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

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.


Abstract 27
Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the  Smith et al. 1996), but also influences the overall Sia 74 transfer and sialidase activities (Smith & Eichinger 1997). Amino acid sequence alignments 75 of a well characterised sialidase from M. viridifaciens (Gaskell et al. 1995) with TcruTS 76 revealed that R572 and E578, which are known to be essential for galactose binding of M. 77 viridifaciens sialidase (Gaskell et al. 1995), are well conserved in TcruTS and TranSA (Smith 78 & Eichinger 1997). Point mutation of one of these residues resulted in reduced sialidase 79 activities for both enzymes and enhanced Sia transfer activity in TcruTS (Smith & Eichinger 80 1997). As a consequence of these findings Smith and Eichinger predicted both amino acid 81 residues (R572 and E578 in TcruTS) to be involved in galactose binding of the acceptor 82 and/or donor substrates, which would necessarily require an overall protein folding that 83 brings the catalytic domain and the region containing the Arg and Glu of Fn3 domain (at 84 least R and E) close together (Smith & Eichinger 1997). However, the resolved crystal found to change Sia transfer and sialidase activities, respectively (Smith & Eichinger 1997). 94 Interestingly, structural and amino acid sequence alignments of TcruTS (EMBL:  First evidence for a more pivotal role of the LDs has come from our phylogenetic analysis 102 done separately on CD and LD of TconTS (Gbem et al. 2013 O-linked glycans on glycoproteins or as part of their glycosylphosphatidylinositol (GPI)-108 anchor on the parasite's surface (Savage et al. 1984;Zamze et al. 1990;Zamze et al. 1991; 109 Bayne et al. 1993;Beecroft et al. 1993;Bütikofer et al. 2002;Thomson et al. 2002). 110 Therefore, these glycans potentially function as ligand structures for TconTS-LD. 111 Furthermore, also TS were found to be glycosylated, predominantly with N-linked glycans   Here we report a strategy to effectively swap CDs and LDs from different TconTS in order 124 to elucidate the functional relationship between these domains and to investigate a potential    343  369  392  422  449  477  501  524  551  577   328  354  377  407  434  462  486  509  537  563   335  361  384  414  441  469  494  517  544  570   398  424  447  477  504  532  556  579  606  632   248  274  297  327  353  381  409  433  460  485   355  381  411  441  468  496  521  544  571  597   270  296  319  349  375  403  431  455  482  507   415  444  474  504  536  564  594  625  654  680 of TconTS1a using the crystal structure of TcruTS (PDB code: 3B69) as template structure as described under 151 Methods. Area 1 and 2, comprising a network of hydrogen bonds formed by amino acid residues at the 152 interface between catalytic domain (CD in grey), interdomain α-helix (blue) and lectin domain (LD in green) 153 are marked. The active centre containing the catalytic tyrosine residue Tyr438 at the CD is labelled with a 154   Table 1. It can be seen that the overall surface area of the contact sites

188
One site (Area 1) is closer to the α-helix connecting both domains ( Figure 1A and B), 189 whereas the second (Area 2) is located opposite of area 1 ( Figure 1A    Energy minimised homology models revealed 13 hydrogen bonds formed at the interface 197 between CD, LD and the α-helix for TconTS3, 12 for TconTS1, and 11 for both, TconTS2  (Table 3). For example,

202
TconTS and TbruTS are highly conserved in these contact areas among each other with no 203 more than three amino acid variations from the consensus sequence (Table 3

459
Interestingly, in the presence of 1,4β-mannotriose the release of free Neu5Ac was about 6-460 fold lower than without this trisaccharide, resulting in a corresponding increase in the ratio 461 of TS over sialidase activity (  These results can be explained by cooperativity between CD and LD in TconTS1a, which 481 catalytic activity is modulated by oligosaccharide binding to its LD as discussed below.

483
In silico structural insights into the contact site between CD and LD of trypanosomal TS 484 Buschiazzo and co-worker first observed that the interface between TcruTS-CD and LD is TconTS family members ( Figure 1D, Table 3), indicating a high evolutionary pressure to 501 maintain these critical amino acids in all TconTS (Gbem et al. 2013). When calculating the hydrogen bond network formed between residues at the interface between CD and LD, 11 503 to 13 potential hydrogen bonds were observed for TconTS1a through TconTS4, which is 504 similar to the number found in TranSA (Amaya et al. 2003). This is in contrast to other 505 sialidases, where only about half of the number have been found. Mainly two separate 506 areas, 1 and 2, comprise the majority of hydrogen bonds formed, evenly distributed over 507 both ( Figure 1A). Area 1 is located at the region where CD, LD and the α-helix are in close 508 contact ( Figure 1A and B), whereas Area 2 is located more closely to the active site of CD 509 ( Figure 1A and C). We assume that the contacts at Area 2 keep the catalytic grove of CD Strikingly, amino acid residues involved in the hydrogen bond network formation at both 513 sites, 1 and 2, are well conserved (Table 3). Since all these amino acid residues are highly 514 conserved among TS family despite their different catalytic activities (e.g. of TranSA), it 515 appears unlikely that their conservation is essential for a specific catalytic activity. to the corresponding holoenzymes, TconTS1a and TconTS3 (Figure 2). Interestingly, when 527 investigating the interface between CD and LD of TconTS1a/TS3, 13 hydrogen bonds were 528 observed formed by amino acid residues, which were predicted to be essential for conformation stability of the wild type TconTS as discussed above. These observations 530 underline the possibility that such an rearrangement of TconTS genes has occurred during 531 evolution, which would provide an explanation for the different phylogenetic relationships of 532 CD and LD in TS from African trypanosomes (Gbem et al. 2013).

533
At first sight, the α-helix itself might present a potential target for the restriction site 534 introduction, but amino acid sequence alignments of TconTS revealed that the majority of 535 amino acid residues of the α-helix are well conserved ( Figure 1D, Figure 3A and Table 3).  (Table 1). In addition, the model 544 also revealed an extended hydrogen bond network between CD and LD of domain swapped 545 TconTS1a/TS3 (Table 2 and (Table 5). Most 553 importantly, the TS/sialidase ratio of TconTS1a* is similar to that of TconTS1a in expression 554 systems (Table 5).

555
The literature shows that TS were predominantly expressed in bacteria (Buschiazzo et al. 2002;Coustou et al. 2012) but also in eukaryotic cells (Coustou et

581
The presence of several putative N-glycosylation sites in TconTS indicates that these 582 enzymes contain N-glycans in both, CD and LD. Interestingly, the TconTS mAb 7/23, binds more strongly to eTconTS1a compared to pTconTS1a ( Figure 5). It is unlikely that this is 584 due to improper folding in the bacteria, since the anti-TconTS mAb 7/23 binds to the SDS-585 denatured protein in this experiment. However, it is likely that for efficient binding of mAb 586 7/23 to TconTS1-LD is supported by at least one N-glycan. Since PNGase treatment of 587 eTconTS1a strongly reduced binding of this antibody ( Figure 5). In this context it is important 588 to note that native glycosylated TconTS was used for immunisation to generate this antibody 589 (Tiralongo et al. 2003). Along this line, it must be keep in mind that the eukaryotic expression  increased more than five-fold, due to a suppression in sialidase activity (Table 5) For example it has been reported that glutamic acid/alanine-rich protein (GARP), a T. 629 congolense stage specific glycoprotein, was co-purified with TS-form 1 but not TS-form 2, 630 both isolated from T. congolense procyclic cultures (Tiralongo et al. 2003). Interestingly, for 631 TS-form 1 significantly higher TS/sialidase ratio was observed, whereas relative sialidase 632 activity was higher in TS-form 2, although for both preparations, TS-form 1 and TS-form 2, 633 the same donor and acceptor substrate preferences were described (Tiralongo et al. 2003).

634
Furthermore, GARP is glycosylated with high-mannose type and galactosyl 635 oligosaccharides ( Thomson et al. 2002) and is shown to be sialylated in TS reactions 636 (Engstler et al. 1995). Together, these findings provide strong evidence for its multivalent binding potential to TconTS-CD and LD, as discussed earlier