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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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Both SRCR and collagenous domains of SR-AI are oAβ- and AcLDL-binding domains. A and B, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. C, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. D, Western blot analysis of total lysates of transfected cells after PNGase F or Endo H cleavage. The experiments were repeated at least three times. E and F, Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI variant-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 6: Both SRCR and collagenous domains of SR-AI are oAβ- and AcLDL-binding domains. A and B, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. C, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. D, Western blot analysis of total lysates of transfected cells after PNGase F or Endo H cleavage. The experiments were repeated at least three times. E and F, Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI variant-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: The collagenous domain has been identified as AcLDL binding domain [33]. Next, we examined whether the SRCR domain also mediates the ligand binding. Variants 341 and Δ273-341 lacked the SRCR and collagenous domain, respectively. Variant 272 lacked both the SRCR and collagenous domains. The protein level of 272 was higher than that of 341 and Δ273-341 in the total cell lysates (Figure 6A). The surface biotinylation assay and Western bolt analysis showed that all these deletion mutants were surface-targeted (Figure 6B). The densitometry analysis indicated similar surface protein levels of 341 and Δ273-341 (Figure 6C). Both Δ273-341 and 272 were predominately Endo H-resistant (Figure 6D). The surface-targeting of SR-AI, 341, and Δ273-341was further confirmed by the confocal images of surface-bound oAβ on the plasma membrane of SR-AI, 341, and Δ273-341-transfected cells (Additional file 4: Figure S3A). 341 and Δ273-341 internalized approximately 50% of the oAβ and AcLDL internalized by SR-AI (Additional file 4: Figure S3B,C and Figure 6E,F). These results indicated that the SRCR domain functioned as a binding domain for oAβ and AcLDL in the absence of the collagenous domain.


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)

Both SRCR and collagenous domains of SR-AI are oAβ- and AcLDL-binding domains. A and B, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. C, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. D, Western blot analysis of total lysates of transfected cells after PNGase F or Endo H cleavage. The experiments were repeated at least three times. E and F, Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI variant-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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750411&req=5

Figure 6: Both SRCR and collagenous domains of SR-AI are oAβ- and AcLDL-binding domains. A and B, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. C, Relative levels of surface-targeted SR-AI variants were quantified by densitometry. D, Western blot analysis of total lysates of transfected cells after PNGase F or Endo H cleavage. The experiments were repeated at least three times. E and F, Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI variant-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: The collagenous domain has been identified as AcLDL binding domain [33]. Next, we examined whether the SRCR domain also mediates the ligand binding. Variants 341 and Δ273-341 lacked the SRCR and collagenous domain, respectively. Variant 272 lacked both the SRCR and collagenous domains. The protein level of 272 was higher than that of 341 and Δ273-341 in the total cell lysates (Figure 6A). The surface biotinylation assay and Western bolt analysis showed that all these deletion mutants were surface-targeted (Figure 6B). The densitometry analysis indicated similar surface protein levels of 341 and Δ273-341 (Figure 6C). Both Δ273-341 and 272 were predominately Endo H-resistant (Figure 6D). The surface-targeting of SR-AI, 341, and Δ273-341was further confirmed by the confocal images of surface-bound oAβ on the plasma membrane of SR-AI, 341, and Δ273-341-transfected cells (Additional file 4: Figure S3A). 341 and Δ273-341 internalized approximately 50% of the oAβ and AcLDL internalized by SR-AI (Additional file 4: Figure S3B,C and Figure 6E,F). These results indicated that the SRCR domain functioned as a binding domain for oAβ and AcLDL in the absence of the collagenous domain.

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