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Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

Mariano VS, Zorzetto-Fernandes AL, da Silva TA, Ruas LP, Nohara LL, Almeida IC, Roque-Barreira MC - PLoS ONE (2014)

Bottom Line: Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6.Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose.Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo, Brasil.

ABSTRACT
TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

Show MeSH
Prediction of N-glycosylation sites in human and murine TLR2 and TLR4.The amino acids sequences of the receptors TLR2 and TLR4 were uploaded to the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and submitted for analysis. Potential sites for N-glycosylation are indicated by lines (blue) above the threshold (red line). The x-axis indicates the position in the protein sequence of potential glycosylation sites.
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pone-0098512-g001: Prediction of N-glycosylation sites in human and murine TLR2 and TLR4.The amino acids sequences of the receptors TLR2 and TLR4 were uploaded to the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and submitted for analysis. Potential sites for N-glycosylation are indicated by lines (blue) above the threshold (red line). The x-axis indicates the position in the protein sequence of potential glycosylation sites.

Mentions: TLR2 is directly involved in the protection against P. brasiliensis infection conferred by ArtinM administration [19], and TLR4 plays an important role in the recognition of fungi [36]. Therefore, we selected TLR2 and TLR4 as possible targets for ArtinM recognition. The sequences of TLR2 and TLR4 were examined for potential N-glycosylation sites (Figure 1). Four N-glycosylation sites were predicted in human and murine TLR2; two of them(Asn414 and Asn442) similarly positioned in both protein sequences. A previous study demonstrated that all four predicted sites are occupied by N-glycans, which are involved in receptor synthesis, secretion, and function (Weber, Morse et al., 2004). Numerous N-glycosylation sites were also predicted in TLR4 but none were positioned similarly in the human and murine proteins (Figure 1).


Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

Mariano VS, Zorzetto-Fernandes AL, da Silva TA, Ruas LP, Nohara LL, Almeida IC, Roque-Barreira MC - PLoS ONE (2014)

Prediction of N-glycosylation sites in human and murine TLR2 and TLR4.The amino acids sequences of the receptors TLR2 and TLR4 were uploaded to the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and submitted for analysis. Potential sites for N-glycosylation are indicated by lines (blue) above the threshold (red line). The x-axis indicates the position in the protein sequence of potential glycosylation sites.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043963&req=5

pone-0098512-g001: Prediction of N-glycosylation sites in human and murine TLR2 and TLR4.The amino acids sequences of the receptors TLR2 and TLR4 were uploaded to the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and submitted for analysis. Potential sites for N-glycosylation are indicated by lines (blue) above the threshold (red line). The x-axis indicates the position in the protein sequence of potential glycosylation sites.
Mentions: TLR2 is directly involved in the protection against P. brasiliensis infection conferred by ArtinM administration [19], and TLR4 plays an important role in the recognition of fungi [36]. Therefore, we selected TLR2 and TLR4 as possible targets for ArtinM recognition. The sequences of TLR2 and TLR4 were examined for potential N-glycosylation sites (Figure 1). Four N-glycosylation sites were predicted in human and murine TLR2; two of them(Asn414 and Asn442) similarly positioned in both protein sequences. A previous study demonstrated that all four predicted sites are occupied by N-glycans, which are involved in receptor synthesis, secretion, and function (Weber, Morse et al., 2004). Numerous N-glycosylation sites were also predicted in TLR4 but none were positioned similarly in the human and murine proteins (Figure 1).

Bottom Line: Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6.Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose.Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo, Brasil.

ABSTRACT
TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

Show MeSH