Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-β-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.

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 highly N-glycosylated, but the biological functions of the glycans remain elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structure stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. MALDI-TOF MS revealed that eight asparagine sites were glycosylated with high-mannose type N-glycans. Deglycosylation of TconTS1 led to a 5-fold decrease in substrate affinity but to the same conversion rate relative to the untreated enzyme. After deglycosylation, no changes in secondary structure elements were observed in circular dichroism experiments. Molecular dynamics simulations revealed interactions between the highly flexible N-glycans and some conserved amino acids belonging to the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and promoting enzyme/substrate complex stability. The here-observed modulation of catalytic activity via the N-glycan shield may be a structure-function relationship intrinsic of several members of the TS family.

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Most work on trypanosomal TS has been done on enzymatic activities of TS from T. cruzi (causing Chagas disease in Latin America), subspecies of T. brucei, (causing human sleeping sickness in Africa) and T. congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS) lectin domain (LD) binds to several carbohydrates, such as 1,4-β-mannotriose. To investigate the influence of TconTS-LD on enzyme activities, we firstly performed in silico analysis on structure models of TconTS enzymes. Findings strongly supports the potential of domain swaps between TconTS without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear sialidase and sialic acid (Sia) transfer activities, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolysis was drastically reduced. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities favouring trans-sialylation over hydrolysis. In summary, this study provides strong evidence that TconTS-LDs play pivotal roles in modulating enzyme activity and biological functions of these and possibly other TS, revising our fundamental understanding of TS modulation and diversity.

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