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Cysteine-rich domain of scavenger receptor AI modulates the efficacy of surface targeting and mediates oligomeric Aβ internalization.

Huang FL, Shiao YJ, Hou SJ, Yang CN, Chen YJ, Lin CH, Shie FS, Tsay HJ - J. Biomed. Sci. (2013)

Bottom Line: The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum.Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer's disease and atherosclerosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy and Cell Biology, National Yang-Ming University, Taipei11221, Taiwan.

ABSTRACT

Background: Insufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer's disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities.

Result: Genetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.

Conclusion: The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer's disease and atherosclerosis.

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Clathrin and dynamin-2 mediate SR-AI-dependent oAβ internalization. A, SR-AI-transfected COS-7 cells were incubated with FAM-oAβ (green) and immunostained with anti-SR-A antibody (red). Representative confocal images showed the co-localization of FAM-oAβ and SR-AI in endocytotic vesicles (left panel). A magnified image of the boxed region was shown in the right panels. Scale bar, 10 μm. B, COS-7 cells were co-transfected with SR-AI and clathrin shRNA or luciferase shRNA (negative control). The confocal images and the quantification of clathrin immunoreactivity showed clathrin shRNA knockdown the expression of clatherin in SR-AI-positive cells. The experiments were repeated at least three times. C. The relative level of internalized oAβ was significantly reduced by clathrin shRNA compared with luciferase shRNA in SR-AI-positive cells. D, COS-7 cells were co-transfected with SR-AI and HA-tagged wild type dynamin-2 or dominant-negative dynamin-2 (K44A). The relative intensity of internalized oAβ was significantly reduced by dynamin-2 (K44A) in SR-AI-positive cells. More than 100 SR-AI-positive cells were analyzed. Bars indicate mean ± SEM of three independent experiments (*p < 0.05).
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Figure 2: Clathrin and dynamin-2 mediate SR-AI-dependent oAβ internalization. A, SR-AI-transfected COS-7 cells were incubated with FAM-oAβ (green) and immunostained with anti-SR-A antibody (red). Representative confocal images showed the co-localization of FAM-oAβ and SR-AI in endocytotic vesicles (left panel). A magnified image of the boxed region was shown in the right panels. Scale bar, 10 μm. B, COS-7 cells were co-transfected with SR-AI and clathrin shRNA or luciferase shRNA (negative control). The confocal images and the quantification of clathrin immunoreactivity showed clathrin shRNA knockdown the expression of clatherin in SR-AI-positive cells. The experiments were repeated at least three times. C. The relative level of internalized oAβ was significantly reduced by clathrin shRNA compared with luciferase shRNA in SR-AI-positive cells. D, COS-7 cells were co-transfected with SR-AI and HA-tagged wild type dynamin-2 or dominant-negative dynamin-2 (K44A). The relative intensity of internalized oAβ was significantly reduced by dynamin-2 (K44A) in SR-AI-positive cells. More than 100 SR-AI-positive cells were analyzed. Bars indicate mean ± SEM of three independent experiments (*p < 0.05).

Mentions: All data were expressed as mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance followed by Tukey’s HSD post hoc tests using SPSS 11.5 software (SPSS Inc., Somers, NY). Values of p < 0.05 were considered statistically significant. Experimental groups labeled with different letters were significantly different from each other. Experimental groups labeled with identical letters were not significantly different from each other. In Figures 1 and 2, asterisks represent statistically significant differences.


Cysteine-rich domain of scavenger receptor AI modulates the efficacy of surface targeting and mediates oligomeric Aβ internalization.

Huang FL, Shiao YJ, Hou SJ, Yang CN, Chen YJ, Lin CH, Shie FS, Tsay HJ - J. Biomed. Sci. (2013)

Clathrin and dynamin-2 mediate SR-AI-dependent oAβ internalization. A, SR-AI-transfected COS-7 cells were incubated with FAM-oAβ (green) and immunostained with anti-SR-A antibody (red). Representative confocal images showed the co-localization of FAM-oAβ and SR-AI in endocytotic vesicles (left panel). A magnified image of the boxed region was shown in the right panels. Scale bar, 10 μm. B, COS-7 cells were co-transfected with SR-AI and clathrin shRNA or luciferase shRNA (negative control). The confocal images and the quantification of clathrin immunoreactivity showed clathrin shRNA knockdown the expression of clatherin in SR-AI-positive cells. The experiments were repeated at least three times. C. The relative level of internalized oAβ was significantly reduced by clathrin shRNA compared with luciferase shRNA in SR-AI-positive cells. D, COS-7 cells were co-transfected with SR-AI and HA-tagged wild type dynamin-2 or dominant-negative dynamin-2 (K44A). The relative intensity of internalized oAβ was significantly reduced by dynamin-2 (K44A) in SR-AI-positive cells. More than 100 SR-AI-positive cells were analyzed. Bars indicate mean ± SEM of three independent experiments (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750411&req=5

Figure 2: Clathrin and dynamin-2 mediate SR-AI-dependent oAβ internalization. A, SR-AI-transfected COS-7 cells were incubated with FAM-oAβ (green) and immunostained with anti-SR-A antibody (red). Representative confocal images showed the co-localization of FAM-oAβ and SR-AI in endocytotic vesicles (left panel). A magnified image of the boxed region was shown in the right panels. Scale bar, 10 μm. B, COS-7 cells were co-transfected with SR-AI and clathrin shRNA or luciferase shRNA (negative control). The confocal images and the quantification of clathrin immunoreactivity showed clathrin shRNA knockdown the expression of clatherin in SR-AI-positive cells. The experiments were repeated at least three times. C. The relative level of internalized oAβ was significantly reduced by clathrin shRNA compared with luciferase shRNA in SR-AI-positive cells. D, COS-7 cells were co-transfected with SR-AI and HA-tagged wild type dynamin-2 or dominant-negative dynamin-2 (K44A). The relative intensity of internalized oAβ was significantly reduced by dynamin-2 (K44A) in SR-AI-positive cells. More than 100 SR-AI-positive cells were analyzed. Bars indicate mean ± SEM of three independent experiments (*p < 0.05).
Mentions: All data were expressed as mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance followed by Tukey’s HSD post hoc tests using SPSS 11.5 software (SPSS Inc., Somers, NY). Values of p < 0.05 were considered statistically significant. Experimental groups labeled with different letters were significantly different from each other. Experimental groups labeled with identical letters were not significantly different from each other. In Figures 1 and 2, asterisks represent statistically significant differences.

Bottom Line: The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum.Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer's disease and atherosclerosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy and Cell Biology, National Yang-Ming University, Taipei11221, Taiwan.

ABSTRACT

Background: Insufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer's disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities.

Result: Genetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.

Conclusion: The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer's disease and atherosclerosis.

Show MeSH
Related in: MedlinePlus