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Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense.

Waespy M, Gbem TT, Elenschneider L, Jeck AP, Day CJ, Hartley-Tassell L, Bovin N, Tiralongo J, Haselhorst T, Kelm S - PLoS Negl Trop Dis (2015)

Bottom Line: Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD.This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself.Experimental evidence for such a scenario is presented.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Interactions Bremen, Faculty for Biology and Chemistry, University Bremen, Bremen, Germany.

ABSTRACT
Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented.

No MeSH data available.


Related in: MedlinePlus

Summary of TconTS-LDs binding to glycans as determined by glycan array analysis.TconTS-LDs binding to the glycan arrays was determined as described under Methods. Black bars indicate glycans bound by the TconTS-LDs. The presence and absence of the α-helix in TconTS-LD constructs is indicated with “+” and “-“, respectively. Further binding data (S2 Fig) and all glycans on the arrays (S1 Table) are available as Supporting Information.
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pntd.0004120.g002: Summary of TconTS-LDs binding to glycans as determined by glycan array analysis.TconTS-LDs binding to the glycan arrays was determined as described under Methods. Black bars indicate glycans bound by the TconTS-LDs. The presence and absence of the α-helix in TconTS-LD constructs is indicated with “+” and “-“, respectively. Further binding data (S2 Fig) and all glycans on the arrays (S1 Table) are available as Supporting Information.

Mentions: Glycan array analysis was performed to identify potential TconTS-LD oligosaccharides binding partners. Recombinant TconTS-LD containing His and MBP fusion tags (Table 1 and Fig 1A) were pre-complexed with anti His mouse polyclonal antibody, anti-mouse-IgG-TexasRed conjugated rabbit polyclonal antibody and anti-rabbit-IgG-TexasRed conjugated donkey polyclonal antibody. These were then applied to glycan arrays printed onto SuperEpoxy2 glass slides comprising 367 diverse biologically relevant glycan structures (S2 Fig). The major subset of glycans bound by TconTS-LD are summarised in Fig 2 (full binding data provided in S2 Fig and S1 Table). As expected, initial glycan array experiments revealed signals associated with maltose, maltotriose, isomaltotriose, maltotretraose, isomaltotetraose and related glycans due to the binding of MBP (S1A Fig). Therefore, 10 mM maltose was added as a competitor during binding and washing steps to inhibit the MBP interaction with maltose and related structures present on the arrays. Under these conditions the majority of maltose related signals disappeared. Only some signals for maltotriose, maltotetraose and other maltodextrins remained. Given that maltotriose has a more than 6-fold higher affinity for MBP (Kd: 0.16 μM) compared to maltose (Kd: 1 μM) [37], 10 mM maltotriose instead of maltose was used during binding and 1 mM in all wash steps. Under these conditions, binding of MBP to all remaining maltose related structures was successfully inhibited (S1B Fig). Another option that could have been used to prevent MBP associated binding to our glycan array would have been a proteolytic cleavage using the TEV protease cleavage site of the recombinant TconTS-LD protein (Fig 1A). However, the removal of the MBP-tag and subsequent purification of TconTS-LD leads to low yield of pure TconTS-LD, since often the protease digest is not complete. Therefore, we choose to inhibit MBP binding to maltose-related structures on the glycan arrays with maltotriose in the analyses of all eight TconTS-LD constructs. Glycan array analysis of TconTS2-αHel-LD and TconTS2-LD showed clear binding to several different galactobiose and lactose containing oligosaccharides, as well as to some of their N-actetylamine derivatives listed in Fig 2. Also several fucosylated, and two sialylated glycans were bound, although the binding to these structures was less pronounced compared to unsubstituted N-acetyllactosamine. Whereas binding to potential TS substrates containing galactose was not unexpected, surprisingly, we also observed binding to α1-6-mannobiose and α1–3,α1-6-mannotriose, which was similar for TconTS2-LD with and without the α-helix. No obvious preference of TconTS2-LD for any of the oligomannose isomers present on the array was identified. The number of glycan structures bound by TconTS1-LD was lower than that observed for TconTS2-LD, and no binding to any glycan structures was observed for either TconTS3-LD or TconTS4-LD under the conditions used.


Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense.

Waespy M, Gbem TT, Elenschneider L, Jeck AP, Day CJ, Hartley-Tassell L, Bovin N, Tiralongo J, Haselhorst T, Kelm S - PLoS Negl Trop Dis (2015)

Summary of TconTS-LDs binding to glycans as determined by glycan array analysis.TconTS-LDs binding to the glycan arrays was determined as described under Methods. Black bars indicate glycans bound by the TconTS-LDs. The presence and absence of the α-helix in TconTS-LD constructs is indicated with “+” and “-“, respectively. Further binding data (S2 Fig) and all glycans on the arrays (S1 Table) are available as Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608562&req=5

pntd.0004120.g002: Summary of TconTS-LDs binding to glycans as determined by glycan array analysis.TconTS-LDs binding to the glycan arrays was determined as described under Methods. Black bars indicate glycans bound by the TconTS-LDs. The presence and absence of the α-helix in TconTS-LD constructs is indicated with “+” and “-“, respectively. Further binding data (S2 Fig) and all glycans on the arrays (S1 Table) are available as Supporting Information.
Mentions: Glycan array analysis was performed to identify potential TconTS-LD oligosaccharides binding partners. Recombinant TconTS-LD containing His and MBP fusion tags (Table 1 and Fig 1A) were pre-complexed with anti His mouse polyclonal antibody, anti-mouse-IgG-TexasRed conjugated rabbit polyclonal antibody and anti-rabbit-IgG-TexasRed conjugated donkey polyclonal antibody. These were then applied to glycan arrays printed onto SuperEpoxy2 glass slides comprising 367 diverse biologically relevant glycan structures (S2 Fig). The major subset of glycans bound by TconTS-LD are summarised in Fig 2 (full binding data provided in S2 Fig and S1 Table). As expected, initial glycan array experiments revealed signals associated with maltose, maltotriose, isomaltotriose, maltotretraose, isomaltotetraose and related glycans due to the binding of MBP (S1A Fig). Therefore, 10 mM maltose was added as a competitor during binding and washing steps to inhibit the MBP interaction with maltose and related structures present on the arrays. Under these conditions the majority of maltose related signals disappeared. Only some signals for maltotriose, maltotetraose and other maltodextrins remained. Given that maltotriose has a more than 6-fold higher affinity for MBP (Kd: 0.16 μM) compared to maltose (Kd: 1 μM) [37], 10 mM maltotriose instead of maltose was used during binding and 1 mM in all wash steps. Under these conditions, binding of MBP to all remaining maltose related structures was successfully inhibited (S1B Fig). Another option that could have been used to prevent MBP associated binding to our glycan array would have been a proteolytic cleavage using the TEV protease cleavage site of the recombinant TconTS-LD protein (Fig 1A). However, the removal of the MBP-tag and subsequent purification of TconTS-LD leads to low yield of pure TconTS-LD, since often the protease digest is not complete. Therefore, we choose to inhibit MBP binding to maltose-related structures on the glycan arrays with maltotriose in the analyses of all eight TconTS-LD constructs. Glycan array analysis of TconTS2-αHel-LD and TconTS2-LD showed clear binding to several different galactobiose and lactose containing oligosaccharides, as well as to some of their N-actetylamine derivatives listed in Fig 2. Also several fucosylated, and two sialylated glycans were bound, although the binding to these structures was less pronounced compared to unsubstituted N-acetyllactosamine. Whereas binding to potential TS substrates containing galactose was not unexpected, surprisingly, we also observed binding to α1-6-mannobiose and α1–3,α1-6-mannotriose, which was similar for TconTS2-LD with and without the α-helix. No obvious preference of TconTS2-LD for any of the oligomannose isomers present on the array was identified. The number of glycan structures bound by TconTS1-LD was lower than that observed for TconTS2-LD, and no binding to any glycan structures was observed for either TconTS3-LD or TconTS4-LD under the conditions used.

Bottom Line: Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD.This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself.Experimental evidence for such a scenario is presented.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Interactions Bremen, Faculty for Biology and Chemistry, University Bremen, Bremen, Germany.

ABSTRACT
Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented.

No MeSH data available.


Related in: MedlinePlus