Cooperativity of catalytic and lectin-like domain of Trypanosoma congolense trans-sialidase modulates its catalytic activity

Abstract

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.

N-glycosylation modulates enzymatic activity ofTrypanosoma congolensetrans-sialidase

“…Whether or not TconTS1 requires N-glycosylation for accurate protein folding cannot be excluded by our experiments, and the role of N-glycans in TconTS folding still needs to be investigated. However, as discussed above our findings on enzymatic activity of bacterially expressed TconTS provide evidence for glycosylation-dependent misfolding of the enzymes (34). Further thermodynamic details of the denaturation processes for the two enzyme forms could be investigated in future works with methods complementary to CD, such as calorimetry.…”

“…Thus, the approach of site-directed glycosylation knockout also has its limitations. Furthermore, for TconTS the orders of magnitude lower specific activity of enzyme expressed in bacteria relative to that expressed by CHO-Lec1 cells provide evidence for misfolding of the enzyme as a consequence of absence of N -glycans (34). Since TconTS1 exhibits nine putative N- glycosylation sites, a first assessment of which sites might be more important than others for enzyme function was desirable and aimed in this study.…”

“…Similar results were found in single-site N -glycosylation mutants of the IBV spike protein by circular dichroism experiments (4). The observed thermal stability of TconTS1 might be explained by the high ß-sheet content of the protein as well as by an extended interface between catalytic and lectin-like domain stabilized by salt bridges and a well-structured hydrogen bond network, making unfolding rather unlikely (34). Although N -glycans do not seem to influence the stability of TconTS1 after successful expression, glycans are known to be required for proper protein folding (56).…”

“…Like all TS, TconTS1 consists of an N -terminal catalytic domain responsible for the transfer of Sia, and of a C-terminal lectin-like domain whose biological function remains unclear. Recently, we demonstrated that the TconTS lectin-like domain can modulate enzymatic activity (34). The catalytic and lectin-like domain are connected via an α-helix (Fig.…”

N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase

“…Like all TS, TconTS1 consists of an N-terminal catalytic domain responsible for the transfer of Sia, and of a C-terminal lectin-like domain whose biological function remains unclear. Recently, we demonstrated that the TconTS lectinlike domain can modulate enzymatic activity (37). The catalytic and lectin-like domain are connected via an α-helix (Fig.…”