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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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Schematic representation of human SR-AI, SR-AII, and SR-AI variants. Constructs 430, 407, 371, and 341 were SR-AI variants with truncated SRCR domains. Construct 341wirh exon 11 fused to the C-terminus of the collagenous domain mimicking SR-AIII is designated as 341-ex 11. * indicates three lysine residues in the collagenous domain that were mutated to alanine at 332, 335, 338 in 341KA and SR-AIKA. Seven putative N-glycosylation sites in the spacer and coiled coil domains of SR-AI were labeled.
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Figure 3: Schematic representation of human SR-AI, SR-AII, and SR-AI variants. Constructs 430, 407, 371, and 341 were SR-AI variants with truncated SRCR domains. Construct 341wirh exon 11 fused to the C-terminus of the collagenous domain mimicking SR-AIII is designated as 341-ex 11. * indicates three lysine residues in the collagenous domain that were mutated to alanine at 332, 335, 338 in 341KA and SR-AIKA. Seven putative N-glycosylation sites in the spacer and coiled coil domains of SR-AI were labeled.

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)

Schematic representation of human SR-AI, SR-AII, and SR-AI variants. Constructs 430, 407, 371, and 341 were SR-AI variants with truncated SRCR domains. Construct 341wirh exon 11 fused to the C-terminus of the collagenous domain mimicking SR-AIII is designated as 341-ex 11. * indicates three lysine residues in the collagenous domain that were mutated to alanine at 332, 335, 338 in 341KA and SR-AIKA. Seven putative N-glycosylation sites in the spacer and coiled coil domains of SR-AI were labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Schematic representation of human SR-AI, SR-AII, and SR-AI variants. Constructs 430, 407, 371, and 341 were SR-AI variants with truncated SRCR domains. Construct 341wirh exon 11 fused to the C-terminus of the collagenous domain mimicking SR-AIII is designated as 341-ex 11. * indicates three lysine residues in the collagenous domain that were mutated to alanine at 332, 335, 338 in 341KA and SR-AIKA. Seven putative N-glycosylation sites in the spacer and coiled coil domains of SR-AI were labeled.
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