypoglycosylated TconTS1 were observed in circular dichroism experiments. Molecular dynamics simulations provided evidence for interactions between monosaccharide units of the highly flexible N-glycans and some conserved amino acids located at the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and enzyme-substrate complex stability. The here-observed modulation of catalytic activity via N-glycans represents a so far unknown structure-function relationship potentially inherent in several members of the TS enzyme family.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵* Shared co-first authorship
We have included the following - new sets of data, including in particular (i) new biological replicates of the central experiment on the enzymatic activity of TconTS1 in glycolsylated and hypoglycosylated form (ii) new SDS-PAGE experiments with subsequent Coomassie staining (iii) extension of one molecular dynamics run up to a longer simulation time - wholly new and revised analyses of the previous data, in particular (i) a comprehensive revision of the MALDI-TOF peak analysis and presentation of the data in a much clearer and more convincing way (ii) a deeper and more substantiated quantitative assessment of the validity of the homology-modelled structures used in the molecular dynamics simulations - restructured the Supplementary Information into a series of sections containing supplementary text, figures and tables that much better support the claims reported in the main text of the manuscript - added several new references, improved the introduction and complemented the discussion with new arguments - adjusted several details of figures and tables - included supplemental tables, raw data. The DOI of the raw data published in the Zenodo database is reported in the manuscript. - Nilima Dinesh Kumar is added as co-author
https://github.com/IsabellGrothaus/Data_repository/raw/main/Rosenau_Grothaus_2021_plumed.zip