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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.


Binding specificity of TconTS2-LD.A: TconTS2-LD concentration dependent binding to immobilised huS2-Fc (5μg/mL). B: Competitive inhibition of TconTS2-LD binding to huS2-Fc in the presents of serially diluted high-mannose N-glycans. Undiluted inhibitor solution was set to 1.0. The maximum increase in relative fluorescence units (RFU) over time was determined as described under Methods. Data points are means ± standard deviation of triplicates.
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pntd.0004120.g004: Binding specificity of TconTS2-LD.A: TconTS2-LD concentration dependent binding to immobilised huS2-Fc (5μg/mL). B: Competitive inhibition of TconTS2-LD binding to huS2-Fc in the presents of serially diluted high-mannose N-glycans. Undiluted inhibitor solution was set to 1.0. The maximum increase in relative fluorescence units (RFU) over time was determined as described under Methods. Data points are means ± standard deviation of triplicates.

Mentions: We established microtitre plate based binding and inhibition assays to further characterise TconTS-LD binding affinity and specificity. Our glycan array and STD NMR experiments revealed TconTS-LD binding to oligo-mannose oligosaccharides. To further investigate how this specificity mediates interactions of TconTS-LD with glycoproteins, recombinant human Siglec 2 (huS2-Fc, described under Methods) expressed in Chinese hamster ovary Lec1 (CHO-Lec1) cells was used as a model glycoprotein. Due to the lack of N-acetylglucosaminyltransferase 1 (GnT1) CHO-Lec1 cells are unable to synthesise complex and hybrid N-glycan structures. Therefore, these proteins contain only high-mannose glycans of the type Man5GlcNAc2-Asn [41]. Purified huS2-Fc immobilised in microtitre plate wells was incubated with different concentrations of TconTS2-LD and binding was detected as described under Methods. TconTS1-catalytic domain (TconTS1-CD) was used as a control for binding specificity. As shown in Fig 4A, concentration-dependent binding of TconTS2-LD to immobilised huS2-Fc was clearly observed, reaching a maximum intensity at approximately 2 μg/mL TconTS2LD due to saturation of the binding sites. Wells without immobilised huS2-Fc were used as a control. No detectable binding to immobilised huS2-Fc was observed for TconTS1-CD at 4 μg/mL (Fig 4A), confirming the specificity of this assay.


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)

Binding specificity of TconTS2-LD.A: TconTS2-LD concentration dependent binding to immobilised huS2-Fc (5μg/mL). B: Competitive inhibition of TconTS2-LD binding to huS2-Fc in the presents of serially diluted high-mannose N-glycans. Undiluted inhibitor solution was set to 1.0. The maximum increase in relative fluorescence units (RFU) over time was determined as described under Methods. Data points are means ± standard deviation of triplicates.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004120.g004: Binding specificity of TconTS2-LD.A: TconTS2-LD concentration dependent binding to immobilised huS2-Fc (5μg/mL). B: Competitive inhibition of TconTS2-LD binding to huS2-Fc in the presents of serially diluted high-mannose N-glycans. Undiluted inhibitor solution was set to 1.0. The maximum increase in relative fluorescence units (RFU) over time was determined as described under Methods. Data points are means ± standard deviation of triplicates.
Mentions: We established microtitre plate based binding and inhibition assays to further characterise TconTS-LD binding affinity and specificity. Our glycan array and STD NMR experiments revealed TconTS-LD binding to oligo-mannose oligosaccharides. To further investigate how this specificity mediates interactions of TconTS-LD with glycoproteins, recombinant human Siglec 2 (huS2-Fc, described under Methods) expressed in Chinese hamster ovary Lec1 (CHO-Lec1) cells was used as a model glycoprotein. Due to the lack of N-acetylglucosaminyltransferase 1 (GnT1) CHO-Lec1 cells are unable to synthesise complex and hybrid N-glycan structures. Therefore, these proteins contain only high-mannose glycans of the type Man5GlcNAc2-Asn [41]. Purified huS2-Fc immobilised in microtitre plate wells was incubated with different concentrations of TconTS2-LD and binding was detected as described under Methods. TconTS1-catalytic domain (TconTS1-CD) was used as a control for binding specificity. As shown in Fig 4A, concentration-dependent binding of TconTS2-LD to immobilised huS2-Fc was clearly observed, reaching a maximum intensity at approximately 2 μg/mL TconTS2LD due to saturation of the binding sites. Wells without immobilised huS2-Fc were used as a control. No detectable binding to immobilised huS2-Fc was observed for TconTS1-CD at 4 μg/mL (Fig 4A), confirming the specificity of this assay.

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.