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Chemosensillum immunolocalization and ligand specificity of chemosensory proteins in the alfalfa plant bug Adelphocoris lineolatus (Goeze).

Sun L, Zhou JJ, Gu SH, Xiao HJ, Guo YY, Liu ZW, Zhang YJ - Sci Rep (2015)

Bottom Line: Further cellular investigation of their immunolocalization revealed their dynamic protein expression profiles among different types of antennal sensilla.In ad`dition, a cooperative interaction was observed between two co-expressed CSPs resulting in an increase of the binding affinities by a mixture of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs.These findings in combination with our previous data for AlinCSP1-3 indicate a possible functional differentiation of CSPs in the A. lineolatus olfactory system.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China [2] Key Laboratory of Tea Plants Biology and Resources Utilization of Agriculture Ministry, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China [3] Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Insect chemosensory proteins (CSPs) are a family of small soluble proteins. To date, their physiological functions in insect olfaction remain largely controversial in comparison to odorant binding proteins (OBPs). In present study, we reported the antenna specific expression of three CSPs (AlinCSP4-6) from Adelphocoris lineolatus, their distinct chemosensillum distribution as well as ligand binding capability thus providing the evidence for the possible roles that they could play in semiochemical detection of the plant bug A. lineolatus. The results of qRT-PCR and western blot assay clearly showed that all of these three CSPs are highly expressed in the adult antennae, the olfactory organ of insects. Further cellular investigation of their immunolocalization revealed their dynamic protein expression profiles among different types of antennal sensilla. In a fluorescence competitive binding assay, the selective ligand binding was observed for AlinCSP4-6. In ad`dition, a cooperative interaction was observed between two co-expressed CSPs resulting in an increase of the binding affinities by a mixture of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs. These findings in combination with our previous data for AlinCSP1-3 indicate a possible functional differentiation of CSPs in the A. lineolatus olfactory system.

No MeSH data available.


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A. Binding curves of 1-NPN to binary mixtures of AlinCSP4-6 and relative Scatchard plot analysis.A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 16 μM. The binary mixtures of AlinCSP4 and AlinCSP5 and of AlinCSP4 and AlinCSP6 showed a regular binding behavior in agreement with that obtained for each single protein. AlinCSP5 and AlinCSP6 indicated a different binding behavior, and the Scatchard plot of this combination resembled an inclined letter “J”. B. Binding curves of selected terpenoids to binary mixtures of AlinCSP5 and AlinCSP6. A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 30 μM. The results showed an increasing binding ability compared with the previous data.
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f9: A. Binding curves of 1-NPN to binary mixtures of AlinCSP4-6 and relative Scatchard plot analysis.A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 16 μM. The binary mixtures of AlinCSP4 and AlinCSP5 and of AlinCSP4 and AlinCSP6 showed a regular binding behavior in agreement with that obtained for each single protein. AlinCSP5 and AlinCSP6 indicated a different binding behavior, and the Scatchard plot of this combination resembled an inclined letter “J”. B. Binding curves of selected terpenoids to binary mixtures of AlinCSP5 and AlinCSP6. A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 30 μM. The results showed an increasing binding ability compared with the previous data.

Mentions: We also estimated potential binary interactions among AlinCSP4-6. The results for the binding of 1-NPN to the binary mixtures of AlinCSP4-6 are shown in Figure 9A. The equimolar mixtures of AlinCSP4/AlinCSP5 and of AlinCSP4/AlinCSP6 revealed binding curves that are not different from those of individual proteins. However, the binary mixtures of AlinCSP5 and AlinCSP6 showed an unusual binding behavior: their Scatchard plots displayed a nonlinear correlation trend. We selected three terpenoids, trans-β-farnesene, α-humulene and β-caryophyllene, all of which exhibited weak or no binding affinities to either AlinCSP5 or AlinCSP6 but strong binding to AlinCSP4 (Figure 8), to estimate their binding possibilities to binary mixtures of AlinCSP5 and AlinCSP6. The results show that the combination of AlinCSP5 and AlinCSP6 increased the affinity to these three terpenoids with estimated IC50 values now less than 30 μM (Figure 9B) relative to the binding affinities of individual proteins (Table 1).


Chemosensillum immunolocalization and ligand specificity of chemosensory proteins in the alfalfa plant bug Adelphocoris lineolatus (Goeze).

Sun L, Zhou JJ, Gu SH, Xiao HJ, Guo YY, Liu ZW, Zhang YJ - Sci Rep (2015)

A. Binding curves of 1-NPN to binary mixtures of AlinCSP4-6 and relative Scatchard plot analysis.A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 16 μM. The binary mixtures of AlinCSP4 and AlinCSP5 and of AlinCSP4 and AlinCSP6 showed a regular binding behavior in agreement with that obtained for each single protein. AlinCSP5 and AlinCSP6 indicated a different binding behavior, and the Scatchard plot of this combination resembled an inclined letter “J”. B. Binding curves of selected terpenoids to binary mixtures of AlinCSP5 and AlinCSP6. A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 30 μM. The results showed an increasing binding ability compared with the previous data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4308698&req=5

f9: A. Binding curves of 1-NPN to binary mixtures of AlinCSP4-6 and relative Scatchard plot analysis.A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 16 μM. The binary mixtures of AlinCSP4 and AlinCSP5 and of AlinCSP4 and AlinCSP6 showed a regular binding behavior in agreement with that obtained for each single protein. AlinCSP5 and AlinCSP6 indicated a different binding behavior, and the Scatchard plot of this combination resembled an inclined letter “J”. B. Binding curves of selected terpenoids to binary mixtures of AlinCSP5 and AlinCSP6. A solution of two proteins in Tris-buffer (pH 7.4), both at the concentration of 2 μM, was titrated with 1 mM solution of 1-NPN in methanol to final concentrations ranging from 2 to 30 μM. The results showed an increasing binding ability compared with the previous data.
Mentions: We also estimated potential binary interactions among AlinCSP4-6. The results for the binding of 1-NPN to the binary mixtures of AlinCSP4-6 are shown in Figure 9A. The equimolar mixtures of AlinCSP4/AlinCSP5 and of AlinCSP4/AlinCSP6 revealed binding curves that are not different from those of individual proteins. However, the binary mixtures of AlinCSP5 and AlinCSP6 showed an unusual binding behavior: their Scatchard plots displayed a nonlinear correlation trend. We selected three terpenoids, trans-β-farnesene, α-humulene and β-caryophyllene, all of which exhibited weak or no binding affinities to either AlinCSP5 or AlinCSP6 but strong binding to AlinCSP4 (Figure 8), to estimate their binding possibilities to binary mixtures of AlinCSP5 and AlinCSP6. The results show that the combination of AlinCSP5 and AlinCSP6 increased the affinity to these three terpenoids with estimated IC50 values now less than 30 μM (Figure 9B) relative to the binding affinities of individual proteins (Table 1).

Bottom Line: Further cellular investigation of their immunolocalization revealed their dynamic protein expression profiles among different types of antennal sensilla.In ad`dition, a cooperative interaction was observed between two co-expressed CSPs resulting in an increase of the binding affinities by a mixture of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs.These findings in combination with our previous data for AlinCSP1-3 indicate a possible functional differentiation of CSPs in the A. lineolatus olfactory system.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China [2] Key Laboratory of Tea Plants Biology and Resources Utilization of Agriculture Ministry, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China [3] Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Insect chemosensory proteins (CSPs) are a family of small soluble proteins. To date, their physiological functions in insect olfaction remain largely controversial in comparison to odorant binding proteins (OBPs). In present study, we reported the antenna specific expression of three CSPs (AlinCSP4-6) from Adelphocoris lineolatus, their distinct chemosensillum distribution as well as ligand binding capability thus providing the evidence for the possible roles that they could play in semiochemical detection of the plant bug A. lineolatus. The results of qRT-PCR and western blot assay clearly showed that all of these three CSPs are highly expressed in the adult antennae, the olfactory organ of insects. Further cellular investigation of their immunolocalization revealed their dynamic protein expression profiles among different types of antennal sensilla. In a fluorescence competitive binding assay, the selective ligand binding was observed for AlinCSP4-6. In ad`dition, a cooperative interaction was observed between two co-expressed CSPs resulting in an increase of the binding affinities by a mixture of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs. These findings in combination with our previous data for AlinCSP1-3 indicate a possible functional differentiation of CSPs in the A. lineolatus olfactory system.

No MeSH data available.


Related in: MedlinePlus