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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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The surface targeting and ligand internalization are abolished by fusing of exon 11 with SR-AI variant 341. A, Surface-targeted SR-A variants were detected by live immunostaining (red). Cytosolic SR-A variants were detected by immunocytochemistry (green). The yellow signal in the merged confocal images indicated that SR-AI and SR-AII were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. D, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. E, Western blot analysis of total cell lysates after PNGase F or Endo H cleavage. F, Lysates of COS-7 cells were immunoprecipitated with anti-SR-A antibody and subjected to Western blot analysis using anti-BiP. The experiments were repeated at least three times. G and H, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-A-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters were significantly different from each other (p < 0.05).
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Figure 5: The surface targeting and ligand internalization are abolished by fusing of exon 11 with SR-AI variant 341. A, Surface-targeted SR-A variants were detected by live immunostaining (red). Cytosolic SR-A variants were detected by immunocytochemistry (green). The yellow signal in the merged confocal images indicated that SR-AI and SR-AII were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. D, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. E, Western blot analysis of total cell lysates after PNGase F or Endo H cleavage. F, Lysates of COS-7 cells were immunoprecipitated with anti-SR-A antibody and subjected to Western blot analysis using anti-BiP. The experiments were repeated at least three times. G and H, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-A-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters were significantly different from each other (p < 0.05).

Mentions: SR-AIII, the splicing isoform of SR-AI with a truncated SRCR domain encoded by exon 11 is intracellularly retained. We have shown that 341 with the collagenous domain only was surface-targeted. The effect of fusing exon 11 with 341 mimicking SR-AIII was examined next. The confocal images of immunostaining confirmed that 341-exon11 was intracellularly retained (Figure 5A). The expression levels of SR-AI, SR-AII, and 341-exon11 in the total cell lysates were comparable (Figure 5B). The surface protein biotinylation assay showed that 341-exon11 was not targeted to the plasma membrane (Figure 5C,D). The surface level of SR-AII was significantly lower than that of SR-AI. Surface-targeted SR-AI and SR-AII were predominantly Endo H-resistant, whereas 341-exon11 was Endo H-sensitive (Figure 5E). It indicated that the fusing of exon 11 with 341 attenuated its N-glycosylation and surface targeting.


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)

The surface targeting and ligand internalization are abolished by fusing of exon 11 with SR-AI variant 341. A, Surface-targeted SR-A variants were detected by live immunostaining (red). Cytosolic SR-A variants were detected by immunocytochemistry (green). The yellow signal in the merged confocal images indicated that SR-AI and SR-AII were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. D, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. E, Western blot analysis of total cell lysates after PNGase F or Endo H cleavage. F, Lysates of COS-7 cells were immunoprecipitated with anti-SR-A antibody and subjected to Western blot analysis using anti-BiP. The experiments were repeated at least three times. G and H, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-A-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters were significantly different from each other (p < 0.05).
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Figure 5: The surface targeting and ligand internalization are abolished by fusing of exon 11 with SR-AI variant 341. A, Surface-targeted SR-A variants were detected by live immunostaining (red). Cytosolic SR-A variants were detected by immunocytochemistry (green). The yellow signal in the merged confocal images indicated that SR-AI and SR-AII were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. D, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. E, Western blot analysis of total cell lysates after PNGase F or Endo H cleavage. F, Lysates of COS-7 cells were immunoprecipitated with anti-SR-A antibody and subjected to Western blot analysis using anti-BiP. The experiments were repeated at least three times. G and H, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-A-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters were significantly different from each other (p < 0.05).
Mentions: SR-AIII, the splicing isoform of SR-AI with a truncated SRCR domain encoded by exon 11 is intracellularly retained. We have shown that 341 with the collagenous domain only was surface-targeted. The effect of fusing exon 11 with 341 mimicking SR-AIII was examined next. The confocal images of immunostaining confirmed that 341-exon11 was intracellularly retained (Figure 5A). The expression levels of SR-AI, SR-AII, and 341-exon11 in the total cell lysates were comparable (Figure 5B). The surface protein biotinylation assay showed that 341-exon11 was not targeted to the plasma membrane (Figure 5C,D). The surface level of SR-AII was significantly lower than that of SR-AI. Surface-targeted SR-AI and SR-AII were predominantly Endo H-resistant, whereas 341-exon11 was Endo H-sensitive (Figure 5E). It indicated that the fusing of exon 11 with 341 attenuated its N-glycosylation and surface targeting.

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