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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

Generated TconTS-LD proteins.A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep: Strep-tag. B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E. coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.
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pntd.0004120.g001: Generated TconTS-LD proteins.A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep: Strep-tag. B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E. coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.

Mentions: To characterise the LDs of TconTS the gene sequences encoding the TconTS1-LD, TconTS2-LD, TconTS3-LD and TconTS4-LD were subcloned into a modified pET28a bacterial expression vector as described under Methods. All proteins comprise a N-terminal poly histidine tag (His-tag) directly attached to maltose binding protein (MBP) as well as C-terminal SNAP- and Strep-tags. In these proteins, the tags flanking TconTS-LD can be enzymatically cleaved using the tobacco etch virus (TEV) and human rhinovirus 3C (HRV 3C) proteases (Fig 1A).


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)

Generated TconTS-LD proteins.A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep: Strep-tag. B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E. coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004120.g001: Generated TconTS-LD proteins.A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep: Strep-tag. B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E. coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.
Mentions: To characterise the LDs of TconTS the gene sequences encoding the TconTS1-LD, TconTS2-LD, TconTS3-LD and TconTS4-LD were subcloned into a modified pET28a bacterial expression vector as described under Methods. All proteins comprise a N-terminal poly histidine tag (His-tag) directly attached to maltose binding protein (MBP) as well as C-terminal SNAP- and Strep-tags. In these proteins, the tags flanking TconTS-LD can be enzymatically cleaved using the tobacco etch virus (TEV) and human rhinovirus 3C (HRV 3C) proteases (Fig 1A).

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