Journal of Biological Chemistry

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 enzymes are N-glycosylated, but the biological functions of their glycans have 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. We expressed the enzyme in Chinese hamster ovary Lec1 cells, which produce high-mannose type N-glycans similar to the TS N-glycosylation pattern in vivo. Our MALDI-TOF mass spectrometry data revealed that up to eight putative N-glycosylation sites were glycosylated. In addition, we determined that N-glycan removal via endoglycosidase Hf treatment of TconTS1 led to a decrease in substrate affinity relative to the untreated enzyme but had no impact on the conversion rate. Furthermore, we observed no changes in secondary structure elements of hypoglycosylated TconTS1 in CD experiments. Finally, our 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.

proteoglycan

molecular dynamics

mass spectrometry

enzyme kinetics

CD

N-glycosylation

protein–glycan interactions

trans-sialidases

trypanosoma

N-acetylglucosamine

glycosylphosphatidylinositol

high-performance anion exchange chromatography

hypoglycosylated TconTS1

pulsed amperometric detection

Trypanosoma congolense trans-sialidase 1

Trypanosoma cruzi trans-sialidase

Trypanosoma rangeli sialidase

© 2022 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.