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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.


STD NMR experiments of TconTS2-LD.STD NMR experiments with 5.5 μM TconTS2-LD were performed as described under Methods. Off-resonance (black lines) and STD NMR (red lines) spectra are shown. A: In the presence of 1.73 mM for 3α,6α-mannotriose; B: In the presence of 3.45 mM lactose; C: In the presence of 1.73 mM 3α,6α-mannotriose and 1.73 mM lactose. D: STD NMR effects for the signals indicated were determined as ratios between the intensities at the indicated ppm in the off-resonance spectra and corresponding STD NMR spectra using the software TopSpin 3.2. M1, M2, M3 and M4 stand for the NMR signals of 3α,6α-mannotriose, and L1, L2, L3 and L4 for those of lactose, for which the STD NMR effects are shown either for the single ligands (spectra shown in A and B) or for the mixture (spectra shown in C). n.d.: not determined, since in the ligands mixture the STD effect for M3 could not be determined from the spectra.
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pntd.0004120.g003: STD NMR experiments of TconTS2-LD.STD NMR experiments with 5.5 μM TconTS2-LD were performed as described under Methods. Off-resonance (black lines) and STD NMR (red lines) spectra are shown. A: In the presence of 1.73 mM for 3α,6α-mannotriose; B: In the presence of 3.45 mM lactose; C: In the presence of 1.73 mM 3α,6α-mannotriose and 1.73 mM lactose. D: STD NMR effects for the signals indicated were determined as ratios between the intensities at the indicated ppm in the off-resonance spectra and corresponding STD NMR spectra using the software TopSpin 3.2. M1, M2, M3 and M4 stand for the NMR signals of 3α,6α-mannotriose, and L1, L2, L3 and L4 for those of lactose, for which the STD NMR effects are shown either for the single ligands (spectra shown in A and B) or for the mixture (spectra shown in C). n.d.: not determined, since in the ligands mixture the STD effect for M3 could not be determined from the spectra.

Mentions: TconTS2-LD showed the highest lectin activity on glycan arrays. Therefore, in further experiments we focused on TconTS2-LD to more fully characterise and confirm the binding of TconTS-LD to both galactose and mannose containing oligosaccharides observed on the glycan array. Several NMR-based methods have been employed to investigate protein carbohydrate interactions on a structural level. For example, line broadening and peak shifts of 1H-NMR signals from amino acid side chains provide information on the type of amino acids involved as well as the occupation of the binding site and thus equilibrium kinetic data, as has been shown for Siglec–1 [38]. Saturation transfer difference (STD) NMR experiments provide important information on the binding epitope of the complexed carbohydrate ligand, since the relative signal intensities of the difference spectra provide direct information on the proximity of the affected protons to the protein [39]. Protein signals are selectively saturated at -1.00 ppm (on-resonance) and subtracted from an off-resonance spectrum (30 ppm) resulting in the final STD NMR spectrum revealing only protons and functional groups of a binding ligand that are in close proximity to the protein surface. Therefore, STD NMR has been widely used to analyse the binding of lectins to their specific carbohydrate ligands. Lactose and α1–3,α1-6-mannotriose were used as ligands for TconTS2-LD as described under Methods. Fig 3A shows the 1H NMR (off resonance) and STD NMR spectra of α1–3,α1-6-mannotriose. The relative signal intensities of the STD spectrum (red line) are almost identical to those of the oligosaccharide 1H NMR spectrum (black line). Binding of lactose to TconTS2-LD was also clearly observed (Fig 3B). It is important to note that relatively strong STD NMR signals at 3.36 ppm (β-GlcH2) and at 3.92 ppm (GalH4) provide good evidence that both monosaccharide units of lactose are in close contact with the protein.


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)

STD NMR experiments of TconTS2-LD.STD NMR experiments with 5.5 μM TconTS2-LD were performed as described under Methods. Off-resonance (black lines) and STD NMR (red lines) spectra are shown. A: In the presence of 1.73 mM for 3α,6α-mannotriose; B: In the presence of 3.45 mM lactose; C: In the presence of 1.73 mM 3α,6α-mannotriose and 1.73 mM lactose. D: STD NMR effects for the signals indicated were determined as ratios between the intensities at the indicated ppm in the off-resonance spectra and corresponding STD NMR spectra using the software TopSpin 3.2. M1, M2, M3 and M4 stand for the NMR signals of 3α,6α-mannotriose, and L1, L2, L3 and L4 for those of lactose, for which the STD NMR effects are shown either for the single ligands (spectra shown in A and B) or for the mixture (spectra shown in C). n.d.: not determined, since in the ligands mixture the STD effect for M3 could not be determined from the spectra.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004120.g003: STD NMR experiments of TconTS2-LD.STD NMR experiments with 5.5 μM TconTS2-LD were performed as described under Methods. Off-resonance (black lines) and STD NMR (red lines) spectra are shown. A: In the presence of 1.73 mM for 3α,6α-mannotriose; B: In the presence of 3.45 mM lactose; C: In the presence of 1.73 mM 3α,6α-mannotriose and 1.73 mM lactose. D: STD NMR effects for the signals indicated were determined as ratios between the intensities at the indicated ppm in the off-resonance spectra and corresponding STD NMR spectra using the software TopSpin 3.2. M1, M2, M3 and M4 stand for the NMR signals of 3α,6α-mannotriose, and L1, L2, L3 and L4 for those of lactose, for which the STD NMR effects are shown either for the single ligands (spectra shown in A and B) or for the mixture (spectra shown in C). n.d.: not determined, since in the ligands mixture the STD effect for M3 could not be determined from the spectra.
Mentions: TconTS2-LD showed the highest lectin activity on glycan arrays. Therefore, in further experiments we focused on TconTS2-LD to more fully characterise and confirm the binding of TconTS-LD to both galactose and mannose containing oligosaccharides observed on the glycan array. Several NMR-based methods have been employed to investigate protein carbohydrate interactions on a structural level. For example, line broadening and peak shifts of 1H-NMR signals from amino acid side chains provide information on the type of amino acids involved as well as the occupation of the binding site and thus equilibrium kinetic data, as has been shown for Siglec–1 [38]. Saturation transfer difference (STD) NMR experiments provide important information on the binding epitope of the complexed carbohydrate ligand, since the relative signal intensities of the difference spectra provide direct information on the proximity of the affected protons to the protein [39]. Protein signals are selectively saturated at -1.00 ppm (on-resonance) and subtracted from an off-resonance spectrum (30 ppm) resulting in the final STD NMR spectrum revealing only protons and functional groups of a binding ligand that are in close proximity to the protein surface. Therefore, STD NMR has been widely used to analyse the binding of lectins to their specific carbohydrate ligands. Lactose and α1–3,α1-6-mannotriose were used as ligands for TconTS2-LD as described under Methods. Fig 3A shows the 1H NMR (off resonance) and STD NMR spectra of α1–3,α1-6-mannotriose. The relative signal intensities of the STD spectrum (red line) are almost identical to those of the oligosaccharide 1H NMR spectrum (black line). Binding of lactose to TconTS2-LD was also clearly observed (Fig 3B). It is important to note that relatively strong STD NMR signals at 3.36 ppm (β-GlcH2) and at 3.92 ppm (GalH4) provide good evidence that both monosaccharide units of lactose are in close contact with the protein.

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.