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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.


Cleavage of N-glycans from TconTS1 and TconTS2.100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoHf glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti-Strep-tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.
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pntd.0004120.g005: Cleavage of N-glycans from TconTS1 and TconTS2.100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoHf glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti-Strep-tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.

Mentions: The enzymatic activities of TconTSs were previously characterised [30,31] using recombinant proteins expressed in CHO-Lec1 cells that therefore contain N-glycans of the high-mannose-type, similar to the recombinant huS2-Fc used here in binding/inhibition assays. N-glycosylation site prediction analysis revealed 8–9 potential sites in TconTSs, and none for the attached SNAP-tag (S3 Fig). In view of the interaction of TconTS-LD with huS2-Fc, we addressed the question of whether TconTS could oligomerise through binding of N-glycans. First, the presence of mannosylated glycans on TconTS1 and TconTS2 expressed in CHO-Lec1 cells was confirmed by lectin blot analysis using concanavalin A (ConA) (Fig 5). To analyse TconTS oligomerisation we used gel permeation chromatography of TconTS expressed in fibroblasts. These proteins contain CD and LD followed by SNAP- and Strep-tags, but no His-MBP at the N-terminus. Fig 6A shows the chromatograms of recombinant TconTS1 and TconTS2 under identical conditions. A double peak of similar intensities was observed for TconTS1, whereas TconTS2 showed a clear single peak with a small shoulder in front of it. The molecular weight (MW) of the TS1 peak eluting at 13 mL (peak 2) was 293 kDa and for that at 16.9 mL (peak 3) was 119 kDa (Fig 6A). This is consistent with peak 3 representing TconTS1 monomers and peak 2 dimers. Furthermore, a molecular mass of 603 kDa calculated for peak 1 eluting at 10 ml is consistent with tetramers of TconTS1. The deviation from the expected monomer (101 kDa without glycosylation) from the calculated MW (119 kDa) can be explained by increased hydrodynamic volumes (Stokes radii) of the oligomeric TconTS1, which is well-known to influence the elution behaviour of a molecule in size-exclusion chromatography [43]. Additionally, glycosylation also influences the protein elution behaviour. For the prominent peak of TconTS2 in Fig 6A eluting at 17 mL (peak 2) a MW of about 113 kDa was calculated, which is consistent with the TconTS2 monomer (100 kDa without glycosylation). The small shoulder at 13.3 mL (peak 1) that represents a MW of about 260 kDa is consistent with the dimeric form of TconTS2 (200 kDa without glycosylation). These findings strongly suggest that both TconTS1 and TconTS2 exist as monomers as well as oligomers in solution, however at different ratios. TconTS1 showed an approximate 1:1 ratio of monomer to dimer, whereas TconTS2 mainly migrates as monomer under the conditions used. To address whether oligomerisation is mediated by N-linked glycans, TconTS1 was enzymatically deglycosylated using EndoHf, and the resulting oligomeric state assessed by size-exclusion chromatography. As shown in Fig 5a clear molecular weight shift as well as a reduction in signal intensity for ConA binding was observed, strongly indicating the release of mannose containing glycans from TconTS1. Subsequent gel permeation chromatography of the deglycosylated TconTS1 resulted in a changed elution profile (Fig 6B, dashed line) compared to the untreated protein (Fig 6B, solid line). Calculating the molecular weight, peak 2 of the deglycosylated protein in Fig 6B is determined as the dimeric form with 260 kDa (elution volume: 13.3 mL) and peak 3 as the monomer with 109 kDa (elution volume: 17.2 mL). The small differences in MW can again be explained by the reduced glycosylation effect on the Stokes radius after EndoHf treatment. Comparing the MW of monomer and dimer from the EndoHf treated sample (peak 2: 260 kDa; 3: 109 kDa, dashed line) to those from the untreated (2: 293 kDa; 3: 119 kDa, solid line), it can be seen that both are decreased due to the loss of high-mannose N-glycans released by EndoHf. Interestingly, it was also observed that EndoHf treatment of TconTS1 reduced the abundance of dimers indicated by the smaller peak 1 (dashed line) in chromatogram B compared to untreated TconTS1 (Fig 6B, solid line). In summary, these results provide strong evidence for oligomerisation of TconTS1 by binding to its N-glycans. Similar, but less pronounced is the oligomerisation of TconTS2.


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)

Cleavage of N-glycans from TconTS1 and TconTS2.100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoHf glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti-Strep-tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004120.g005: Cleavage of N-glycans from TconTS1 and TconTS2.100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoHf glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti-Strep-tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.
Mentions: The enzymatic activities of TconTSs were previously characterised [30,31] using recombinant proteins expressed in CHO-Lec1 cells that therefore contain N-glycans of the high-mannose-type, similar to the recombinant huS2-Fc used here in binding/inhibition assays. N-glycosylation site prediction analysis revealed 8–9 potential sites in TconTSs, and none for the attached SNAP-tag (S3 Fig). In view of the interaction of TconTS-LD with huS2-Fc, we addressed the question of whether TconTS could oligomerise through binding of N-glycans. First, the presence of mannosylated glycans on TconTS1 and TconTS2 expressed in CHO-Lec1 cells was confirmed by lectin blot analysis using concanavalin A (ConA) (Fig 5). To analyse TconTS oligomerisation we used gel permeation chromatography of TconTS expressed in fibroblasts. These proteins contain CD and LD followed by SNAP- and Strep-tags, but no His-MBP at the N-terminus. Fig 6A shows the chromatograms of recombinant TconTS1 and TconTS2 under identical conditions. A double peak of similar intensities was observed for TconTS1, whereas TconTS2 showed a clear single peak with a small shoulder in front of it. The molecular weight (MW) of the TS1 peak eluting at 13 mL (peak 2) was 293 kDa and for that at 16.9 mL (peak 3) was 119 kDa (Fig 6A). This is consistent with peak 3 representing TconTS1 monomers and peak 2 dimers. Furthermore, a molecular mass of 603 kDa calculated for peak 1 eluting at 10 ml is consistent with tetramers of TconTS1. The deviation from the expected monomer (101 kDa without glycosylation) from the calculated MW (119 kDa) can be explained by increased hydrodynamic volumes (Stokes radii) of the oligomeric TconTS1, which is well-known to influence the elution behaviour of a molecule in size-exclusion chromatography [43]. Additionally, glycosylation also influences the protein elution behaviour. For the prominent peak of TconTS2 in Fig 6A eluting at 17 mL (peak 2) a MW of about 113 kDa was calculated, which is consistent with the TconTS2 monomer (100 kDa without glycosylation). The small shoulder at 13.3 mL (peak 1) that represents a MW of about 260 kDa is consistent with the dimeric form of TconTS2 (200 kDa without glycosylation). These findings strongly suggest that both TconTS1 and TconTS2 exist as monomers as well as oligomers in solution, however at different ratios. TconTS1 showed an approximate 1:1 ratio of monomer to dimer, whereas TconTS2 mainly migrates as monomer under the conditions used. To address whether oligomerisation is mediated by N-linked glycans, TconTS1 was enzymatically deglycosylated using EndoHf, and the resulting oligomeric state assessed by size-exclusion chromatography. As shown in Fig 5a clear molecular weight shift as well as a reduction in signal intensity for ConA binding was observed, strongly indicating the release of mannose containing glycans from TconTS1. Subsequent gel permeation chromatography of the deglycosylated TconTS1 resulted in a changed elution profile (Fig 6B, dashed line) compared to the untreated protein (Fig 6B, solid line). Calculating the molecular weight, peak 2 of the deglycosylated protein in Fig 6B is determined as the dimeric form with 260 kDa (elution volume: 13.3 mL) and peak 3 as the monomer with 109 kDa (elution volume: 17.2 mL). The small differences in MW can again be explained by the reduced glycosylation effect on the Stokes radius after EndoHf treatment. Comparing the MW of monomer and dimer from the EndoHf treated sample (peak 2: 260 kDa; 3: 109 kDa, dashed line) to those from the untreated (2: 293 kDa; 3: 119 kDa, solid line), it can be seen that both are decreased due to the loss of high-mannose N-glycans released by EndoHf. Interestingly, it was also observed that EndoHf treatment of TconTS1 reduced the abundance of dimers indicated by the smaller peak 1 (dashed line) in chromatogram B compared to untreated TconTS1 (Fig 6B, solid line). In summary, these results provide strong evidence for oligomerisation of TconTS1 by binding to its N-glycans. Similar, but less pronounced is the oligomerisation of TconTS2.

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.