Limits...
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.

Show MeSH

Related in: MedlinePlus

The SRCR domain is critical for the surface targeting and N-glycosylation of SR-AI. COS-7 cells were transfected with SR-AI and variants with truncated SRCR domains. A, Surface-targeted SR-AI variants were detected by live immunostaining (green). Cytosolic SR-AI variants were detected by immunocytochemistry (red). The yellow signal in the merged confocal images indicated that SR-AI, 371, and 341 were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 20 μm. B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. β-actin and transferrin receptor (TfR) served as loading controls. 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 and G, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. The experiments were repeated at least three times. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters (a, b, c) were significantly different from each other (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750411&req=5

Figure 4: The SRCR domain is critical for the surface targeting and N-glycosylation of SR-AI. COS-7 cells were transfected with SR-AI and variants with truncated SRCR domains. A, Surface-targeted SR-AI variants were detected by live immunostaining (green). Cytosolic SR-AI variants were detected by immunocytochemistry (red). The yellow signal in the merged confocal images indicated that SR-AI, 371, and 341 were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 20 μm. B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. β-actin and transferrin receptor (TfR) served as loading controls. 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 and G, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. The experiments were repeated at least three times. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters (a, b, c) were significantly different from each other (p < 0.05).

Mentions: Next, we assessed the role of the SRCR domain in the protein trafficking of SR-AI by expressing mutated variants with serial truncations of the SRCR domain in COS-7 cells (Figure 3). The comparable enzymatic activities of co-transfected β-galactosidase across variants suggest that their transfection efficiencies were similar (data not shown). Merged confocal images of live immunostaining and immunocytochemistry showed that full-length SR-AI and deletion variants 371 and 341 were surface-targeted, whereas deletion variants 430 and 407 were retained intracellularly (Figure 4A). The molecular weight of nascent SR-AI is approximately 50 kDa. In the total cell lysates while SR-AI was in the process of protein modifications, a diffuse block was detected by Western bolt analysis. To quantify the expression level of SR-A variants, cell lysates were incubated with PNGase F, which cleaves N-acetylglucosamine from asparagine at N-glycosylation sites. In SR-AI-transfected cell lysate, we detected one major band at 55 kDa and a second band close to 50 kDa (Figure 4B). To investigate the identity of these bands, we performed tandem mass spectrometry analyses after enriching the proteins by immunoprecipitation (data not shown). Although we found that these two bands exhibited partial SR-A sequences, our data was not sufficient to determine the cause of the two bands detected in the cell lysates after PNGase F cleavage. The expression levels of SR-AI variants in the total cell lysates were comparable.


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 SRCR domain is critical for the surface targeting and N-glycosylation of SR-AI. COS-7 cells were transfected with SR-AI and variants with truncated SRCR domains. A, Surface-targeted SR-AI variants were detected by live immunostaining (green). Cytosolic SR-AI variants were detected by immunocytochemistry (red). The yellow signal in the merged confocal images indicated that SR-AI, 371, and 341 were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 20 μm. B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. β-actin and transferrin receptor (TfR) served as loading controls. 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 and G, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. The experiments were repeated at least three times. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters (a, b, c) were significantly different from each other (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The SRCR domain is critical for the surface targeting and N-glycosylation of SR-AI. COS-7 cells were transfected with SR-AI and variants with truncated SRCR domains. A, Surface-targeted SR-AI variants were detected by live immunostaining (green). Cytosolic SR-AI variants were detected by immunocytochemistry (red). The yellow signal in the merged confocal images indicated that SR-AI, 371, and 341 were surface-targeted. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 20 μm. B and C, Western blot analysis of total cell lysates and avidin pull-down of biotinylated lysates after PNGase F cleavage. β-actin and transferrin receptor (TfR) served as loading controls. 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 and G, Transfected cells were incubated with fluorescent oAβ and AcLDL followed by immunostaining using anti-SR-A antibody. The experiments were repeated at least three times. Relative fluorescence intensities of internalized oAβ and AcLDL for more than 100 SR-AI-positive cells were quantified using MetaMorph software. Bars indicate mean ± SEM of three independent experiments. Experimental groups labeled with different letters (a, b, c) were significantly different from each other (p < 0.05).
Mentions: Next, we assessed the role of the SRCR domain in the protein trafficking of SR-AI by expressing mutated variants with serial truncations of the SRCR domain in COS-7 cells (Figure 3). The comparable enzymatic activities of co-transfected β-galactosidase across variants suggest that their transfection efficiencies were similar (data not shown). Merged confocal images of live immunostaining and immunocytochemistry showed that full-length SR-AI and deletion variants 371 and 341 were surface-targeted, whereas deletion variants 430 and 407 were retained intracellularly (Figure 4A). The molecular weight of nascent SR-AI is approximately 50 kDa. In the total cell lysates while SR-AI was in the process of protein modifications, a diffuse block was detected by Western bolt analysis. To quantify the expression level of SR-A variants, cell lysates were incubated with PNGase F, which cleaves N-acetylglucosamine from asparagine at N-glycosylation sites. In SR-AI-transfected cell lysate, we detected one major band at 55 kDa and a second band close to 50 kDa (Figure 4B). To investigate the identity of these bands, we performed tandem mass spectrometry analyses after enriching the proteins by immunoprecipitation (data not shown). Although we found that these two bands exhibited partial SR-A sequences, our data was not sufficient to determine the cause of the two bands detected in the cell lysates after PNGase F cleavage. The expression levels of SR-AI variants in the total cell lysates were comparable.

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